PROTHROMBIN AND FIBRINOGEN

Technical field of the invention

The present invention relates to a method for the production of a human coagulation factor. Specifically, the coagulation factor is produced in a human host cell. The invention also relates to the use of the recombinant human coagulation factor in medicine.

Background of the invention

Coagulation (thrombogenesis) is the process by which blood forms clots. It is an important part of hemostasis, the cessation of blood loss from a damaged vessel, wherein a damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop bleeding and begin repair of the damaged vessel. Disorders of coagulation can lead to an increased risk of bleeding (hemorrhage) or obstructive clotting (thrombosis)

Thrombin is the key to coagulation. If thrombin is present then clotting will proceed. Thrombin is derived from an inactive precursor called prothrombin.

There are two pathways that lead to the conversion of prothrombin to thrombin; (1) the intrinsic pathway and (2) the extrinsic pathway.

The intrinsic and the extrinsic pathway are triggered by different elements.

However, at a certain point the intrinsic and extrinsic pathways converge - i.e. by the activation of factor X (Stuart- Prower factor). Activated factor X is an enzyme that converts prothrombin to thrombin. Thrombin converts fibrinogen to fibrin monomers, which then polymerize into fibrin fibers. The mechanical stability of the fibrin clot depends on the properties of the fibrin monomer. In some cases, depending on the quality of the fibrinogen, fibrin fibers may form a loose meshwork that is stabilized by crosslinks created by factor XIII. The clot traps red blood cells and platelets and thus stops the flow of blood . Commercial prothrombin products are purified from pooled human and animal blood products (blood or plasma) and the risk of of contamination is therefore present. Besides from comprising contaminating proteins such products may also comprise antigen and may therefore induce autoimmunity. Moreover prothrombin products purified from blood pruducts are less stable and must be stored at low temperatures in order to avoid degradation.

Coagulation factors such as fibrinogen and prothrombin has been produced in e.g. HEK cells, however, such cells have a significant limitation due to their co- production of proteases. The abundance of proteases limits the yield of recombinant protein obtained in HEK cells and the production of recombinant proteins in HEK cells therefore requires addition of a broad range of protease inhibitors. Additonally the HEK cells cannot be grown to high cell densities high enough to provide a yield wich is commercially relevant.

Hence, an improved method for the production of one or more coagulation factors without the above disadvantages would be advantageous and a valuable contribution to the art.

Summary of the invention

Thus, an object of the present invention relates to a method for the production of a recombinant coagulation factor. It is furthermore an object ot the present invention to provide a recombinant coagulation factor wich is safe to use in medicine and which can be obtained in sufficient quantities and thereby satisfy the requirements for industrial size production. Thus, one aspect of the invention relates to a method for the production of a coagulation factor, said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with at least one exogenous nucleic acid sequence encoding said coagulation factor, wherein the the human host cell, is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic membrane and an immortalised cell from the amniotic fluid.

Another aspect of the present invention relates to a human host cell selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amnion and an immortalised cell from the amniotic fluid, said cell comprising an exogenous nucleotide sequence encoding a coagulation factor.

Yet another aspect of the present invention is to provide a recombinant coagulation factor obtainable by the method according to the present invention.

Still another aspect of the present invention is to provide a protein composition comprising a recombinant coagulation factor, characterised in that (i) said protein composition comprises substantially no synthetic protease inhibitors, and/or (ii) said protein composition is serum free.

A further aspect of the present invention is to provide a kit comprising (i) a scaffold comprising prothrombin and (ii) CaCI ₂ . Yet another aspect of the present invention is to provide a scaffold, comprising prothoombin and CaCI ₂ , wherein said prothrombin and said CaCI ₂ is present in different parts of the scaffold.

An aspect of the present invention is also to provide a recombinant coagulation factor according to the present invention, or a protein composition according to the present invention, for use as a medicament, as a tissue sealant or to facilitate tissue adherence.

A further aspect of the present invention is the use of a recombinant coagulation factor according to the present invention, or a protein composition according to the present invention, for the preparation of a medicament for the treatment or prevention of bleedings.

An aspect of the present invention includes a method for preventing, treating and/or alleviating bleedings comprising administering to a subject in need thereof a recombinant coagulation factor according to the present invention, or a protein composition according to the present invention.

The present invention will now be described in more detail in the following .

Detailed description of the invention

Definitions

In the present context the terms:

(i) prothrombin is to be understood as a glycoprotein comprising at least a Gla domain and two kringle domains (kringle 1 and kringle 2) and a serine protease domain. Such prothrombin may be WT or M400A - the latter a prothrombin comprising a mutation.

Prior to discussing the present invention in further details, the following terms and conventions will first be defined :

In the context of the present invention, the term "fibrinogen" may refer to any of the forms of fibrinogen and includes variants which have arisen through genetic polymorphisms, differences in glycosylation and phosphorylations, (partial) proteolysis of the carboxy-terminal part of the Aa chain and alternative splicing .

In the context of the present invention, the term "prothrombin" may refer to any of the forms of prothrombin and includes variants which have arisen through genetic polymorphisms, differences in glycosylation and phosphorylations and alternative splicing .

An Expression Vector is usually a DNA molecule, which contains, among others, a DNA sequence, which is encoding a protein of interest together with a promoter and other sequences, such as a transcription terminator and polyadenylation signal, that facilitate expression of the protein. The expression vectors contain genetic information which provides for replication in a host cell, either by autonomous replication or by integration into the host genome. It is evident to one skilled in the art that such information that provides for the autonomous replication of an expression vector in a host cell encompasses known yeast and bacterial origins of replication.

Transformation is generally applied to microorganisms.

Transfection is generally applied to cells from multicellular organisms.

Both processes consist of stably and hereditably changing the genotype of a recipient cell by introduction of purified DNA, a part selected from a DNA library.

Cultured cell : A cell capable of being cultured in medium over a number of generations. In the case of cells derived from multicellular organisms, a cultured cell is a cell isolated from the organism as a single cell, a tissue, or an explant from a tissue.

A DNA Construct is a DNA molecule, or it could be a clone of such a molecule, and could be either single or double-stranded. This molecule will have been modified through human manipulation of certain segments of DNA combined and juxtaposed in a manner, which normally does not exist in nature. A DNA construct may contain operably linked elements, which direct the transcription and translation of the DNA sequence encoding the polypeptides that are of interest. Such elements include promoters, enhancers and transcription terminators. If such a DNA construct encompass the encoding of a polypeptide of interest, which contains a secretory signal sequence, the DNA construct such elements will be considered to be capable of directing the secretion of the polypeptide. HPC 4 is defined as an attached marker, to which one has a monoclonal antibody to identify the molecule during the process of splitting the prothrombin to preprothrombin.

By the prefix "h" of proteins is intended to mean the human wild type variant of the protein. Wild type is normally prefixed by wt. By the prefix "r" of proteins is intended to mean a recombinant protein, which has been prepared by recombinant expression. By the prefix "rh" of proteins is intended to mean the human wild type variant of the protein which has been prepared by recombinant expression.

By the term "sequence identity", such as "nucleic acid sequence identity" or "amino acid sequence identity" in this context equals quantitative measure of the degree of homology between two amino acid sequences or between two nucleic acid sequences that are optimally aligned. The two sequences shall the insertion of gaps or, alternatively, truncation at the ends of the polypeptide sequences or nucleotide sequences. Sequence identity as used herein is the number of aligned amino acids which are identical, divided by the total number of amino acids in the shorter of the two sequences being compared.

By the term "more efficiently" in relation to clotting of fibrinogen by recombinant human thrombin as compared to human natural thrombin is intended to mean that a smaller amount of recombinant human thrombin is needed to provide a similar response to human natural thrombin.

The following definitions are adapted from the US patent application

#20120040400. The term "permanent cell lines", as used herein, relates to cells being genetically modified in such a way that they may continue to grow permanently in cell culture under suitable culture conditions. Such cells are also called immortalized cells.

The term "polypeptide" or "recombinant polypeptide", as used herein, relates to peptides consisting of at least 2 amino acids. The polypeptide can be modified co- and/or post-translationally, e.g., by the attachment of sugar residues or by modification of amino acid residues. The polypeptide can be linear, circular or branched. Furthermore, the polypeptide can consist of more than one amino acid chain, wherein the chains may adopt more or less complex three-dimensional structures by intra- and/or intermolecular bonds (e.g ., secondary, tertiary, quaternary structure). If the polypeptide consists of one amino acid chain it can adopt more or less complex three-dimensional structures also by intramolecular bonds. The polypeptides can be pharmacologically or immunologically active polypeptides or polypeptides used for diagnostic purposes. The term "primary cells", as used herein, relates to cells that were obtained by direct removal from an organism or a tissue and put in culture. Primary cells exhibit only a very limited life span.

The term "production cell lines", as used herein, relates to permanent cell lines that were genetically stable modified by the introduction of a transgene encoding the desired polypeptide to be produced.

The term "CAP" as used herein, relates to a permanent human amniocytic cell line generated by immortalization of primary human amniocytes with adenoviral gene functions E1A and E1B.

The term "CAP-T", as used herein, relates to CAP-cells that are in addition stably transfected with a nucleic acid molecule containing the sequence of the SV40 large T-antigen.

The term "transfection", as used herein, relates to any method suitable for the introduction of the mentioned nucleic acid(s) into the cells. As examples the classical calcium phosphate method, electroporation, liposomal systems of any kind and combinations of these methods are to be mentioned.

The term "transient expression" as used herein, relates to any method in which nucleic acid(s) are introduced into the cell by transfection without the selection of stable cell lines by a suitable selection method, said stable cell lines can be onwards cultured in cell culture permanently.

The term "stabile expression", as used herein, relates to the expression of a transgene in production cell lines.

The term "transgene", as used herein, relates to the nucleic acid sequence encoding a recombinant polypeptide. Coagulation factor

In one embodiment the the coagulation factor may be selected from the group consisting of Fibrinogen (Factor I), Prothrombin (Factor II), Thrombin, Tissue factor (Factor III), Labile factor (Factor V), Proconvertin (Factor VII),

Antihaemophilic factor (Factor VIII), Christmas factor (Factor IX), Stuart-Prower factor (Factor X), Plasma thromboplastic (Factor XI), Hageman factor (Factor XII), Fibrin-stabilizing factor (transamidase or Factor XHIa), von Willebrand factor, Prekallikrein (Fletcher factor) and high-molecular-weight kininogen (HMWK) and Fibronectin.

In a preferred embodiment the coagulation factor may be selected from the group consisiting of Fibrinogen (Factor I), Prothrombin (Factor II), Thrombin, Tissue factor (Factor III), Labile factor (Factor V), and Fibrin-stabilizing factor

(transamidase or Factor XHIa).

In a preferred embodiment the coagulation factor is Fibrinogen (Factor I).

Fibrinogen pay an important role in managing major bleedings. Fibrinogen concentrate can reduce blood transfusion when given as intraoperative, targeted, first line hemostatic therapy in bleeding patents undergoing aortic replacement surgery (Rahe-Meyer et al. 2013).

It may be contemplated that the recombinant human coagulation factors of the present invention is compared to wild type human coagulation factor.

Prethrombin, Prothrombin and thrombin

In a preferred embodiment the coagulation factor is thrombin (Factor II).

Prothrombin is defined as a two chain, disulphide-bonded, glycosylated

polypeptide that cleaves specific bonds in fibrinogen to produce fibrin monomers that self-assemble to form a fibrin clot, when activated to thrombin.

When mixing prothrombin, activated to thrombin with fibrinogen the final stage of the natural fibrin clotting cascade takes place. In addition to providing hemostasis, this fibrin sealant also provides ideal environment for fibroblastic proliferation as well as proliferation of other mesenchymal cells.

It may be preferred that the recombinant human prothrombin of the present invention is gla-domain-prothombin. The gla-domain containing prothrombin is connected to the post-translational modifications of prothrombin to thrombin. More specifically the gamma-carboxyglutamyl residues, which bind calcium ions, result from the carboxylation of glutamyl residues by a microsomal enzyme, the vitamin K-dependent carboxylase. The modified residues are necessary for the calcium-dependent interaction with a negatively charged phospholipid surface, which is essential for the conversion of prothrombin to thrombin. It is therefore contemplated that the prothrombin of the present invention is gla-domain- prothombin since it is possible to obtain activated thrombin after activation using calcium chloride.

One objective of the present invention is to provide methods for producing prothrombin using recombinant methods in human host cells to primarily produce prothrombin in placenta derived cell lines and/or in amnion cells, among those in immortalized amnion cell lines such as Cap/CAP-T cells.

The thrombin mutants may retain comparable fibrinogen cleavage activity, but have higher expression level and may have the advantage of being produced at lower manufacturing cost. It is also within the scope of this invention that prothrombin as well as

prothrombin mutants retain comparable expression level but display higher fibrinogen cleavage activity and thus these mutants may lower therapeutic dosage. Further, it is within the scope of this invention that the prothrombin mutants that display reduced autolysis, which are an integrated part of this invention, may facilitate purification process and storage.

As described above it is further envisioned in this invention that mutants that lack protein C activation may have improved coagulation activity. Mutation can be identified by sequence comparison among prothrombin family members, rational site-directed mutagenesis or alanine-scanning. It is envisioned genes can be modified via insertions, substitutions, deletions or domain swapping . The methods within the scope of this invention is built on producing recombinant mammal prothrombin or more specifically recombinant human prothrombin and even more specific recombinant human prothrombin analogue(s), and yet even more specific a recombinant human prothrombin analogue, called M84A, also called M400A; when all of the above recombinant human thrombins or thrombin analogue(s) are made from "gla domain prothrombin" gene, meaning that the deduced protein of recombinant human prothrombin contains signal peptide, propeptide, Gla domain, two kringle domains and a (two-chain) protease domain, the M84A or M400A analogue would be called "prothrombin analogue M400A. In a specific embodiment of the present invention the gene sequence expressed according to the invention is any molecule that exhibit prothrombin activity.

In a specific embodiment of the present invention the gene sequence expressed according to the invention is any sequence, which encodes a molecule that exhibit prothrombin activity. The gene sequence expressed according to the invention can encode natural human prothrombin or any mutant thereof. Thus, in one embodiment of the invention, prothrombin encoded by the polynucleotide has a sequence at least 80% identical to the human prothrombin sequence. Fibrinogen

In this invention the fibrinogen is co-expressed by the fibrinogen human genes encoding α, β and γ chains.

Fibronectin

Fibronectin has emerged as player in platelet thrombus formation and diseases associated with thrombosis including vascular remodelling, atherosclerosis, and cardiac repair following a myocardial infarct or infarction (Maurer et al. 2010).

It is contemplated that the recombinant human coagulation factor has a specific activity in the range from 10 - 5000 U/mg protein, such as in the range from 100- 4000 U/mg protein, e.g . in the range from 500 - 3000 U/mg protein, such as in the range from 1000 - 2000 U/mg protein. For fibrinogen to induce a clotting in minor but crucial bleedings, as for instance during surgery performed in small organ parts as eyesurgery, and intervention into small areas of the brain the need of fibrinogen for the clotting may be theoretically be down to 1 mg of fibrinogen per ml, which may demand as little as 10 to 100 U/ mg protein of the coagulation factor per mg fibrinogen. Therefore the amount of the coagulation factor needed is very much depending upon how much proteins such as for instance fibrinogen that shall produce the clotting or hemostasis. This could of course be necessary to enable dosage of the coagulation factor to the amount of protein that needs to be converted, without being too large and over-filling amount in areas where microsurgery application is performed. Therefore the range of the coagulation factor needed may theoretically range from 10 units to 5000 units. Human host cell

In one aspect the present invention pertains to a method for the production of a coagulation factor, said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with at least one exogenous nucleic acid sequence encoding said coagulation factor, wherein the the human host cell, is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic membrane and an immortalised cell from the amniotic fluid.

In an embodiment the human host cell may be artificially immortalized.

The human host cell of the present invention are non-embroynic and include all placenta cells (incl. the tissue and fluid) but excluding the baby and umbilical cord it self. In a further embodiment the human host cell may be a transformed primary human amniocyte. The transformed primary human amniocyte may be a CAP or CAP-T cell line. The CAP and CAP-T cells are immortalized by a function not oncogenic in human Such CAP or CAP-T cell lines and the production of such cell lines have been descriped by Schiedner et al. 2008a and 2008b and Bernd Voedisch 2012.

In an embodiment the transformed primary human amniocyte is the cell line which is deposited in the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) Mascheroder Weg lb, D-38124 Braunschweig on 26 October 1999DSM under accession number DSM ACC2416.

Protein Glycolysation

Posttranslational modification performed in human cells, influence several physical properties and chemical properties such as glycosylation, phosphorylation, carbocylation, palmitoylation, and neuraminoglycosilation, etc. are of significant importance for the properties of the coagulation factor. Thus, It is an object of the present invention to provide recombinant human coagulation factor essentially comprising a glycosylation patterns substantially identical to that of the natural human coagulation factor.

Thus, in an embodiment at least 90% of the recombinant coagulation factor has a glycolysation pattern that results in an immunogenicity response substantially identical to that of the natural human coagulation factor.

Thus, in one embodiment at least 90% of the recombinant coagulation factor has a glycolysation pattern that results in an immunogenicity response substantially identical to that of the natural human coagulation factor, such as at least 91%, at least 92%, least 93%, at least 94%, least 95%, at least 96%, least 97%, at least 98%, least 99%, or 100%, e.g . in the range from 90-100%, such as in the range from 92-99%, such as in the range from 93-98%, such as in the range from 94-97%, such as in the range from 95-96%.

In a preferred embodiment the coagulation factor is selected from the group consisting of fibrinogen, prothrombin, thrombin, tissue factor, Labile factor and fibrin stabilizing factor. Suitable expression systems

The uniqueness of the invention described in this patent application is the fact that it is possible to produce considerable amount of recombinant human coagulation factors utilizing the plasmid vector HZec6. HZec6 is used for HEK cells but can be amplified by adding an amplifying vector unique for instance for amnion cells such as CAP and CAP-T cells, which increase the yield of protein produced and secreted to significantly higher amount than can be obtained by HEK cells, and at the same time apparently do not amplify cytologic and secreted proteases to a significant degree.

Construction of the expression vector

In one embodiment the human host cell may comprise an expression vector comprising a nucleotide sequence encoding a coagulation factor.

Thus, the expression vector may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4 (fibrinogen alpha chain), 8 (fibrinogen beta chain), 12 (fibrinogen gamma chain), 14 (human prothrombin M400A coding sequence), 16 (human prothrombin WT coding sequence), 18 (human

prethrombin M400A coding sequence) and 20 (human prethrombin WT coding sequence).

More specifically the human host cell e.g . the CAP/CAP-T cells may be transfected by cloning of the three different Fibrinogen chains (alpha, beta, gamma) :

•Fibrinogen alpha into pStbl-Neo-CMV-MCS (-) (G418/Neormycin Resistance) •Fibrinogen beta into pStbl-Bsd-CMV-MCS (-) (Blasticidin Resistance)

"Fibrinogen gamma into pStbl-Hygro-CMV-MCS (-) (Hygromycin Resistance).

Culturing conditions

In order to obtain such high yields the oxygen tension must be closely regulated. Method for the preparation of recombinant coagulation factor

The recombinant human coagulation factors produced by the method of the present invention may be less heterogenic, but on the contrary, more

homogenous, enabling a better consistency in the final protein product, more reproducible from lot to lot, functionally more active and less immunogenic due to the lesser difference in the sugar molecules, which otherwise may be obtained in cells such as insect cells or CHO cells. At the same time the final product will also be virus free. According to the method as claimed in this invention we are capable of generating stable cell lines and easily make cells adapted to serum-free suspension.

The host cells of the present inventions allows a stable and high yield expression of the coagulation factor(s) of the present invention. They may be grown in suspension under serum-free to high densities in different types of bioreactos.

Mammalian cells are generally cultured in commercially available serum- containing or serum-free media. Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art. Serum-free culture medium for placenta and for amnion cell cultures or cells lines and their suitable growth and expression media may be used to further improve protein production yields.

Selection of optimal media is within the level of the skill in the art. Certain thrombin precursors may preferably be produced from transfectants by the addition of heparin or thrombin. The activation of thrombin precursors containing a thrombin cleavage site in place of the wild-type thrombin activation site (Arg- Ile) may be enhanced by heparin added to the medium . Preferably, in this scenario, between 0.5 and 5.0 U/ml of heparin is added to the serum-free medium, more preferably between 1 and 5 U/ml and most preferably 1 U/ml of heparin is added to the serum-free medium according to some authors. To activate the protein produced from human placenta cell lines or from amnion cells more preferably will have an anticipated activity between 1 and 2 mu.g/ml of thrombin, with 1 mu.g/ml of thrombin added to the serum-free medium as being particularly preferred.

Thus, in an embodiment of the present invention the human host cell may be cultured to a cell density of above 10 ³ cells per ml. Traditionally used HEK cells may grow to cell densities of at the most 10 ³ cells per ml. It is therefore indeed surprising that the host cell of the present invention may grow to densities as high as 10 ⁹ in suspension under serum-free conditions. Thus, in an embodiment of the present invention the human host cell may be cultured to a cell density of above 10 ³ cells per ml, such as above 10 ⁴ cells per ml, e.g. above 10 ⁵ cells per ml, such as above 10 ⁶ cells per ml, e.g . above 10 ⁷ cells per ml, such as above 10 ⁸ cells per 5 ml, e.g . above 10 ⁹ cells per ml, e.g . above 10 ¹⁰ cells per ml, the cell count of which may be further improved, and thereby have a pronounced influence on the yield of protein produced.

Initially 500.000 cells per ml is seeded in serum free medium and after 1 week 10 10 ³ - 10 ¹⁰ cells per ml may be obtained .

The amniotic cell line of the present invention e.g . the CAP or CAP-T cell lines, does, unlike many embryonal cell, not need to be maintained by Zeocin for killing none producing cells during processing the transfected cell line and retaining the

15 protein production according to the transfected gene or genes. Unlike the HEK cell line the inventors of the present invention surprisingly found that the human host cell line(s) of the present invention does not produce or secrete matriptase-2, which in HEK cells exists both as single monomers and as complexes in cellular membranes. Matriptase-2 is one of the many proteases secreted into the culture

20 medium thus, cutting the proteins - such proteins may e.g . be sectreted

coagulation factors, such as fibrinogen or prothrombin. The abundant protease expression in HEK cells virtually abort an industrial production of e.g. fibrinogen or prothrombin.

25 Another important ability of the human host cell of the present invention such as the CAP and/or CAP-T cells is the fact that they (in contrast to HEK cells) possess inherent capabilities to express and secrete complex and difficult target molecules such as fibrinogen thus, adding an attractive alternative and advantage over HEK cells.

30

Additionally, the different subtypes of HEK cells have identical production of a broad spectrum of proteases that make it almost impossible to obtain measurable amounts of e.g. recombinant fibrinogen. It was found that the crude recombinant HEK cell medium first of all were at levels averaging 15 mg per liter cells (with a 35 cell density of 3 to 4 million HEK cells per liter). Afterwards one had to quickly cool the harvested medium down for the purification procedure, and even during such cold purifications, the harvest of final crude recombinant fibrinogen was down to 2- 10% of the original amount of 15 mg/liter of recombinant fibrinogen. Furthermore, even when performing the first purification of the HEK produced 5 fibrinogen, a major part of the harvested purified small amount of recombinant fibrinogen, showed extremely low polymerization when tested. As described this first part of the fibrinogen harvested was eluted with 20mM arginine, whereas the very small part of the purified HEK fibrinogen, showing polymerization at the level of CHO cell recombinant fibrinogen was eluted when increasing the arginine from 10 20 mM to 50 mM. This part was an even smaller part of the HEK recombinant fibrinogen that could be purified. These small amounts of fibrinogen that could polymerize, will only have academic interest, and can not be brought to any significant industrial producion level at all even when using a mixture of proteases already added during the cessation of the HEK cell production run.

15

The host cells of the present invention (e.g. the CAP or CAP-T cells) on the other hand, live up to industrial size production without being significantly hurt by any proteases, which if at all present, most probably only occur in such small amount that one does not even have to add proteases to the crude harvest of for instance 20 recombinant fibrinogen made in these cells.

Another very important difference between the HEK cell production versus the production of the present invention, is that the host cells of the present invention does not need any necessary selection process during culturing the transfected

25 cells for production, unlike what you have to do when handling transfected HEK cell for production for instance adding an antibiotic or a compound such as Zeocin, where HEK cells without Zeocin resistants is killed, whereas HEK cells transfected with pHZsec vector where this vector is resistant against Zeocin. Zeocin would need to be added to the cell culture and/or be part of the serum-free medium

30 used for HEK cell culturing. The Zeocin will kill out the non pHZsec vector a

protein producing HEK cells, to maintain recombinant producing HEK cells to maintain an acceptable amount of recombinant protein producing HEK cells, while killing off or reducing the non producing HEK cells. This selection has to be performed even if one starts up with a single HEK cell culturing. This selection

35 process is completely unnecessary when culturing transfected the host cells of the present invention (e.g. amnion cells) because no significant selection is needed after the cells have been started as single cell cultures,

In an embodiment the present invention pertains to a human host cell selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amnion and an immortalised cell from the amniotic fluid, said cell comprising an exogenous nucleotide sequence encoding a coagulation factor. It may be contemplated that the human host cell produces at least 50 mg/L such as at least 60 mg/L, e.g. at least 70 mg/L, such as at least 80 mg/L, e.g. at least 90 mg/L, such at at least 100 mg/L, e.g. in the range from 50- 100 mg/L coagulaton factor per liter cell at a cell concentration of 3-10 millions of cells per ml.

Therefore this provides the resulting recombinant human prothrombin and especially gla-domain containing prothrombin with a yield of over 300 mg to 1000 mg recombinant human glycosylated prothrombin/liter cells at a cell concentration of 3 - 10 millions of cells per ml.

The method of the present invention may further comprise a step of obtaining the recombinant coagulation factor. More specifically the recombinant coagulation factor may be obtained by (i) harvesting said human host cell from the culture medium; and (ii) purifying said recombinant coagulation factor from the culture medium . The host cell may be harvested by decantering, filtration, centrifugation and any combination thereof.

In a further embodiment the recombinant coagulation factor may be obtained by (I) harvesting the cell culuture medium, (II) filtering the cell culture medium, (III) obtaining a retentate and a filtrate, (IV) purifying the filtrate and

(V) obtaining the recombinant coagulation factor.

If a filtration step is present it may be contemplated that the filter is having a pore size og at least 60μ. Such a step of filtration allows a upstream use of the cells (i.e. re-use of the cells), whereas the the coagulation factor is purified downstream of the process.

The purification may be selected from the group consisting of affinity

chromoatography, ion-exhange chromatography, gel chromatography and any combination thereof. The chromatography may be 2-step chromatography.

In an embodiment of the present invention substantially no synthetic protease inhibitor(s) may need to be added according to the method of the present invention. If synthetic protease inhibitor(s) are added, this is in very small amounts, and to the crude harvested medium comprising the coagulation factor.

One would perform a testing sample, down to a prefrozen tube, to check the concentration, when freezed in a tube, placed in dry ice, additional samples is then withdrawn and checked for loss of activity and change in the protein bands as measured using SDS PAGE electrophoresis, at different time points, at room temperature, such as for instance time 0, time 1 hour, time 2, 4 8 and 16 hours. A fibrinogen sample where the activity is known is used as external control. The loss is then distributed on a chart to identify the loss using these intervals and compare this to the content of the first tube, kept in dry ice and then quick thawed within 1-2 minutes. This testing on time intervals are then done 3 repetitive times from 3 different batches of crude protein harvested from the cell cultures. One can then titrate the amount of a protein inhibitor such as for instance peptidase-A.

In a HEK cell culture system the HEK cells must be harvested around 0 degrees due to the abundance of proteases in the cell culture medium . Even though harvested at around 0 degrees, the proteases present will interfere and eventually destroy the protein making it virtually impossible to purify the harvested protein before it is destroyed. The biggest loss when obtaining protein from HEK cultures happens during purification - and this, in spite of the addition of protease inhibitors to prevent further decay.

In an experiment performed the expected yield from approximately 33.9 Liter HEK cells with a titer of approximately 15 mg on average, should have yielded approx. 500 mg. A loss of 50% of the material during a two step column chromatography, should have yielded approx. 250 mg. However, the end result was 46 mg, meaning merely a 9% yield using HEK cells even though considerable efforts were done using protease inhibitors and cooling during purification.

Using a HEK cell line for the generation of a coagulation factor one needs to add at least 1 mL 100 mM PMSF (a protease inhibitor) to 1000 mL of the media in order to obtain a final concentration of 100 μΜ PMSf in the media.

Protease inhibitors (synthetic or natural) added to a media are not easily removed - it is therefore indeed a great advantage of the present invention that substantially no synthetic protease inhibitors may need to be added in order to obtain a high yield of recombinant coagulation factor.

In the production of fibrinogen for instance the alpha chain, in particular the c- terminal part of the alpha chain, is the part proteolytic enzymes degrade first. Obviously such degradation of the alpha chain impair fibrinogen polymerization to some degree and is undesired.

It may therefore be contemplated that the addition of synthetic/artificial protease inhibitors are avoided. This on the other hand allows the handling of larger volumes in bioreactors (4000-5000L) with the human host cells of the present invention e.g. CAP/CAP-T, since the volume must not be immediately cooled down during harvesting of crude proteins in order to avoid protease activity. Such bioreactors may e.g . be GE-wave bioreactors and Xcellerix bioreactors (GE). Filtering of e.g . of fibrinogen is not possible and it is therefore crucial that the process is kept sterile.

In the method of the present invention there is also no need to add zeocin wich is added to HEK cell cultures in ordet to "kill" non-transfected cells.

Human recombinant preprothrombin can be prepared using two different routes, one starting from a point mutated prothrombin with Gla, Kringle 1 and Kringle 2 domains, and maintaining these domaing during the process; the other one sarting with WT prothrombin (non-mutated) or alternatively use the path via a preprothrombin with a HPC4-kringle 2 domain and subjecting this preprothrombin to a point mutation.

In an embodiment of the present invention the the exogenous nucleic acid is selected from the group consisting of SEQ ID NOs: 4 (fibrinogen alpha chain), 8 (fibrinogen beta chain), 12 (fibrinogen gamma chain), 14 (prothombin), 18 (prethrombin M84A), 20 (prethrombin M84A with HCP) 22 (tissue factor), 24 (Labile factor), 26 (fibrin stabilizing factor, A polypeptide), 28 (fibrin stabilizing factor, B polypeptide).

In a further embodiment the human host cell transfected with three exogenous nucleic acid sequences encoding said coagulation factor.

In an embodiment it may be contemplated that the human host cell is transfected with an exogenous nucleic acid sequence encoding a fibrinogen apha chain, an exogenous nucleic acid sequence encoding a fibrinogen beta chain and an exogenous nucleic acid sequence encoding a fibrinogen gamma chain.

Thus, in one embodiment the fibrinogen alpha chain comprises an amino acid sequence selected from the group consisting of:

i) SEQ ID NO. : 3 (fibrinogen alpha chain)

ii) a subsequence of the sequence defined in i);

iii) a sequence which has at least 85% sequence identity with the sequence set forth in i) or ii).

In a further embodiment the fibrinogegn beta chain comprises an amino acid sequence selected from the group consisting of:

i) SEQ ID NO. : 7 (fibrinogen beta chain)

ii) a subsequence of the sequence defined in i);

iii) a sequence which has at least 85% sequence identity with the sequence set forth in i) or ii).

In en even further embodiment the fibrinogen gamma chain comprises an amino acid sequence selected from the group consisting of:

i) SEQ ID NO. : 11 (fibrinogen gamma chain) ii) a subsequence of the sequence defined in i);

iii) a sequence which has at least 85% sequence identity with the sequence set forth in i) or ii). Thus, the exogenous nucleic acid may be selected from the group consisting of: i) SEQ ID NOs. : 4 (fibrinogen alpha chain), 8 (fibrinogen beta chain) or 12 (fibrinogen gamme chain),

ii) a subsequence of any of the sequences defined in i);

iii) a sequence which has at least 85% sequence identity with anyone of the sequences set forth in i) or ii).

In a further embodiment the exogenous nucleic acid sequence may encode a polypeptide comprising an amino acid sequence selected from the group consisting of:

i) SEQ ID NO: 13 (human prothrombin M400A), SEQ ID NO: 15 (human prothrombin WT), SEQ ID NO: 17 (human prethrombin M400A) and SEQ ID NO: 19 (human prethrombin WT);

ii) A subsequence of any one of the sequences defined in i); and

iii) A sequence which has at least 85% sequence identity with any one of the sequences in i) and ii).

It may be contemplated that said amino acid sequence comprises a gla domain , kringle 1, and/or kringle 2. In an embodiment the exogenous nucleic acid may be selected from the group consisting of:

i) SEQ ID NOs. : 14 (human prothrombin M400A coding sequence), 16 (human prothrombin WT coding sequence), 18 (human prethrombin M400A coding sequence) and 20 (human prethrombin WT coding sequence)

ii) a subsequence of any one of the sequences defined in i);

iii) a sequence which has at least 85% sequence identity with anyone of the sequences set forth in i) or ii).

In a further embodiment of the present invention the exogenous nucleic acid may encodes an amino acid sequence selected from the group consisting of: i) SEQ ID NOs. : 21;

ii) a subsequence of any one of the sequences defined in i);

iii) a sequence which has at least 85% sequence identity with anyone of the sequences set forth in i) or ii).

In a further embodiment of the present invention the exogenous nucleic acid encodes an amino acid sequence selected from the group consisting of:

i) SEQ ID NOs. : 23;

ii) a subsequence of any one of the sequences defined in i);

iii) a sequence which has at least 85% sequence identity with anyone of the sequences set forth in i) or ii).

In a further embodiment of the present invention the human host cell is transfected with an exogenous nucleic acid sequence encoding a fibrinogen stabilizing factor Al subunit, and an exogenous nucleic acid sequence encoding a fibrinogen stabilizing factor B subunit.

In particular, the said fibrinogen stabilizing factor Al subunit comprises an amino acid sequence selected from the group consisting of:

i) SEQ ID NOs. : 25

ii) a subsequence of the sequence defined in i);

i) a sequence which has at least 85% amino acid identity with the sequence set forth in i) or ii);

Further, said fibrinogen stabilizing factor B subunit may comprise an amino acid sequence selected from the group consisting of:

i) SEQ ID NOs. : 27

ii) a subsequence of the sequence defined in i);

iii) a sequence which has at least 85% amino acid identity with the sequence set forth in i) or ii).

In an embodiment it may be contemplated that any one of the sequences stated above has at least 85% sequence identity with anyone of the sequences set forth in i) or ii), such as at least 86%, e.g. at least 87%, such as at least 88%, e.g. at least 89%, such as at least 90%, e.g. at least 91%, such as at least 92%, e.g. at least 93%, such as at least 94%, e.g. at least 95%, such as at least 96%, e.g. at least 97%, such as at least 98%, e.g. at least 99%, such as 100%.

It may be contemplated the the recombinant coagulation factor obtained by the method of the present invention is lyophilized.

Thus, in one embodiment the lyophilized recombinant fibrinogen may retain at least 80% of the initial fibrinogen polymerization activity after one week of storage at room temperature (or lower), such as at least 85% of the initial fibrinogen polymerization activity after one week of storage at room temperature (or lower), such as at least 90% of the initial fibrinogen polymerization activity after one week of storage at room temperature (or lower), such as at least 95% of the initial fibrinogen polymerization activity after one week of storage at room temperature (or lower), such as at least 99% of the initial fibrinogen

polymerization activity after one week of storage at room temperature (or lower), such as 100% of the initial fibrinogen polymerization activity after one week of storage at room temperature (or lower).

There may always be contaminants in plasma fibrinogen that would - again decrease the polymerization of the fibrinogen, whereas it is less likely that an optimally purified recombinant fibrinogen would decrease in the same fast manner.

It may be preferred that the product is not stored above 25 °C, that is is not freezed, and/or that it is protected from light. After reconstitution the physico chemical stability may be intact for 8 hours at room temperature (max. +25 °C). From a microbiological point of view the reconstituted product should preferably be used immediately. If it is not administered immediately, storage should preferably not exceed 8 hours at room temperature. In a further embodiment the lyophilized recombinant prothrombin may retain at least 80% of the initial prothrombin polymerization activity after one week of storage at room temperature (or lower), such as at least 85% of the initial prothrombin polymerization activity after one week of storage at room

temperature (or lower), e.g. at least 90% of the initial prothrombin polymerization activity after one week of storage at room temperature (or lower), such as at least 95% of the initial prothrombin polymerization activity after one week of storage at room temperature (or lower), e.g. 100% of the initial prothrombin polymerization activity after one week of storage at room temperature (or lower). Preferably prothrombin will have several month of stability in a freeze dried

(lyophilized) condition, most probably up to between 0.5 to 1 year. The activity of thrombin to activate fibrinogen may be with the area of 10- 100 IU/mg fibrinogen, such as in the range from 20-90 IU/mg, e.g. in the range from 30-80 IU/mg, such as in the range from 40-70 IU/mg, e.g. in the range from 50-60 IU/mg.

In an embodiment according to the present invention the recombinant human prothrombin clots fibrinogen at a faster rate than the natural human prothrombin. It is believed that because of its purity, not contaminated with impure contents of proteins or peptides, the clotting time will be somewhat faster with recombinant clotting proteins (recombinant prothrombin activated) than plasma clotting proteins in relation to fibrinogen or recombinant fibrinogen.

Since the product is essentially serum free it is possible to carry the products in a e.g. a medipack.

Thus, it may be contemplated that the lyophilized recombinant fibrinogen retains at least 80% of the clotting activity of the initial fibrinogen at room temperature, such as at least 85%, e.g. at least 90%, such as at least 95%, e.g. 100% of the clotting activity of the initial fibrinogen at room temperature.

It may further be contemplated that the lyophilized recombinant prothrombin retains at least 80% of the clotting activity of the natural prothrombin at room temperature, such as at least 85%, e.g. at least 90%, such as at least 95%, e.g. 100% of the clotting activity of the initial fibrinogen at room temperature.

In the present context room termperature is to be understood as 20 to 25°C.

Figure 22 shows the recombinant fibrinogen with prolin instead of alanine in the beta chain. It can be seen that the clotting is approximately 1 Log higher concentration of this prolin rec. fibrinogen called rH Fbg M400a (green) rH Fbg HTI (blue). Figure 22b shows recombinant fibrinogen (Prolin to Alanin change in the beta chain), indicated by the orange rH mutant Fbg HTI, and the black rH mutant Fbg M400a. In an embodiment according to the present invention the recombinant human prothrombin has a significantly lower autolytic activity than human natural prothrombin.

Applications of human recombinant proteins according to the present invention

A most probable approach within the scope of this invention is the usage of one single recombinant human coagulation factor such as for instance certain recombinant human prothrombin analogue(s) and/or recombinant prothrombin mutant(s), produced in the human cell lines of the present invention intended to be either used individually as sole product(s), when appropriately activated to thrombin without other combinations for local hemostasis or in certain bleeding areas in the organism where the applied recombinant human thrombin can react with fibrinogen present in the bleeding area, and thus prevent or stop the bleeding.

In one embodiment the concentration of prothrombin according to the present invention, such as recombinant human prothrombin or recombinant human prothrombin with one or more point mutations, may be less than 20 NIH units/ml, such as 1-20 NIH units/ml, such as less than 15 NIH units/ml, less than 10 NIH units/ml, less than 5 NIH units/ml, or less than 1 NIH unit/ml.

In one embodiment the concentration of fibrinogen according to the present invention, such as recombinant human fibrinogen, may be less than around 50 mg/ml, such as 1-50 mg/ml, such as less than 40 mg/ml, less than 30 mg/ml, less than 20 mg/ml, less than 10 mg/ml, or less than 2 mg/ml.

Recombinant human prothrombin, e.g., activated as thrombin has a wide range of uses ranging from the treatment of coagulation disorders in humans to act as enzymatic initiator of clotting, treatment of burns, of skin grafting, both in minor and in major surgery either alone or as part of a product, such as tissue sealants. The effective doses of recombinant human prothrombin may vary considerably ranging from 100 to 15,000 units depending on the manner in which prothrombin is used (the specific activity of pure prothrombin activated as thrombin is 3,000 units per pg protein). If the recombinant prothrombin or the recombinant prothrombin activated to thrombin is used in scaffolds or microcarriers such as MPEG PLGA, or in e.g. a polyurethane foam like Biatain, the units which may be needed may be in the range from 25 - 150 per cm2, such in the range from 30 - 145 per cm2, e.g in the range from 40 - 140 per cm2, such in the range from 50 - 130 per cm2, such in the range from 60 - 120 per cm2, e.g. in the range from 70 - 110 per cm2, such in the range from 80 - 100 per cm2.

Quite another model of this invention could be the use of some or one of the proteins which are within the scope of this invention that conveniently could be incorporated in substances which again could react with substances added to provoke gelation, adherence or gluing effect, as well as hemostatic effects.

Recombinant human prothrombin or thrombin according to the present invention may be formulated with any known pharmaceutically acceptable excipients.

Additional compositions, kits, ingredient assemblies are included within the scope of the present invention for use in connection with the methods herein described as the follows. Obviously the present invention also pertains to a recombinant coagulation factor obtainable by the method according to the present invention.

The invention also pertains to a protein composition comprising a recombinant coagulation factor, characterised in that

i) said protein composition comprises substantially no synthetic protease inhibitors, and/or

ii) said protein composition is serum free.

The human host cell of the present invention e.g. the CAP or CAP-T cell lines, does unlike many embryonal cell not need to be maintained by Zeocin for killing none producing cells during processing the transfected cell line and retaining the protein production according to the transfected gene or genes.

Therefore, it is possible to obtain a protein composition essentially free or completely free of Zeocin, protease inhibitors and serum, which has a positive effect on the possible ways of applying the protein composition.

In an embodiment of the present invention the protein composition according to the present invention may be used for internal traumatic bleedings caused by severe trauma, excessive bleedings during planned surgery (hip, aorta, major arterial bleedings, brain vessel bleedings, aneurisms, bleedings occurring during orthopaedic surgery, bleedings occurring during organ transplants, etc.), postpartum haemorrhages, army related IEDs, bombardement suicide bomber attacks, heavy artillery shelling, flight accidents, helicopter accidents or shooting towards flights and helicopters, rocket target casualties, civil major traumas, car accidents, flight accidents, accidents onboard ships both civil and during combat, surgical bleedings when operating on major organs, such as but not limited to the liver and the brain. In the majority of cases the fibrinogen would be administered after the kit containing dried recombinant fibrinogen diluted in adequate solvens for intravenous or other infusions/injections, such as including intrathecal and lumbar spinal infusions, in major joints apart from IV use, sometimes during orthopaedic bleedings during major joint or back surgery, heart surgery.

In a further embodiment the protein composition may comprise recombinant fibrinogen and recombinant prothrombin and may be used for the prevention of possible excessive bleedings when planning or performing major surgery. Such surgery may be where vessels have to be connected before unclamping, when unclamping is released should be efficient to.

The protein composition may further comprise factor V and factor XIII. Factor V may activate clotting and factor XIII may increase the clotting strength. The protein composition comprising recombinant fibrinogen and recombinant protrombin, may be for topical use and for preventing bleeding which might occur after repairing vessels, muscles for instance related to aorta, other vessels feeding organs. Such organs may be the liver, kidney, female organs, such as surgery in 5 uterus, ovarian or other female organs where it would be and advantage to prepare areas that may eventually start excessive bleedings which otherwise are difficult to control.

Accordingly, such a protein composition may be part of a glue, a powder or 10 present in a scaffold. The powder may be MPEG PLGA whereas the scaffold may be a haemostatic selected from the group consisting Tacosil or Hema-Seal and products resembling these products and which may be selected from the group consisting of FloSeal, Surgiflo, Surgicel, Spongostan, Hemopatch

Traumastem, Apatec, Aarista, Avitene, other gel foam or coated scaffold.

15

In a further embodiment a protein composition comprising recombinant recombinant prothrombin may also be for topical use. Also there the composition may be a part of a powder (such as but not limited to MPEG PLGA + CaCI ₂ ), a gel or present in a scaffold. Such a scaffold may e.g. be Biatain (see e.g . WO

20 2013/007266) which is polyurethane foam.

In on aspect the present invention also pertains to a kit comprising :

(i) a scaffold comprising prothrombin, and

(M) CaCI ₂ .

25

In on aspect the present invention also pertains to a kit comprising :

(i) a gel granulate comprising prothrombin, and

(N) CaCI ₂ .

30 In one embodiment the kit may further comprise recombinant human fibrinogen.

In kits comprising recombinant human prothrombin, the prothrombin (most probably freeze-dried) can not be mixed with calcium chloride until immediately before use (probably not exceeding one or two hours before) due to the

35 activation. On the other hand a kit comprising recombinant human fibrinogen and

prothrombin if both ingrediences are freeze-dried, could be activated by adding calcium chloride immediately, so there might be one vial containing freeze-dried fibrinogen and freeze-dried prothrombin, which then can be activated by a vial containing calcium chloride solvens, which one can add to the freeze dried proteins.

Snake poison like Ecarin may also be used as an activator however, such activators are somewhat more cumbersome to work with, because one can not add it to an immediate prothrombin conversion to thrombin.

By adding CaCI ₂ to recombinant human prothrombin it activates the prothombin. In order to properly activate prothrombin and thrombin it is contemplated the solution comprises a concentration of CaCI ₂ in the range from 30-500 pmol is used, such as in the range from 50-400 pmol, e.g. in the range from 100-300 μιηοΙ, such as in the range from 200-250 pmol.

Thus in practice a kit may comprise recombinant human prothrombin, a solution or powder comprising CaCI ₂ and fibrinogen.

If CaCI ₂ is present in a solution the prothrombin and the CaCI ₂ solution are mixed in order to activate prothrombin to thrombin right before use. The mix comprising prothrombin or thrombin may be mixed with recombinant human fibrinogen and used e.g. to "glue" a scaffold onto a cartilage defect. It may be contemplated that the bleeding area to which the activated prothrombin and scaffold is applied with stop the bleeding within 1-3 minutes. It is important that the two mixing steps are performed immediately prior to use. If CaCI ₂ is present as a powder it may be mixed with prothrombin and stored . It may be applied to a scaffold and when exposed to a fresh bleeding, providing an instant clotting effect.

Thus, recombinant CAP or CAP-T produced human prothrombin may react with fibrinogen as such as well as certain human recombinant fibrinogens to create a stable product, with an optimal fibrinogen cleavage activity and with a low effect on activation of protein C. The activation of recombinant prothrombin from CAP and CAP-T cells with CaCI ₂ makes it possible to build it into or coat it onto scaffolds such as for instance Coloplast's Biatain Foam, whereby prothrombin powder with calcium chloride powder is activated to thrombin when exposed to a fresh bleeding, providing instant clotting effect.

The ideal matrix for wound healing, hemostatic, has to be capable of adhering to any mammal tissue surfaces including bleeding surfaces to prevent bleeding during surgery and to conserve the patient's own blood volume, or at least minimize the necessity for the usage of natural blood products on the patient. Prothrombin or prothrombin (activated by endogenous factors present in the sieving blood, added to a bleeding surface will form a clot and work as a hemostatic.

The scaffold may be selected from the group consisting of a dressing, a patch, a sponge, such as Tacosil, Hema-Seal, Biatain, MPEG-PLGA , Aseed, Spongostan and Surgifoamm Surgiflo. The following are examples of kits in which prothrombin either activated prior to mixing with scaffold/powder, or a kit where the if the scaffold e.g. is biatain it may also be used as a scaffold-foam or be granulated. The prothrombin (most likely freeze-dried or powdered)may either be coated into the biatain scaffold- foam, and with a spray like a syringe distributing the activating component such as for instance calcium chloride or calcium chloride in physiological salt, or in a buffer that will activate prothrombin to thrombin, at the moment when the coated biatain scaffold also called foam is applied directly on the bleeding area. Otherwise prothrombin may be pre-mixed separately with activating calcium chloride or buffers discussed above, prior to spraying the activated thrombin on the scaffold or foam, or mixing the prothrombin activated to thrombin immediately prior mixing the activated thrombin with the biatain powder or any powder such as for instance MPEG-PLGA powder, or for instance combining the freeze-dried or "powdered" prothrombin with the powder, and thereafter add the solvent (calcium chloride solvent or alike, as activator of prothrombin), and then applying either the activated scaffold-foam or powder Becton Dickinson has among others available a product called BD Hypak Physioilois system which would most probably be feasible for use in the prothrombin - scaffold/powder kit.

The amount of recombinant coagulation factor may vary depending on the intended use of the product, the more severe bleeding, the higher a

comcentration of the one or more coagulation factor is needed. Suitably, the amount of prothrombin is in the range from 70 -1000 to 5000 U/cm ² or a comparable measured amount taking into account the measurement cm ³ . It is envisaged that an amount of 10-1000 U/cm ³ may be suitable in a device for stopping severe bleedings.

Several conventions for activity units are used in the literature and conversions from one convention to another may be needed : 1 IOWA unit = 0.84 NIH unit; 1 WHO unit = 0.56 NIH unit; 1 NIH unit = 0.324 +/-0.073 pg; and 1 NIH unit = 1 USP unit.

The kit may also comprise CaCI ₂ present in a spray. CaCI ₂ activates prothrombin and is therefore easy applicable on the scaffold before use. This is a great advantage if e.g. such a scaffold is carried as part of a medipack.

The invention also pertains to a scaffold, comprising prothrombin and CaCI ₂ , wherein said prothrombin and said CaCI ₂ is present in different parts or compartments of the scaffold. Clearly, the invention also pertains to a recombinant coagulation factor according to the present invention, or a protein composition comprising one or more recombinant coagulation factors, for use as a medicament, as a tissue sealant or to facilitate tissue adherence

The invention therefore also pertains to the use of a recombinant coagulation factor according to the present invention, or a protein composition comprising one or more recombinant coagulation factors, for the preparation of a medicament for the treatment or prevention of bleedings. The invention therefore also pertains to the use of a recombinant coagulation factor according to the present invention, or a protein composition comprising one or more recombinant coagulation factors, for the preparation of a medicament for the treatment or prevention of infections.

The invention also pertains to a a method for preventing, treating and/or alleviating bleedings comprising administering to a subject in need thereof one or more recombinant coagulation factors according to the present invention, or a protein composition according to the present invention.

Items

Prothrombin

Abstract

Prothrombin protein and derivates thereof made in placenta cell lines

The present invention discloses a novel method for the preparation of one or more compositions of human recombinant proteins such as recombinant prothrombin, and recombinant prothrombin, a gla-domain containing precursor of recombinant human thrombin from which is activated by components in the coagulation system or activated by factors such as snake venom proteins, among others, ecarin or recombinant ecarin, and more specifically recombinant human prothrombin and even more specifically recombinant human prothrombin analogues or recombinant prothrombin mutants (e.g., M84A (M400A) or R77aA), in which, according to this invention is relying upon the stabilization of these prothrombins, which is either obtained by high salt concentration, which is stabilizing proteins such as for instance recombinant prothrombins, thrombins especially obtained by freeze- drying (lyophilisation).

The prothrombins and derivates thereof is according to this invention made in placenta derived cells among those, specifically, the present invention relates to novel methods for the transfection of gla domain containing prothrombin in novel systems from mammal or human placenta derived cell cultures (human host cells) or more specifically from a novel system consisting of amnion cells, amniocytes, immortalized amniocytes together with comparable vector systems such as CAP and CAP-T cells, which are high yield producer cells originated from CEVEC Pharmaceuticals, Cologne in Germany. The CAP cells relates to a permanent human amnion cell or amniocytic cell line generated by immortalization of primary amniocytes generated by adenoviral gene E1A and E1B, thus being amnion cells and among those immortalized amnion cells from a specific time during the gestation, removed from amnion fluid obtained during a certain time of the pregnancy by amniocentesis as described in US patent applications:

US20070111312 and US20120040400. The CAP-T cell line relates to CAP cells that in addition are stably transfected with nucleic acid molecule containing the sequence of the SV40 large T-antigen. The term "transfection" mentioned herein relates to any method suitable for introduction by other methods such as the classical calcium phosphate method, the eletroporation, and liposomal systems of any kind capable of being used for the transfection. The transfection method used in relation to CAP cells consists of 1. transfection used in CAP system consists of two transfections, namely of a 1. transfection with E1A and E1B, and of 2.

Transfection consisting of SV40 large T-antigen.

Moreover, the method employs serum-free culturing conditions for the placenta derived cells and the amnion cells or more specifically, immortalized amnion cells especially those placenta derived cells or amnion cells that can be cultured and which produce the human prothrombin, in which case the prothrombin gene(s) are transfected via vector(s), that could be amplified to fit placenta cells or amnion cells, so that even vectors for instance used to transfect in HEK 293T or HEK 293TS cells, is amplified with a second transfection using the amplification technology used and done by CEVEC Pharmaceutical AG, Cologne, Germany for placenta like cells such as immortalized CAP and CAP-T . Any other amnion cells or immortalized amnion cells, thus being a new manner which is started by constructing vectors such as HZ sec vector(s) amplified by Cevec vectors to produce a higher yield of prothrombin or derivates thereof.

Even more important according to this invention is a stabilizing method of the prothrombins and even more specifically recombinant human prothrombins irrespectively of the mutant or type of analogue, and even how unstable the prothrombin, or more specifically the recombinant prothrombin may be in a solvens. There will of course be a loss of recombinant human prothrombin or recombinant human thrombin during the lyophilization process, which may variate from 40 - 80%, however the remaining amount of recombinant prothrombin is indeed stable after the lyophilization procedure. The reason for the loss is still unknown, but it appears that adjustments in the process will minimize the loss during the lyophilization process. It is on the other hand possible to optimized the lyophilization process by researchers who are skilled in the art, and thus the loss of activity of prothrombins and in particular recombinant prothrombins by lyophilization is minimized .

Moreover, the method employs serum-free culturing conditions and therefore provides recombinant human proteins expressed in human cells of increased safety to the patient when used in human medical treatments. In addition, the immunogenic response to the recombinant human proteins expressed in human cells may be lower. Recombinant human prothrombin or recombinant human thrombin is expressed in the human placenta cell system or in human amnion cells and amplified using a combination of vectors originally used as vectors for HEK293T or HEK 293TS. These transfected amnion cells are from Cevec (from Cevec, Cologne Germany).

The protein can be prepared using two different routes, one starting from a point mutated prothrombin with gla and Kringle 1 and 2 domains, and maintaining these domains during the process; the other one starting with prothrombin (non- mutated - WT), via a preprothrombin with a HPC4-Kringle 2 domain and subjecting this preprothrombin to a point mutation, however the route via preprothrombin using HPC4, etc. is not so straight forward . Human recombinant fibrinogen is expressed in the human placenta cell lines or more specifically in human amnion cells or human immortalized amnion cells from CEVEC.

Stable expression

Stable expression describes the production of recombinant proteins due to integration of the respective gene expression cassette into the chromosome of the producer cell line. In most cases, the gene of interest is integrated into the chromosome via non-homologous recombination in varying copy numbers.

Using CEVEC ^' s CAP (immortalized amnionic cells or immortalized amniocytes) for stable expression, stable integration of your gene is achieved with : · Optimized expression cassettes • Optimized selection cassettes

• Fast selection procedure

• Efficient transfection The process of developing a high yield producer CAP cell line includes several important steps:

• Development of stably expressing pools

• Efficient single cell cloning with a selected pool

• Volumetric upscaling and optimization of fermentation process parameters · Cell line development in suspension and serum-free medium

The present invention relates to a novel concept and method for preparing compositions of recombinant human proteins, each prepared in a novel manner including the glycosylation that create authentic or natural human proteins produced in human cells, human cell lines, human stem cells, or human precursor cells. Posttranslational modifications caused by human cells and particularly the human cells derived from a human placenta, or placenta related fluid, umbilical cord blood stem cells, umbilical cord cells such as mesenchymal cells from

Wharton's jelly, harvested by using the cell harvesting technique described by Osther et al. (Osther K, et al. The Rationale to Use Explants for ACI in "Basic Science and Clinical Repair of Articular Cartilage defects", Current Status and Prospects" in Fundamentals of Tissue Engineering and Regenerative Medicine, (editors, Zanasi S, Brittberg M, Nehrer S, Marcacci M), 2006, pp 313-316), and human cells such as amnion cell, amniocytes, CAP or CAP-T cells (previously defined, originated from CEVEC Pharmaceuticals AG, Cologne, Germany). These posttranslational modifications caused by these human cells, which are actually not embryonic cells, influence several physical and chemical properties, such as glycolysation, phosphorylation, carbocylation, palmitoylation, or specific cleavages unique to translated type of the human cells as described above, are of significant importance for different properties of the expressed product or production or protein, originated from the expression of the transfected cell. The glycosylation patterns arede decided by the nature of the transfected placenta - related cell, e.g., amnion cells, CAP and CAP-T cells (Cevec), rather than by the gene, which was transfected into these cells, being placenta related cells, amnion cells, CAP and CAP-T cells. So, physical and chemical properties , e.g., molecular weight, pi, stability of the expressed protein, folding of the protein and, of utmost importance the biological activity of the protein.

Specifically, the present invention relates to novel methods for the preparation of human recombinant prothrombin and human recombinant fibrinogen using non- embryonic cells, but instead focusing on human placentar cells or any cells from amnion meaning cells that have nothing to do with embryonic cells or embryonic stem cells. Human recombinant prothrombin can be prepared using two different routes, one starting from a point mutated prothrombin with gla and Kringle 1 and 2 domains, and maintaining these domains during the process; the other one starting with wild type prothrombin (non-mutated) or alternatibvely use the path via a preprothrombin with a HPC4-Kringle 2 domain and subjecting this preprothrombin to a point mutation. The preprothrombin is not that interesting and is in many ways significantly different from gla domain containing

prothrombin.

Stable expression describes the production of recombinant proteins due to integration of the respective gene expression cassette into the chromosome of the producer cell line. In most cases, the gene of interest is integrated into the chromosome via non-homologous recombination in varying copy numbers.

Using CEVEC ^' s CAP (immortalized amnionic cells or immortalized amniocytes) for stable expression, stable integration of your gene is achieved with :

• Optimized expression cassettes

• Optimized selection cassettes

· Fast selection procedure

• Efficient transfection

The process of developing a high yield producer CAP cell line includes several important steps:

• Development of stably expressing pools

· Efficient single cell cloning with a selected pool

• Volumetric upscaling and optimization of fermentation process parameters

• Cell line development in suspension and serum-free medium

The present invention relates to a novel concept and method for preparing compositions of recombinant human proteins, each prepared in a novel manner including the glycosylation that create authentic or natural human proteins produced in human cells, human cell lines, human stem cells, or human precursor cells. Posttranslational modifications caused by human cells and particularly the human cells derived from a human placenta, or placenta related fluid, umbilical cord blood stem cells, umbilical cord cells such as mesenchymal cells from

Wharton's jelly, harvested by using the cell harvesting technique described by Osther et al. (Osther K, et al. The Rationale to Use Explants for ACI in "Basic Science and Clinical Repair of Articular Cartilage defects", Current Status and Prospects" in Fundamentals of Tissue Engineering and Regenerative Medicine, (editors, Zanasi S, Brittberg M, Nehrer S, Marcacci M), 2006, pp 313-316), and human cells such as amnion cell, amniocytes, CAP or CAP-T cells (previously defined, originated from CEVEC Pharmaceuticals AG, Cologne, Germany). These posttranslational modifications caused by these human cells, which are actually not embryonic cells, influence several physical and chemical properties, such as glycolysation, phosphorylation, carbocylation, palmitoylation, or specific cleavages unique to translated type of the human cells as described above, are of significant importance for different properties of the expressed product or production or protein, originated from the expression of the transfected cell. The glycosylation patterns arede decided by the nature of the transfected placenta - related cell, e.g., amnion cells, CAP and CAP-T cells (Cevec), rather than by the gene, which was transfected into these cells, being placenta related cells, amnion cells, CAP and CAP-T cells. So, physical and chemical properties , e.g., molecular weight, pi, stability of the expressed protein, folding of the protein and, of utmost importance the biological activity of the protein.

Specifically, the present invention relates to novel methods for the preparation of human recombinant prothrombin and human recombinant fibrinogen using non- embryonic cells, but instead focusing on human placentar cells or any cells from amnion meaning cells that have nothing to do with embryonic cells or embryonic stem cells. Human recombinant prothrombin can be prepared using two different routes, one starting from a point mutated prothrombin with gla and Kringle 1 and 2 domains, and maintaining these domains during the process; the other one starting with wild type prothrombin (non-mutated) or alternatibvely use the path via a preprothrombin with a HPC4-Kringle 2 domain and subjecting this

preprothrombin to a point mutation. The preprothrombin is not that interesting and is in many ways significantly different from gla domain containing

prothrombin.

Background

Prothrombin is defined as a two chain, disulfide-bonded, glycosylated polypeptide that cleaves specific bonds in fibrinogen to produce fibrin monomers that self- assemble to form a fibrin clot.

When mixing prothrombin, activated to thrombin (for instance by using and the fibrinogen together, a final stage of the natural fibrin clotting cascade takes place. In addition to providing hemostasis, this fibrin sealant also provides ideal environment for fibroblastic proliferation as well as proliferation of other mesenchymal cells. The Wild type (wt) Recombinant human Prothrombin kit made from recombinant human wild type prothrombin obtained from transfected CAP and/or CAP-T cells could for instance consist of the following :

• The recombinant prothrombin is lyophilized for instance with 2500 units in a vial, (sterile filtered

· (The recombinant prothrombin could be vapor heated and treated with solvent detergent) sterile filtered

• The Calcium chloride consists of 5 ml (total 200 pmol CaCI2) or 40 pmol/ml Calcium chloride, sterile filtered .

• The two mixed components, the rec. HU prothrombin mixed with the Calcium chloride can then be sterile filtered .

A support material, such as for instance gel matrix, or a biatain foam (Coloplast A/S, Humlebaek, Denmark) either in granulated form in a 5 ml syringe, or as a solid foam to which the prothrombin/Calcium Chloride mixture can be applied as described in EVERLAND, Hanne, OSTHER, Kurt, VANGE, Jakob, and NIELSEN, Lene Feldskov patent application #WO2013007266.

• The biatain foam (Coloplast A/S) can be pretreated using the sterilizing method that Coloplast is using.

• The recombinant human wild type prothrombin, thereby activated and mixed as described above can then be applied directly into the bleeding area of a patient and will stop bleedings within a short time (e.g., 1 or 3 minutes). It is anticipated that the wild type recombinant human prothrombin has a specific activity of ~3000 units/mg protein. The modified recombinant human M84A, hereinafter called M400A recombinant human prothrombin has a specific activity of ~5000 units/mg protein. This M400A recombinant human prothrombin appears to be far more gentle to live cells, and could for instance be used as part of the recombinant human M400A prothrombin (activated to M400A thrombin) mixed with recombinant fibrinogen could conveniently be used when performing orthopaedic repair such as when applying a scaffold such as Cencor Biotech's MPEG-PLGA membrane in a cartilage defect in the knee, when the membrane is used to cover a microfractured cartilage defect, thus being glued onto the cartilage defect (e.g ., in the knee or in the ankle). The recombinant M400A Human prothrombin, activated by calcium chloride to thrombin would then enhance or promote the cell ingrowth and cell survival without altering the mesenchymal appearance of these stem cells appearing in the defect sieving from the microfractured bleeding bottom of the defect coming from the bone marrow into which the microfracture has been opened for. This M400A originated recombinant fibrin (prepared as a product made by the inventor or the assigned Cencor Biotech, which consists of two recombinant clotting proteins, e.g., recombinant M400A human prothrombin (activated by calcium chloride) mixed prior to application with recombinant human fibrinogen made in CAP or CAP-T cells. The recombinant human M400A prothrombin can also be used as a hemostatic as follows:

When preparing the M400A recombinant human prothrombin, transfected from CAP and/or CAP-T cells could for instance consist of the following :

· The recombinant M400A human prothrombin is lyophilized for instance with 5000 units in a vial, (sterile filtered)

• (The recombinant M400A prothrombin could be vapor heated and treated with solvent detergent) sterile filtered

• The Calcium chloride consists of 5 ml (total 400 pmol CaCI2) or 80 pmol/ml Calcium chloride, sterile filtered . • The two mixed components, the rec. HU M400A prothrombin mixed with the Calcium chloride can then be sterile filtered.

• A support material, such as for instance gel matrix, or a biatain foam (Coloplast A/S, Humlebaek, Denmark) either in granulated form in a 5 ml syringe, or as a solid foam to which the prothrombin/Calcium Chloride mixture can be applied.

• The biatain foam (Coloplast A/S) can be pretreated using the sterilizing method that Coloplast is using.

• The recombinant human M400A prothrombin, thereby activated and mixed as described above can then be applied directly into the bleeding area of a patient and will stop bleedings within a short time (e.g., 1 or 3 minutes).

The tissue sealant and modification of the individual amount of fibrinogen, and thrombin and possibly factor XIII decides the speed by which the clotting is taking place and the density of the clot. The tissue sealant combined in the manner that Tisseel is used, was tested by Mats Brittberg et a I., in his dissertation from 1994, where he showed that for instance chondrocytes did in his studies not infiltrate the fibrin clot obtained using Tisseel, but the cells were merely lining up against the clot, which again might be theorized as appearing as a blocking mechanism, which would entrap chondrocytes in for instance cartilage defects, but not allowing the cells to infiltrate into the fibrin clot, also called fibrin glue or tissue sealant. Fibrin is routinely used as tissue sealant when autologous chondrocyte implantation are performed on patients, when either periosteal grafting are used as cover over the implanted chondrocytes or collagen type I/III

(ChondroGide. RTM.) , synthetic PLGA or MPEG PLGA membranes as cover or carrier of cells coated onto the membranes. The tissue sealant (also called fibrin glue) such as for instance Tisseel is added to the cover either as a cover over the implanted chondrocytes or as carrier, coated with chondrocytes. However, Mats Brittberg also found that autologous fibrin clot, made from the spontaneous clotting of the blood in the cartilage defects in the animals appeared to allow the chondrocytes to infiltrate and, contrary to Tisseel, be cultured in the fibrin clot. Recently it has been described that Tisseel. RTM. is used in a method called Matrix Assisted Chondrocyte Implantation (Marlovits S, et al. Knee Sure Sports Traumatal Arthrosc. (2005) 13 : 451-7; Bartlett W, et al. J Bone Joint Surg Br. (2005) 87: 640-5). Tissue sealant has also been used in animals to study a possible enforcement of a myocardial infarction as a scaffold capable of preserving the myocardial wall (Christman K L, et al. Tissue Eng. (2004) 10:403). Fibrinogen and thrombin combined components, in general, appear to be significantly better for the use as sealant of tissue, such as for instance in controlling bleeding on the cut surface of organs difficult to place sutures in organs such as kidneys, where Tisseel, when compared to CoSeal (Baxter), which is made from polyethylene glycol components, showed superior advantages in the form of clotting and adhering to surfaces, where CoSeal appeared to be significantly inferior. CoSeal could not adhere or prevent sieving of blood from a cut surface, which make it non-useable when compared to fibrinogen- prothrombin, where prothrombin is converted instantly during the process, or fibrinogen - thrombin as combinations of tissue sealant (Bernie J E, et al. J.

Endourol. (2005) 19: 1122-6).

FLOSEAL (Baxter), which consists of collagen particles and plasma prothrombin appearing as prothrombin activated to thrombin, which was capable of preventing more extensively bleeding areas such as tested on kidneys by L'Esparance et at (L'Esparance J O, et al. J. Endourol. (2005) 19: 1114-21) and has proven to be significantly better than suturing or stapling (Langrehr J M, Rozhl Chir. (2005) 84: 399-402)

The ideal matrix for wound healing, hemostatic, has to be capable of adhering to any mammal tissue surfaces including bleeding surfaces to prevent bleeding during surgery and to conserve the patient's own blood volume, or at least minimize the necessity for the usage of natural blood products on the patient, thrombin or prothrombin (activated by endogenous factors present in the sieving blood, added to a bleeding surface will form a clot and work as a hemostatic.

The natural prothrombin or plasma prothrombin has been used clinically in several years to control bleeding during surgery, for burns and in certain trauma situations (Nakamura at al. The Amer. Surgeon (1991) 57: 226-230; Thompson et al. Opthalmology (1986) 93 : 279-282; Harris at al., J. Bone Joint Surg. Am.

(1978) 60: 454-456; Craig and Asher, Spine (1997) 2: 313-317; Prasad et al. ; Burns (1991) 17: 70-71). Bovine thrombin has also been used quite a lot in the US to prevent bleeding (Angiotech Pharmaceuticals, Inc. Vancouver, BC, Canada, Kings Pharmaceuticals, Pfizer) This clotting factor constists of or may even contain bovine collagen.

5

Commercial thrombin therapeutics are purified from pooled human and animal blood products and as such run the risk of contamination with viruses such as the HIV and hepatitis viruses. In comparing three commercial thrombin preparations, Suzuki and Sakuragawa (Suzuki and Sakuragawa, Thromb. Res. (1989) 53 : 271-

10 278) found that the preparations contained contaminating proteins, and the

human preparation contained immunoglobulin G, hepatitis B surface antigen antibodies and human immunodeficiency antibodies. Xenogeneic immunization with bovine thrombin has been reported in patients who have developed self- reactive antibodies to both human thrombin and human factor V (factor V is a

15 contaminant in the bovine thrombin preparation) (Strieker et al., Blood (1988) 72 : 1375-1380); Flaherty and Weiner, Blood (1989) 73: 1388); Flaherty et al., Ann Int. Med. (1989) 111 : 631-634); Zehnder and Leung, Blood (1990) 76: 2011- 2016); Lawson et al., Blood (1990) 76: 2249-2257); Strieker et al., Blood (1988) 72 : 1375-1380); Berguer et al., J. Trauma (1991) 31 :408-411).

20

In 139 patients treated with the first Floseal product from Baxter, 25 out of 139 (18%) of the patients presented with an increased titer over baseline for bovine thrombin antibodies. Whereas in a control group, consisting of patients treated with a gelatine-containing bovine thrombin sponge, 26/131 (20%) of the patients 25 presented with an increased titer over baseline for bovine thrombin. The

corresponding numbers for bovine Factor Va were 39/139 (28%) and 43/131 (33%) for the Floseal (Baxter and Control groups, respectively. Since that time Baxter has stopped using bovine thrombin and instead use human plasma thrombin.

30

The differences in the frequency of developing either bovine thrombin or bovine Factor Va antibodies between the 2 groups were not statistically significant (Winterbottom, N. et al., J. Applied Res. (2002) Vol. 2, Number 1). Results recently published by Wai Y et al. (Wal Y, Tsui V, Peng Z, Richardson R, Oreopoulos D, Tarlo S M, Clin. Exp. Allergy. (2003) 33: 1730) indicate the potential for sensitization and clinical allergic responses to bovine thrombin when used for haemostasis topically in patients in hemodialysis and suggest that other 5 haemostatic methods should be considered.

Bovine thrombin was recently used as an aid to hemostasis in medical and surgical procedures in the US. At least 500,000 Americans are exposed to this therapeutic annually and reports suggest that exposure is associated with the

10 development of autoreactive antibodies, which ended up with FDA employing a black box warning against the use of bovine thrombin. To determine whether bovine thrombin can induce pathological autoimmunity Schoenecker et al.

exposed non-autoimmune-prone galactose-. alpha .1-3-galactose-deficient mice to the two bovine thrombin preparations currently approved for use in the United

15 States, and found that, like humans exposed to bovine thrombin, mice developed an immune response against the therapeutic and the xenogeneic carbohydrate galactose-. alpha. 1-3-galactose, and some mice developed autoantibodies against clotting factors. Further, unexpectedly, a single exposure to this therapeutic also induced autoimmunity in mice with features characteristic of systemic lupus

20 erythematosus including antibodies against nuclear antigens, native DNA, double- stranded DNA, and cardiolipin.

High levels of these autoantibodies were correlated with glomerulonephritis in all mice evaluated. This autoimmune syndrome was detected in mice 15 weeks after

25 a secondary exposure to bovine thrombin and female mice were found to develop the syndrome at a significantly greater frequency than males. Thus, these studies indicate that exposure to bovine thrombin preparations can induce a pathological systemic autoimmune syndrome with lupus-like serology (Schoenecker J G, Johnson R K, Lesher A P, Day J D, et al., American J. of Pathology, (2001)

30 159: 1957).

Therapeutic human blood products are also subject to contamination by viral particles such as the hepatitis virus and the human immunodeficiency virus as well as other adventigious infectioius particles as well as pathogenic prions.

35 In other expression systems such as bacteria (e.g., E. coli), yeast systems, to which a DNA sequence is transmitted or in insect cells, avian cells, and other animal cells to which a DNA sequence is transfected, the post translation of many human proteins, especially of the types described above, such as fibrinogen, thrombin, collagens, and other cross-linking proteins such as recombinant factor XIII, which is actually, as described in U.S. Pat. No. 6,780,411 is made in transmitted yeast, --actually expressed cytoplasmically by yeast (natural fibrinogen purified from blood contains recombinant factor XIII as the cross- linking protein stabilizing the coagulation process) referring to the U.S. patents, mainly described in U.S. Pat. No. 6,780,411, does not completely resemble the authentic natural protein as much as the recombinant human protein (e.g., fibrinogen, prothrombin, construction of Prothrombin Expression Units and Expression in Mammalian Cells, thrombin, factor XIII, collagen(s) produced in a system consisting of transfected human placenta cells or amnion cell lines maintained and producing the said protein or proteins in a serum free synthetic cell culture medium system.

While recombinantthrombin may be produced in a variety of hosts, the most preferred recombinant prothrombin that can be activated as thrombin when needed, until this invention was the production in CHO cells which actually, contrary to this invention requires the presence of foetal bovine serum, and thereby adding the possible disadvantages to the proteins made first of all from a source like the CHO cells, and next relying on the addition of foetal bovine serum as a significant contaminant both in regards to non human proteins added and in regards to the inherent danger of the presence of pathogenic prions derived from diseases such as mad cow disease. Furthermore, recombinant prothrombin has been made in HEK cells (K. Osther, Inspicos pat. Application 17499). The significant advantage using placenta derived cells and such cells as amnion cells or immortalized amnion cells is that the glycosylation is more resembling human glycosylation, and - unlike Human embryonic stem cells, which can not produce exact sialyzation, the placenta derived cells and amnion cells produce exact sialyzation, and the resulting protein production with human posttranslational modifications and include sialylation for improved half life time. Unlike the prothrombin produced according to this present invention, other recombinant prothrombin molecules have been prepared through recombinant means, where approximately 14% of the protein was abnormally carboxylated . According to U.S. Pat. No. 5,502,034, the prothrombin made according to this invention was prepared in suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc. Natl. Acad, Sci. USA 76: 1035-1039 (1978)), YEpl3 (Broach et al., Gene 8: 121- 133 (1979)), POT vectors (Kawasaki et al, U.S. Pat. No. 4,931,373, which is incorporated by reference herein), pJDB249 and pJDB219 (Beggs, Nature 275: 104-108 (1978)) and derivatives thereof. The prothrombin made in yeast will not be identical to authentic natural human prothrombin. Furthermore, unlike the prothrombin or thrombin made in HEK cells where sialyation is not correctly translated, the sialyation is correctly translated in the recombinant human prothrombin made in non-embryonic cells such as amnion cells (e.g., CAP or CAP-T, CEVEC) or in other non-embryonic placentar or umbilical cord (Whartons' jelly) or even from umbilical cord blood and thus also resulting in a correct prothrombin significantly of higher quality than the prothrombin made in HEK cells, resulting in an improved half life time. At the same time using the placenta cells and the amnion cells respectively, these grow at much higher density in bioreactors, where HEK cells grow up to 3 million per ml suspension, the equivalent volume using placenta or amnion cell concentratoin is much higher under identical conditions, e.g., up to 10 million cells per ml. Therefore, the yield of protein concentration, in this case prothrombin, will be much higher than compared to HEK cells. Another important issue is that contrary to the HEK cell concentration normally obtained in shaker flasks, the number of non-embryonic cells such as for instance transfected amnion cells (e.g., CAP and/or CAP-T) can easily grow to a density of more than 8 million cells per ml, which is a significant difference in cell density those two different system compared.

Biopharmaceutical sialylation refers to the type and distribution of sialic acids in the glycans of therapeutic glycoproteins. Sialylation can significantly influence the safety and efficacy profiles of these drugs. In particular, the in vivo half-life of some biopharmaceuticals correlates with the degree of oligosaccharide sialylation. Furthermore, the sialylation pattern can be a very useful measure of product consistency during manufacturing. Advances in our understanding of these issues have led the FDA, EMEA and other regulators to tighten their rules on glycoprofiling throughout the drug life cycle. These rules are being harmonized under the ICH programme and the ICH Q6B and Q5E guidelines cover

requirements for characterisation of biopharmaceutical sialylation during normal biomanufacturing and after biomanufacturing changes.

Sialic acids are a family of 9-carbon monosaccharides with heterocyclic ring structures. They bear a negative charge via a carboxylic acid group attached to the ring as well as other chemical decorations including N-acetyl and N-glycolyl groups. The two main types of sialyl residues found in biopharma-ceuticals produced in mammalian expression systems are N-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). These usually occur as terminal structures attached to galactose (Gal) residues at the non-reducing terminii of both N- and O-linked glycans. Summary of the invention

The present invention provides recombinant human proteins, notably

prothrombin, preprothrombin, thrombin, vectors and human expression systems for use in the preparation of the proteins with much higher secretion output per liter cells. Furthermore, the amnion cell line(s) and most probably also the placenta type cell lines in general, can easily be cultured in General Electric Wave Bioreactors. Moreover, the recombinant human prothrombin provided in an embodiment of the invention comprises a mutation, (M84A or M400A), or in a more exact and descriptive manner according to this invention, the prothrombin analogue previously called M84A, will be "Prothrombin Analogue M400A", and the preprothrombin analogue also previously called M84A, will be "Preprothrombin Analogue M256A". The invention also relates to the use of the recombinant human proteins in medicine as described herein for the corresponding non-recombinant proteins. Two different routes for obtaining prothrombin is described, one starting from a point mutated prothrombin with gla and Kringle 1 and 2 domains, and maintaining these domains during the whole process; the other one starting with prothrombin (non-mutated), via a preprothrombin with a HPC4-Kringle 2 domain and subjecting this preprothrombin to a point mutation.

The uniqueness of the invention described in this patent application to produce considerable amount of human recombinant authentic proteins building upon the other uniqueness that one can utilize the plasmid vector HZec6 used for HEK cells can be amplified by adding an amplifying vector with 2. Transfection system unique for instance for amnion cells such as CAP and CAP-T cells which increase the yield of protein produced and secreted to significantly higher amount than can be obtained by HEK cells. In this invention human cells such as human placenta type cell lines as well as amnion cell lines as well as immortalized amnion cell lines such as those produced by Cevec are unique in this regard

This system can in this invention also be used for producing proteins such as recombinant human fibrinogen and recombinant human collagen, or more specifically recombinant human collagen of one of the around 19-20 types of human collagens types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX as well as other known human collagens. Detailed description

The present invention relates to a novel concept and method for combining certain recombinant human prothrombin produced by amnion cells (e.g., CAP and CAP-T) (for instance activated in the presence of calcium chloride 0.1% vol/vol or of 40 μιηοΙ will activate recombinant prothrombin to recombinant thrombin to be used as hemostatic product either with gel granulates or coated into an appropriate scaffold such as biatain scaffold, also called biatain foam (e.g., a product under development together with Coloplast A/S), alternatively the same composition can be applied onto another scaffold, a MPEG-PLGA scaffold, called Aseed™, which Cencor Biotech is in the process of obtaining global license from Coloplast A/S, who has hold the IP rights to Aseed™.

Another part of the present invention is creation of a recombinant fibrin kit product, the first recombinant human fibrin kit that has been developed. This product consists of recombinant prothrombin, to be activated for instance with Calcium chloride as described above, immediately prior to use and then mixed together with recombinant human fibrinogen produced in amnion cells (e.g., CAP and CAP-T cells) to obtain recombinant fibrin. It is emphasized that - unlike any other known recombinant prothrombin and recombinant fibrinogen that either are non-human cells or especially human embryonic transfected cells. One of the significant and important difference is that the recombinant proteins such as recombinant prothrombin and recombinant fibrinogen according to my invention is that it is made in placenta originated cells and especially amnion cells (e.g., CAP and/or CAP-T cells from CEVEC) or from stem cells made from umbilical cord blood or from umbilical cord in the area called Wharton's gel, non of these cells are of embryonic origin.

Another important point with many of these placenta related or amnion cell related cells is their ability to produce large amount of recombinant prothrombin or recombinant fibrinogen or recombinant collagen, because this particular system can be transfected with at least two vector systems, one being a step 1 transfection the next, a step 2 transfection as described in CEVEC

Pharmaceutical's patent application regarding CAP and CAP-T (vide: patent application 20120040400 owned by CEVEC). This recombinant CAP or CAP-T produced human prothrombin can react with fibrinogen as such as well as certain human recombinant fibrinogens to create a stable product, with an optimal fibrinogen cleavage activity and with a low effect on activation of protein C. The activation of recombinant prothrombin from CAP and CAP-T cells with calciumchloride has been developed to build into scaffolds such as for instance Coloplast's Biatain Foam, whereby prothrombin powder with calciumchloride powder is activated to thrombin when exposed to a fresh bleeding, providing instant clotting effect.

All the other methods described by the use of human DNA library from which cDNA libraries are produced by inserting into an expression vector driven by a strong promoter, described under "Construction of the expression vector" can be used. A stable human cell will under then produce said proteins, in certain individual amounts of each of said human recombinant proteins from one or more of the cell tines, or some of the recombinant proteins, and will create a fast working end product consisting of proteins involved in the controllable gelling or coagulation, and will be free of contaminating proteins for instance derived from any form of use of mammal cells as described in U.S. Pat. Nos. 6,780,411, 5,476,777, 5,502,034 and 5,572,692, where it, on the contrary, is described that when using a transfecting method for instance recombinant human prothrombin is produced either in inactive form and activated as part of the purification process. Processes for the production of rhProthrombin in this way are disclosed in U.S. Pat. Nos. 5,476,777, 5,502,034 and 5,572,692, the teachings of which are incorporated herein by reference. In all these cases described, where mammal cells or other cells are used for obtaining either prothrombin such as gal prothrombin, where it is stated that the cells used to produce these proteins and actually also recombinant human proteins such as for instance recombinant human fibrinogen, as well as cross linking recombinant human proteins such as recombinant human factor XIII or other cross linking recombinant human proteins such as collagens and in particular recombinant human collagen type I and/or recombinant human collagen type III.

The proteins produced in this manner will be less heterogenic, but on the contrary more homogenous, enabling a better consistency in the final protein product, more reproducible from lot to lot, functionally more active and less immunogenic due to the lesser difference in the sugar molecules, which otherwise may be obtained in cells such as insect cells or CHO cells. At the same time the final product will also be virus free. According to the method as claimed in this invention we are capable of generating stable cell lines and easily make cells adapted to serum-free suspension. The advantage using placenta type cells or amnion cells or even more specific immortalized amnion cells (e.g., CAP or CAP-T from Cevec, Cologne, Germany), is that based upon the already present H

So far, secreted, membrane-bound and intracellular proteins could be produced by transient gene expression. Currently, mammalian cells are the commonly used expression systems for a lot of complex proteins, in particular if said proteins should be used for therapeutic purposes, since prokaryotic and simple eukaryotic cell systems (e.g. yeasts) are clearly disadvantaged in respect of posttranslational modifications. So far, four mammalian cell lines have basically been used for transient protein expression : COS-1 and COS-7 cells, respectively, deriving from the CV-1 cell line derived from kidney cells of the African green monkey; BHK cells deriving from baby hamster kidney cells; CHO cells deriving from the ovary of the Chinese hamster; and HEK293 cells, a human embryonic kidney cell line having neuronal characteristics (Pham et al., Molecular Biotechnology 34, 225-237, 2006; Wurm et Bernard, Current Opinion in Biotechnology 10, 156-159, 1999). Yields averaged of about 10-20 mg/liter are generated by said transient expression of recombinant proteins by using such mammalian cell systems (Baldi et al., Biotechnol. Lett. 29, 677-684, 2007).

In contrast, yields in the range of one to several grams per liter are normal by using stable, permanent production cell lines, as mentioned above, however, with a quite significant higher expense of time and money as described previously in US patent application 20120040400. As also pointed out in US patent application 20120040400, a further disadvantage of cell systems used for recombinant protein expression so far is that some cell lines are indeed suitable for transient expression due to their ability to be easily transfected and to allow episomal plasmid amplification, but other cell lines are preferably used for the production of stable cell lines due to their properties in cultivation and yields (e.g. CHO cells). However, since cell systems differ from each other in several aspects of posttranslational modification, data of structure and function of said gene products obtained for a specific cell system after transient expression (mostly in an earlier phase of the development of therapeutic protein products) can only be transferred in a highly limited way to the structure and function of said gene products after expression in stable cell lines of a distinct cell system (mostly in the later phase of development, for clinical studies and market supply).

Posttranslational modifications, such as glycosylation, phosphorylation, carboxylation, palmitoylation or specific cleavages are of great importance for different properties of the expression products for many candidates of therapeutic products. They can have an influence on the activity, solubility, half life, stability or immunogenicity. Thus, human cell systems play an increasingly rule for the production of therapeutic proteins; only human cells as production facilities provide an authentic, human modification of the expression products and reduce therefore the risk of affected product quality or undesired side effects (US patent application 20120040400). Thus, according to patent application 20120040400 the object of the invention is the provision of a human cell system being comparably well suited for the transient expression of polypeptides and the production of stable production cell lines.

It is anticipated that the proteins according to this invention are as near as human natural authentic proteins so that they do not induce immunogenicity in man. The tissue sealant disclosed in this invention, made of for instance recombinant human prothrombin or recombinant prothrombin mutants or analogues with the previously described pro-coagulation effect (enzymatically cleaving fibrinogen or activating fibrinogen to build fibrin), which again has showed to have a low enzymatic effect against protein C.

A subject matter of the present invention relates to a method for producing a permanent human cell line comprising the following steps: [0032] a) Transfecting primary human cells with a nucleic acid molecule comprising a nucleic acid sequence encoding the adenoviral gene functions EIA and E1B; so called 1.

transfection, and [0033] b) subsequently transfecting the permanent human cell line with a nucleic acid molecule comprising a nucleic acid sequence encoding the SV40 large T-antigen, so called 2. transfection.

Preferably, said nucleic acid molecule of step b) of the method for the production of a permanent human cell line according to the present invention comprises a nucleic acid sequence encoding a non secreted form of the SV40 large T-antigen. During the transfection in step b) of the method according to the present invention the permanent human cell line is alternatively transfected with a nucleic acid molecule comprising a nucleic acid sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), so called 2. transfection. Preferably, said nucleic acid molecule comprises a nucleic acid sequence encoding a non secreted form of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA- 1).

By the transfections performed in the method according to the present invention said primary human cells are preferably transfected stably, i.e. the transfected DNA is integrated into the genome of the cell.

The cells are immortalized by the transfection of said primary human cells with the nucleic acid molecule comprising the nucleic acid sequences encoding EIA and E1B. The nucleic acid molecule used for the immortalization of said primary human cells comprises nucleic acid sequences of EIA and E1B preferably deriving from human adenoviruses, in particular of human adenovirus serotype 5. In a preferred embodiment the nucleic acid molecule used for the immortalization comprises the nucleic acid sequence encoding the adenoviral gene function pIX in addition to the nucleic acid sequences encoding EIA and EIB. The pIX

polypeptide, a viral structural protein, acts as a transcriptional activator on different viral and cellular promoters such as the thymidine kinase and the beta- globin promoter. An exemplary sequence can be found in GenBank acc. no.

X02996. In particular, nucleic acid molecules comprise nucleotides 1 to 4344 comprises nucleic acid sequences encoding EIA, EIB and pIX), 505 to 3522 comprises nucleic acid sequences encoding EIA and EIB) or the nucleotides 505 to 4079 comprises nucleic acid sequences encoding EIA, EIB and pIX) of human adenovirus serotype 5.

In particular, the human cells are transfected with the nucleic acid sequences encoding the desired gene function, which is to be expressed, in form of an expression cassette. Said expression cassette comprises a nucleic acid molecule containing a regulatory element or promoter being positioned in front of the coding region, a coding region and an open reading frame, respectively, as well as a transcriptional termination element lying behind the coding region. In the present invention, it is anticipated that cells from one or more of the chimeric layers of cells in the placenta could be changed and immortalized.

According to Cevec, Cologne, Germany, the primary human cells could be obtained by direct removal from the organism or a tissue removed from the organism and put in culture. Preferred are such primary human cells, which can be well turned into permanent human cell lines by expression of adenoviral EIA and EIB, in particular amniocytic cells, embryonic retina cells and embryonic cells of neuronal origin. Preferably permanent human amniocytic cell lines are produced by the method according to the invention licensed from Cevec for the use in the production of recombinant fibrinogen, and which is anticipated to be used in this invention to also produce the prothrombin and also the thrombin, made in the Cevec originated cell line, according to the patent application #20120040400. The term, "placenta cell types or placenta derived cell types from any of the 3 chimeric layers of cells, and harvested and processed from placenta at delivery is an integrate part of the present invention. According to US patent application #20120040400 the term "amniocytes", as used in that patent application, relates in the broadest sense to all cells that are present in amniotic liquor and may be obtained by amniocentesis. They originate either from amnion or from fetal tissue that is in contact with the amniotic liquor. Three main classes of amniocytes have been described that can be distinguished based on morphological criteria : fibroblast like cells (F cells), epitheloid cells (E cells) and amniotic fluid cells (amniotic fluid cells, AF cells) (Hohn et al., Pediat. Res. 8 : 746-754, 1974). AF cells are the predominant cell type.

Usage of filtrate through MWCO provides dialysis membrane pore sizes that are characterized by the molecular weight at which 90% of the solute will be retained (prevented from permeating) by the membrane. The permeability of a solute is dependent upon the shape of the molecule, its degree of hydration and its charge. Each of these may be influenced by the nature of the solvent, the pH and the ionic strength. As a general rule, it is best to choose a MWCO that is half the molecular weight of the solute to be retained. For effective separation, please refer to the following formula as a guide:

Protein glycosylation

Also pointed out in the US patent application # 20120040400 the glycosylation pattern of complex recombinant proteins, so the structure and arrangement of sugar moieties in the molecule, will reproduce the pattern of the authentic human polypeptide substantially better in the production in human cells than in the production in non human production systems. Said glycosylation pattern is often of crucial importance for important properties of the polypeptide such as biological activity, stability, solubility and immunogenicity.

It is an important part of this invention to assure that the recombinant human proteins are glycosylated correctly. The glycosylation of recombinant proteins in general, especially those destined for potential administration to human subjects, is of much larger critical importance than previously believed. Glycosylation profoundly affects biological activity, function, clearance from circulation, and crucially, antigenicity (Brooks, S A, Molecular Biotechnology, (2004) Vol 28, 241- 256(16)) This Brooks Review gives a brief overview of human N- and O-linked protein glycosylation, summarizes what is known of the glycosylation potential of the cells of nonhuman species, and presents the implications for the biotechnology industry.

The cells of non-human species do not glycosylate their proteins in the same way as human cells do. In many cases it has been found that the differences between "human" glycosylation and "animal" glycosylation are profound . Overall, it is known that the more distant, in evolutionary terms, such as bacteria, yeasts, fungi, insects, and plants, the species used most commonly in expression systems have glycosylation repertoires least like humans, and even cells originated from animals such as for instance in family with rodents, hamsters, and actually up to the animals nearest humans, the pig, there are significant differences in the glycosylation which possibly also can explain that the apparent activity of even proteins, such as for instance recombinant human prothrombin in produced hamster cells show less biological activity, different function, clearance, and, again crucially, the antigenicity. Therefore, in regards to recombinant human

prothrombin described in the following patents: in U.S. Pat. No. 5,527,692, in which BHK 570 cells and yeast were described as selected for the recombinant prothrombin production, and in U.S. Pat. No. 5,502,034 in which BHK 570 cells are used for the transfection of Gla-domainless prothrombin, activated to recombinant thrombin (wild type), was claimed to react with fibrinogen, but not specifically with recombinant human fibrinogen or recombinant human authentic fibrinogen. The placenta cell types and the amnion cell line and especially immortalized amnion cell lines these cell lines far more quantities (up to 1 gram of complex glyhcoproteins, much higher yield compared to what could be obtained using HEK 293.

It is within the scope of this invention to produce recombinant human authentic prothrombin, due to the fact that the prothrombin was transfected in to human embryonic kidney cells in synthetic medium, where we in the invention also have significantly purposely focused on ending up with the correct glycosylation as possible for human use, meaning that we avoided using animal cells, insect cells, yeast or bacteria as host for our two products, recombinant human prothrombin or more specifically recombinant human prothrombin analogue with a site mutation of Chy 84 from methionin to alanin, which we believe will not cause any antigenecity in humans, and the glycosylation will be correct, because the prothrombin or prothrombins claimed in this invention are produced using transfection of the plasmid into human cells, e.g., human placenta cell lines or amnion cell lines. Furthermore, the cells were cultured in serum free synthetic medium as a suspension culture, which appeared to give significantly higher yields, than previously described to be possible to achieve.

Many proteins in eukaryotic cells are glycoproteins because they contain oligosaccharide chains covalently linked to certain amino acids. Glycosylation is known to affect protein folding, localization and trafficking, protein solubility, antigenicity, biological activity and half-life, as well as cell-cell interactions.

Protein glycosylation can be divided into four main categories mainly depending on the linkage between the amino acid and the sugar. These are N-linked glycosylation, O-linked glycosylation, C-Mannosylation, sialyation and GPI anchor attachments. N-glycosylation is characterized by the addition of a sugar to the amino group of an asparagine. In O-glycosylation, a sugar is attached to the hydroxyl group of a serine or threonine residue.

Complex carbohydrates are involved in multiple biological processes, from protein folding, oligomerization and stability, to the immune response and host-pathogen interactions (Varki, 1993). Glycoconjugates also play important roles in developmental processes, as revealed by the pathology of human diseases caused by abnormal glycosylation (Freeze and Aebi, 2005) and genetic studies in model organisms (Haltiwanger and Lowe, 2004). There is a significant difference in glycosylation between different species. Human glycoproteins are glycosylated with a extremely diverse heterogeneous array of complex N- and O-linked glycans, which are the product of the coordinated activity of enzymes resident in the endoplasmic reticulum and Golgi apparatus of the cell. Glycosylation of proteins is highly regulated and changes during differentiation, development, under different physiological--and cell culture species from which the cell cultures derive, meaning that there is pronounced differences between the complex glycosylation in human cells versus animal cells, which wilt most certainly affect biological activity function, clearance in the organism, and of course antigenicity (Brooks S A, Mol. Biotechnol. (2004), Vol 28, 241-255). In many cases, the differences are profound . Overall, the species most distant to humans in evolutionary terms, such as bacteria, yeasts, fungi, insects and plants— the species used most commonly in expression systems— have glycosylation repertoires least like our own. Immunogenetic properties of proteins are determined both by their amino acid sequence and their glycosylation pattern.

Recombinant Prothrombin and Recombinant Thrombin, made by transfecting placenta cells, amnion cells (e.g., CAP and/or CAP-T)

Host cells containing DNA constructs of the present invention are then cultured to produce prothrombin, which in this particular invention will be activated to thrombin during purification by proteases such as factor Xa. The placenta cell lines and the amnion cell lines with incorporated cDNA are cultured according to proprietary methods in a serum-free synthetic medium containing nutrients required for the growth of the host cells and at the same time the section of the post translated protein or polypeptide.

Prothrombin is defined as a two chain, disulfide-bonded, glycosylated polypeptide that cleaves specific bonds in fibrinogen to produce fibrin monomers that self- assemble to form a fibrin clot.

Host cells containing DNA constructs of the present invention are unique because unlike the methods used by Zymogenetics and others aimed at producing prothrombin, is in this particular invention preprothrombin (which is different from prothrombin) will be activated to thrombin.

Host cells containing DNA constructs of the present invention are unique because unlike the Gla-domainless preprothrombin methods used by Zymogenetics and others aimed at producing prothrombin with gla-domainless prothrombin-, is in this particular invention gla-domain-prothrombin (which is completely and significantly different from gla-domainless prothrombin) will be activated to thrombin. The presence of the gla-domain can be evidenced by using a

monoclonal antibody that specifically recognize gamma-carboxyglutamyl (Gla) residues in proteins such as gla-domain containing prothrombin using a method consisting of monoclonal antibodies specific for Gla residues, as described by Brown et al. (Brown M A, Stenberg L M, Persson U, Stenflo J, J. Biol. Chem.

(2000), 275: 19795-1980). The recombinant human prothrombin mutants, that specifically have proven to be procoagulant prothrombin mutants (with high procoagulation effect) and with the same signalling enzyme effects as the Wild Type of prothrombin on cells, such as for instance mesenchymal cells. Of the procoagulant prothrombin mutants which also have PARI signal enzyme effect on cells when compared and low

anticoagulant effect—either as low as wild type prothrombin or even lower. Among those recombinant prothrombin mutants are for instance the type called W215P.

Of other recombinant human prothrombin mutants that are within the scope of this invention, are such types as M84A, also called "Prothrombin Analogue M400A" (when made from prothrombin analogue M400A), and "Prothrombin Analogue M256A (when made from preprothrombin analogue M256A), which clots fibrinogen at a faster rate than the wild type and the arg77A (or R77aA) mutant that lacks autolytic activity. Using the above nomenclature for Prothrombin analogue M400A, when made from prothrombin analogue M400A, and

Prothrombin analogue M256A, when made from preprothrombin analogue M256A, one can always easily distinguish, by which method the prothrombin analogue in question was made, instead of having M84A as the common denominator for both activated prothrombins. In the case of R77aA, the prothrombin analogue would be "Prothrombin analogue R393aA", and the R77aA analogue from preprothrombin would be "Preprothrombin Analogue R249aA".

Also within the scope of this invention, recombinant human prothrombin mutants showing low grade— or no autolysis may be selected among the types of recombinant prothrombin mutants as for instance arg77A (or R77aA). The specific aim is to produce a recombinant prothrombin mutant, which include at least one or more of the recombinant human prothrombin mutants described above, that can be stored refrigerated and still retain their enzymatic activity to polymerize fibrinogen and especially recombinant human fibrinogen, for instance made in mammal cells.

One object of the present invention is to provide methods for besides producing recombinant thrombin, but also producing recombinant prothrombin using recombinant methods in host cells to primarily produce precursor to prothrombin called preprothrombin, in placenta derived cell lines and/or in amnion cells, among those in immortalized amnion cell lines such as those owned by Cevec, Cologne, Germany.

These recombinant human prothrombin types will be compared to wild type human prothrombin and will be recombinant human prothrombin analogues or mutants.

The prothrombin mutants that are part of this invention that retain comparable fibrinogen cleavage activity, but have higher expression level may have the advantage of being produced at lower manufacturing cost.

It is also within the scope of this invention that prothrombin mutants retain comparable expression level but display higher fibrinogen cleavage activity and thus these mutants may lower therapeutic dosage. Further, it is within the scope of this invention that the prothrombin mutants that display reduced autolysis, which are an integrated part of this invention, may facilitate purification process and storage.

As described above it is further envisioned in this invention that mutants that lack protein C activation may have improved coagulation activity.

Mutation can be identified by sequence comparison among prothrombin family members, rational site-directed mutagenesis or alanine-scanning. It is envisioned genes can be modified via insertions, substitutions, deletions or domain swapping. The methods within the scope of this invention is built on producing recombinant mammal prothrombin or more specifically recombinant human prothrombin and even more specific recombinant human prothrombin analogue(s), and yet even more specific a recombinant human prothrombin analogue, called M84A, also called M400A; when all of the above recombinant human prothrombins or prothrombin analogue(s) are made from "gla domain prothrombin" gene, meaning that the deduced protein of recombinant human prothrombin contains signal peptide, propeptide, Gla domain, two kringle domains and a (two-chain) protease domain, the M84A or M400A analogue would be called "prothrombin analogue M400A. The Gla domain is seen in the prothrombin depicted in figure 1.

In a specific embodiment of the present invention the gene sequence expressed according to the invention is any molecule that exhibit prothrombin activity. In a specific embodiment of the present invention the gene sequence expressed according to the invention is any sequence, which encodes a molecule that exhibit prothrombin activity. The gene sequence expressed according to the invention can encode natural human prothrombin or any mutant thereof. Thus, in one embodiment of the invention, prothrombin encoded by the polynucleotide has a sequence 100% identical to the human prothrombin sequence.

Suitable Expression Systems

Host cells for use in practicing this present invention can primarily include a well defined human cell lines, known to secrete proteins or peptides, which closely resembles the natural counterpart of proteins or peptides.

The present invention is applying protein informatics to accurately identify proteins and isozymes or isoforms from the human genome, identifying the gene or genes in question, and using a novel vector system the gene or genes in question is transfected into a well defined mammal or human placenta cell line(s) and/or amnion cell line.

Construction of the Expression Vector

Human prothrombin cDNA (BC051332) was purchased from OpenBiosystems . Human prothrombin is consisted of a signal peptide (AA 1-24 of SEQ ID NO. : 13), a pre-pro leader (AA 26-43 of SEQ ID NO. : 13), activation peptide fragment-1 (AA 44- 198 of SEQ ID NO. : 13) containing gla-domain and kringle 1 domain, activation peptide fragment-2 (AA 199-327 of SEQ ID NO. : 13) containing kringle 2 domain, prothrombin light chain (AA 328-363 of SEQ ID NO. : 13), and prothrombin heavy chain (AA 364-622 of SEQ ID NO. : 13).

M400A prothrombin and M84A preprothrombin are illustrated in SEQ ID NOs 15 and 17, respectively. Point mutation of M400A (or M84A: ATG-GCC) was performed using the primers below:

- M400A-mF: 5 '- G AAAAG ATATCCGCCTTGG AAAAG ATC- 3 ' (SEQ ID NO: 29) - M400A-mR: 5'-GATCTTTTCCAAGGCGGATATCTTTTC-3' (SEQ ID NO: 30)

(B) HPC4 moiety (18 AA) for preprothrombin was added on N-terminus by PCR method using the known sequence in Steams DJ et al. paper. Full cloning map

All the genes were cloned into HumanZyme's pHZsec vector at Srf I site by a gene cloning contractor, Shanghai Genomics <http://www.shanghaigenomics.com > . Since all the secreted proteins have their own signal peptides may yield different levels of their secretion pHZsec vector (a modified vector of Invitrogen's pSecTag2C ) was employed with its own signal peptide from murine Ig kappa leader sequence. Then the prothrombin sequence starting with gla-domain and the preprothrombin starting with HPC4 epitope were inserted at Srf I site of 973 (Figure 5). Entire sequence of pHZsec vector is given in Figure 5. Characterization of cloning

Both prothrombin and preprothrombin of wild type and mutant M400A/M84A were cloned into the pHZsec vector at Srf I site (Figure 6).

Mammalian expression vectors for use in carrying out the production of the proteins, claimed in this invention will include a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Viral promoters could for instance be the immediate early cytomegalovirus promoter (Boshart et al., Cell (1985) 41 : 521- 530) or the SV40 promoter (Subramani et al., Mol. Cell. Biol. (1981) 1 : 854-864). These vectors are amplified together with the vectors used by CEVEC and described in patent application # 20120040400.

The cloned DNA sequences may be introduced into cultured placenta derived cells and/or amnion cell lines such as those amnion and immortalized amnion cell lines from Cevec, Cologne, Germany, using a variety of methods, for example, calcium phosphate-mediated transfection, described by several authors is one preferred method (Wigler M, Pellicer A, Silverstein 5, Axel R. Cell (1978), 14: 725; Corsaro C M, Pearson M L, Somatic Celt Genetics (1981), 7: 603; Graham F L, van der Eb A J, Virology (1973) 52 :456). Electroporation is another technique used for introducing cloned DNA sequences into mammalian cells as previously mentioned (Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider P H., EMBO J. (1982) 1 : 841-845). Cloned DNA sequences from DNA libraries may be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Graham F L and Van der Eb A J, Virology (1973) 52 :456; Wigler M, Pellicer A, Silverstein S, Axel R., Cell (1978) 14: 725; Corsaro C M and Pearson M L, Somatic Cell Genetics (1981) 7: 603).

Neuman et al's technique introducing DNA sequences into mammalian cells by the use of electroporation may also be used (Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider P H., EMBO J. (1982) 1 : 841-845), such as Electric impulses (8 kV/cm, 5 microseconds), which were found to increase greatly the uptake of DNA into mouse lyoma cells by electroporation in high electric fields.

A selectable marker is normally used to identify cells along with the gene or cDNA of interest. Such preferred selectable markers for use in cultured mammalian cells could for instance include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The choice of selectable markers is within the level of ordinary skill in the art. Selectable markers can be introduced into a mammalian cell together with the gene of interest, or introduced, incorporated on the same plasmid. This type of constructs are known in the art (for example, Levinson and Simonsen, U.S. Pat. No. 4,713,339). Transfected mammalian cells are allowed to grow for a period of time, typically a few days, to begin expressing the DNA sequence(s) introduced, Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased stepwise to select in order to increase the copy number of the cloned sequences, thereby increasing expression levels.

Methods thought to be useful for introducing expression vectors encoding gla- domainless prothrombin, is not used in this present invention due to the fact that, in the present invention encompasses the methodology in which prothrombin will be activated during purification by proteases such as factor Xa .

Culturing Conditions

Mammalian cells are generally cultured in commercially available serum- containing or serum-free media. Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art.

Serum-free culture medium for placenta and for amnion cell cultures or cells lines and their suitable growth—and expression media may be used to further improve protein production yields. Variants of commercially available expression vectors including different promoters, secretion signals, transcription enhancers, etc., may also be used to improve protein production yields. Al. pHZsec vector sequence (SEQ ID NO: 31)

1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC

51 TGCTCTGATG CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT

101 GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG

151 GCTTGACCGA CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG

201 CTGCTTCGCG ATGTACGGGC CAGATA ACG CGTTGACATT GATTATTGAC

251 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA

301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG

351 CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT

401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAC TATTTACGGT

451 AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC

501 CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 551 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA

601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA

651 TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA

701 TGGGAGTTTG TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA

751 ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG

801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG

851 GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC

901 CACCATGGAG ACAGACACAC TCCTGCTATG GGTACTGCTG CTCTGGGTTC

951 CAGGTTCCAC TGGTGACGCG CCCGGGCCGG CCAGGCGCGC GCGCCGTACG

1001 TACGAAGCTT GGTACCGAGC TCGGATCCAC TCCAGTGTGG TGGAATTCTG

1051 CAGATATCCA GCACAGTGGC GGCCGCTCGA GGAGGGCCCG AACAAAAACT

1101 CATCTCAGAA GAGGATCTGA ATAGCGCCGT CGACCATCAT CATCATCATC

1151 ATTGAGTTTA AACCCGCTGA TCAGCCTCGA CTGTGCCTTC TAGTTGCCAG

1201 CCATCTGTTG TTTGCCCCTC CCCCGTGCCT TCCTTGACCC TGGAAGGTGC

1251 CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC

1301 TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG

1351 GGGGAGGATT GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTC

1401 TATGGCTTCT GAGGCGGAAA GAACCAGCTG GGGCTCTAGG GGGTATCCCC

1451 ACGCGCCCTG TAGCGGCGCA TTAAGCGCGG CGGGTGTGGT GGTTACGCGC

1501 AGCGTGACCG CTACACTTGC CAGCGCCCTA GCGCCCGCTC CTTTCGCTTT

1551 CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT CAAGCTCTAA

1601 ATCGGGGCAT CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC

1651 CCCAAAAAAC TTGATTAGGG TGATGGTTCA CGTAGTGGGC CATCGCCCTG

1701 ATAGACGGTT TTTCGCCCTT TGACGTTGGA GTCCACGTTC TTTAATAGTG

1751 GACTCTTGTT CCAAACTGGA ACAACACTCA ACCCTATCTC GGTCTATTCT

1801 TTTGATTTAT AAGGGATTTT GGGGATTTCG GCCTATTGGT TAAAAAATGA

1851 GCTGATTTAA CAAAAATTTA ACGCGAATTA ATTCTGTGGA ATGTGTGTCA

1901 GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA

1951 GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC

2001 CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT

2051 AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG CCCAGTTCCG

2101 CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC

2151 GAGGCCGCCT CTGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT

2201 TGGAGGCCTA GGCTTTTGCA AAAAGCTCCC GGGAGCTTGT ATATCCATTT

2251 TCGGATCTGA TCAGCACGTG TTGACAATTA ATCATCGGCA TAGTATATCG

2301 GCATAGTATA ATACGACAAG GTGAGGAACT AAACCATGGC CAAGTTGACC

2351 AGTGCCGTTC CGGTGCTCAC CGCGCGCGAC GTCGCCGGAG CGGTCGAGTT

2401 CTGGACCGAC CGGCTCGGGT TCTCCCGGGA CTTCGTGGAG GACGACTTCG

2451 CCGGTGTGGT CCGGGACGAC GTGACCCTGT TCATCAGCGC GGTCCAGGAC 2501 CAGGTGGTGC CGGACAACAC CCTGGCCTGG GTGTGGGTGC GCGGCCTGGA

2551 CGAGCTGTAC GCCGAGTGGT CGGAGGTCGT GTCCACGAAC TTCCGGGACG

2601 CCTCCGGGCC GGCCATGACC GAGATCGGCG AGCAGCCGTG GGGGCGGGAG

2651 TTCGCCCTGC GCGACCCGGC CGGCAACTGC GTGCACTTCG TGGCCGAGGA

2701 GCAGGACTGA CACGTGCTAC GAGATTTCGA TTCCACCGCC GCCTTCTATG

2751 AAAGGTTGGG CTTCGGAATC GTTTTCCGGG ACGCCGGCTG GATGATCCTC

2801 CAGCGCGGGG ATCTCATGCT GGAGTTCTTC GCCCACCCCA ACTTGTTTAT

2851 TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA

2901 ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC

2951 AATGTATCTT ATCATGTCTG TATACCGTCG ACCTCTAGCT AGAGCTTGGC

3001 GTAATCATGG TCATAGCTGT TTCCTGTGTG AAATTGTTAT CCGCTCACAA

3051 TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGTGCC

3101 TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT

3151 CCAGTCGGGA AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG

3201 CGGGGAGAGG CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC CTCGCTCACT

3251 GACTCGCTGC GCTCGGTCGT TCGGCTGCGG CGAGCGGTAT CAGCTCACTC

3301 AAAGGCGGTA ATACGGTTAT CCACAGAATC AGGGGATAAC GCAGGAAAGA

3351 ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG

3401 TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA

3451 TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC

3501 AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG

3551 CCGCTTACCG GATACCTGTC CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT

3601 TTCTCAATGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG GTCGTTCGCT

3651 CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC

3701 TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC

3751 GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG

3801 GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA

3851 AGGACAGTAT TTGGTATCTG CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA

3901 AAGAGTTGGT AGCTCTTGAT CCGGCAAACA AACCACCGCT GGTAGCGGTG

3951 GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA AGGATCTCAA

4001 GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA

4051 CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT

4101 AGATCCTTTT AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT

4151 GAGTAAACTT GGTCTGACAG TTACCAATGC TTAATCAGTG AGGCACCTAT

4201 CTCAGCGATC TGTCTATTTC GTTCATCCAT AGTTGCCTGA CTCCCCGTCG

4251 TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC CAGTGCTGCA

4301 ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT CAGCAATAAA

4351 CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG

4401 CCTCCATCCA GTCTATTAAT TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG 4451 CCAGTTAATA GTTTGCGCAA CGTTGTTGCC ATTGCTACAG GCATCGTGGT

4501 GTCACGCTCG TCGTTTGGTA TGGCTTCATT CAGCTCCGGT TCCCAACGAT

4551 CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC

4601 TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT

4651 CATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA

4701 GATGCTTTTC TGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG

4751 TGTATGCGGC GACCGAGTTG CTCTTGCCCG GCGTCAATAC GGGATAATAC

4801 CGCGCCACAT AGCAGAACTT TAAAAGTGCT CATCATTGGA AAACGTTCTT

4851 CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC CAGTTCGATG

4901 TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA CTTTCACCAG

4951 CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA

5001 TAAGGGCGAC ACGGAAATGT TGAATACTCA TACTCTTCCT TTTTCAATAT

5051 TATTGAAGCA TTTATCAGGG TTATTGTCTC ATGAGCGGAT ACATATTTGA

5101 ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA TTTCCCCGAA

5151 AAGTGCCACC TGACGTC

A2. M84A Pre prothrombin gene cloning report

Gene cloning Report

Clone Name : HZsec-M84A-4B

Vector : pHZsec (come from Humanzyme), or from an amplifying vector from Cevec, Cologne, Germany

Cloning site : Srf I

Gene : Pre- prothrombin M84A mutation

Primer :

HPC4epitope:

5'-TCCACTGGTGACGCGCCCGCAGCAAAGCTTGAAGACCAAGTAGATCCGCGGCTCATT G-3' (SEQ

ID NO: 32)

Pre-prothrombin-F: 5'-

AGTAGATCCGCGGCTCATTGATGGGAAGGTCGACCTGTCACCTCCATTGGAGCAGTG T-3' (SEQ ID NO: 33)

prothrombin-R: 5'-GGCGCGCCTGGCCGGCCCTCACTACTCTCCAAACTGATCAATGAC- 3'(SEQ ID NO: 34)

Pre-prothrombin-M84A-mF: 5'-GAAAAGATATCCGCCTTGGAAAAGATC-3' (SEQ ID NO: 35)

Pre-prothrombin-M84A-mR: 5'-GATCTTTTCCAAGGCGGATATCTTTTC-3' (SEQ ID NO: 36) Sequencing Primer :

HZsec-seqF: A GGAGACAGACACACTCCTGC-3' (SEQ ID NO: 37)

HZsec-seqR2 : TGGTCGACGGCGCTATTCAG-3' (SEQ ID NO: 38)

Gene Sequence (SEQ ID NO: 39):

GCAGCAAAGCTTGAAGACCAAGTAGATCCGCGGCTCATTGATGGGAAGGTCGACCTGTCA CCTCCATTG GAGCAGTGTGTCCCTGATCGGGGGCAGCAGTACCAGGGGCGCCTGGCGGTGACCACACAT GGGCTCCCC TGCCTGGCCTGGGCCAGCGCACAGGCCAAGGCCCTGAGCAAGCACCAGGACTTCAACTCA GCTGTGCAG CTGGTGGAGAACTTCTGCCGCAACCCAGACGGGGATGAGGAGGGCGCGTGGTGCTATGTG GCCGGGAAG CCTGGCGACTTTGGGTACTGCGACCTCAACTATTGTGAGGAGGCCGTGGAGGAGGAGACA GGAGATGGG C GGA GAGGAC CAGAC GGGCCA CGAAGGGCGTACCGCCACCAG GAGTACCAGAC C CAAT CCGAGGACCTTTGGCTCGGGAGAGGCAGACTGTGGGCTGCGACCTCTGTTCGAGAAGAAG TCGCTGGAG GACAAAACCGAAAGAGAGCTCCTGGAATCCTACATCGACGGGCGCATTGTGGAGGGCTCG GATGCAGAG ATCGGCATGTCACCTTGGCAGGTGATGCTTTTCCGGAAGAGTCCCCAGGAGCTGCTGTGT GGGGCCAGC CTCATCAGTGACCGCTGGGTCCTCACCGCCGCCCACTGCCTCCTGTACCCGCCCTGGGAC AAGAACTTC ACCGAGAATGACCTTCTGGTGCGCATTGGCAAGCACTCCCGCACCAGGTACGAGCGAAAC ATTGAAAAG ATATCCGCCTTGGAAAAGATCTACATCCACCCCAGGTACAACTGGCGGGAGAACCTGGAC CGGGACATT GCCCTGATGAAGCTGAAGAAGCCTGTTGCCTTCAGTGACTACATTCACCCTGTGTGTCTG CCCGACAGG GAGACGGCAGCCAGCTTGCTCCAGGCTGGATACAAGGGGCGGGTGACAGGCTGGGGCAAC CTGAAGGAG ACGTGGACAGCCAACGTTGGTAAGGGGCAGCCCAGTGTCCTGCAGGTGGTGAACCTGCCC ATTGTGGAG CGGCCGGTCTGCAAGGACTCCACCCGGATCCGCATCACTGACAACATGTTCTGTGCTGGT TACAAGCCT GATGAAGGGAAACGAGGGGATGCCTG GAAGG GACAG GGGGGACCC TTGTCATGAAGAGCCCCT T AACAACCGCTGGTATCAAATGGGCATCGTCTCATGGGGTGAAGGCTGTGACCGGGATGGG AAATATGGC TTCTACACACATGTGTTCCGCCTGAAGAAGTGGATACAGAAGGTCATTGATCAGTTTGGA GAGTAGTGA

Protein Sequence (SEQ ID NO: 40):

AAKLEDQVDPRLI DGKVDLSPPLEQCVPDRGQQYQGRLAVTTHGLPCLAWASAQAKALSKHQDFNSAVQ LVENFCRNPDGDEEGAWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDRAIEGRTA TSEYQTFFN PRTFGSGEADCGLRPLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQVMLFRKS PQELLCGAS LISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIEKISALEKIYIHPRYN WRENLDRDI ALMKLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGKGQPSVL QWNLPIVE RPVCKDSTRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMGIVSWGE GCDRDGKYG FYTHVFRLKKWIQKVIDQFGE

Restriction Map(Nde I & Hind III) (figure 7) A3. M400A prothrombin gene cloning report

Gene cloning Report

Clone Name : HZsec-Pro M84A-H1

Vector : pHZsec (come from Humanzyme)

Cloning site : Srf I

Gene : Pro- prothrombin M84A mutation

Primer :

Prothrombin-F:

5'-TCCACTGGTGACGCGCCCGCCAACACCTTCTTGGAGGAGGTGCG-3' (SEQ ID NO: 41)

prothrombin-R2 : 5'-

CGCGCCTGGCCGGCCCTCACTACTCTCCAAACTGATCAATGAC-3' (SEQ ID NO: 42) M84A-mF: 5 ' - G AAAAG ATATCCGCCTTGG AAAAG ATC- 3 ' (SEQ ID NO: 43)

M84A-mR: 5'-GATCTTTTCCAAGGCGGATATCTTTTC-3' (SEQ ID NO: 44)

Sequencing Primer :

HZsec-F: 5'-ATGGAGACAGACACACTCCTGC-3' (SEQ ID NO: 45)

HZsec-R2 : 5'-TGGTCGACGGCGCTATTCAG-3' (SEQ ID NO: 46) prothrombin-wl : 5'-CGAAGGCTCCAGTGTGAATC-3 ' (SEQ ID NO: 47)

Gene Sequence (SEQ ID NO: 48) :

GCCAACACCTTCTTGGAGGAGGTGCGCAAGGGCAACCT GGAGCGAGAGTGCGTGGAGGAGACGTGCAGC TACGAGGAGGCCTTCGAGGCTCTGGAGTCCTCCACGGCTACGGATGTGTTCTGGGCCAAG TACACAGCT TGTGAGACAGCGAGGACGCCTCGAGATAAGCTTGCTGCATGTCTGGAAGGTAACTGTGCT GAGGGTCTG GG ACGAACTACCGAGGGCA G GAACAT CACCCGGTCAGGCATTGAGTGCCAGC A GGAGGAGTCGC TACCCACATAAGCCTGAAATCAACTCCACTACCCATCCTGGGGCCGACCTACAGGAGAAT TTCTGCCGC AACCCCGACAGCAGCACCACGGGACCCTGGTGCTACACTACAGACCCCACCGTGAGGAGG CAGGAATGC AGCATCCC GTCTG GGCCAGGATCAAGTCAC GTAGCGA GACTCCACGCTCCGAAGGCTCCAG GTG AATCTGTCACCTCCATTGGAGCAGT GTGTCCCTGATCGGGGGCAGCAGTACCAGGGGCGCCTGGCGGTG ACCACACATGGGCTCCCCTGCCTGGCCTGGGCCAGCGCACAGGCCAAGGCCCTGAGCAAG CACCAGGAC TTCAACTCAGCTGTGCAGCTGGTGGAGAACTTCTGCCGCAACCCAGACGGGGATGAGGAG GGCGCGT GG TGCTATGTGGCCGGGAAGCCTGGCGACTTTGGGTACTGCGACCTCAACTATTGTGAGGAG GCCGTGGAG GAGGAGACAGGAGATGGGCTGGAT GAGGACTCAGACAGGGCCATCGAAGGGCGTACCGCCACCAGT GAG TACCAGACTTTCTTCAATCCGAGGACCTTTGGCTCGGGAGAGGCAGACTGTGGGCTGCGA CCTCTGTTC GAGAAGAAGTCGCTGGAGGACAAAACCGAAAGAGAGCTCCTGGAATCCTACATCGACGGG CGCATTGTG GAGGGCTCGGAT GCAGAGATCGGCATGTCACCTTGGCAGGTGATGCTTTTCCGGAAGAGTCCCCAGGAG C GC GTGTGGGGCCAGCCTCAT CAGTGACCGC GGGTCCTCACCGCCGCCCACTGCCTCCTGTACCCG CCCTGGGACAAGAACTTCACCGAGAATGACCTTCTGGTGCGCATT GGCAAGCACTCCCGCACCAGGTAC GAGCGAAACATT GAAAAGATATCCgccTT GGAAAAGATCTACATCCACCCCAGGTACAACTGGCGGGAG AACCTGGACCGGGACATTGCCCTGATGAAGCTGAAGAAGCCT GTTGCCTTCAGTGACTACATTCACCCT GTGTGTCTGCCCGACAGGGAGACGGCAGCCAGCTTGCTCCAGGCTGGATACAAGGGGCGG GTGACAGGC TGGGGCAACCTGAAGGAGACGTGGACAGCCAACGTTGGTAAGGGGCAGCCCAGTGTCCTG CAGGTGGTG AACCTGCCCATTGTGGAGCGGCCGGTCTGCAAGGACTCCACCCGGATCCGCATCACTGAC AACATGTTC TGTGCTGGTTACAAGCCTGATGAAGGGAAACGAGGGGATGCCTGTGAAGGTGACAGTGGG GGACCCTTT GTCATGAAGAGCCCCTTTAACAACCGCTGGTATCAAATGGGCATCGTCTCATGGGGTGAA GGCTGT GAC CGGGATGGGAAATATGGCTTCTACACACATGTGTTCCGCCTGAAGAAGTGGATACAGAAG GTCATTGAT CAGTTTGGAGAGTAG GA

Protein Sequence(SEQ ID NO: 49):

ANT FLEEVRKGNLERECVEETCSYEEAFEALESSTATDVFWA YTACETARTPRDKLAACLEGNCAEGL GTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTT DPTVRRQEC SIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQYQGRLAVTTHGLPCLAWASAQ AKALSKHQD FNSAVQLVENFCRNPDGDEEGAWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDRA IEGRTATSE YQTFFNPRTFGSGEADCGLRPLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQV MLFRKSPQE LLCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSRTRYERNIEKISALEKI YIHPRYNWRE NLDRDIALMKLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVT GWGNLKETWTANVGKGQPSVLQW NLP IVERPVCKDSTRI RITDNMFCAGYKPDEGKRGDACEGDSGGP VMKSPFNNRWYQMGIVSWGEGCD RDGKYGFYTHVFRLKKWIQKVIDQFGE

Restriction Map(Nde I & Hind III) (figure 8)

A4. Wild type prothrombin gene cloning report

Gene cloning Report

Clone Name : HZsec-Pro-21

Vector : pHZsec (come from Humanzyme)

Cloning site : Srf I

Gene : prothrombin

Primer :

Pro-prothrombin-F:

5'-TCCACTGGTGACGCGCCCGCCAACACCTTCTTGGAGGAGGTGCG-3' (SEQ ID NO: 50) prothrombin-R: 5'-GGCGCGCCTGGCCGGCCCTCACTACTCTCCAAACTGATCAATGAC- 3'(SEQ ID NO: 51)

Sequencing Primer :

HZsec-seqF: 5'-ATGGAGACAGACACACTCCTGC-3' (SEQ ID NO: 52)

HZsec-seqR2: 5'-TGGTCGACGGCGCTATTCAG-3' (SEQ ID NO: 53) prothrombin-wl: S'-CGAAGGCTCCAGTGTGAATC-S' (SEQ ID NO: 54)

Gene Sequence: GCCAACACCTTCTTGGAGGAGGTGCGCAAGGGCAACCTGGAGCGAGAGTGCGTGGAGGAG ACGTGCAGC TACGAGGAGGCCTTCGAGGCTCTGGAGTCCTCCACGGCTACGGATGTGTTCTGGGCCAAG TACACAGCT TGTGAGACAGCGAGGACGCCTCGAGATAAGCTTGCTGCATGTCTGGAAGGTAACTGTGCT GAGGGTCTG GGTACGAACTACCGAGGGCATGTGAACATCACCCGGTCAGGCATTGAGTGCCAGCTATGG AGGAGTCGC TACCCACATAAGCCTGAAATCAACTCCACTACCCATCCTGGGGCCGACCTACAGGAGAAT TTCTGCCGC AACCCCGACAGCAGCACCACGGGACCCTGGTGCTACACTACAGACCCCACCGTGAGGAGG CAGGAATGC AGCA CCC GTC G GGCCAGGATCAAG CAC GTAGCGA GAC CCACGCTCCGAAGGC CCAG G G AATCTGTCACCTCCATTGGAGCAGTGTGTCCCTGATCGGGGGCAGCAGTACCAGGGGCGC CTGGCGGTG ACCACACATGGGCTCCCCTGCCTGGCCTGGGCCAGCGCACAGGCCAAGGCCCTGAGCAAG CACCAGGAC TTCAACTCAGC GTGCAGC GG GGAGAACTTC GCCGCAACCCAGACGGGGA GAGGAGGGCGCG GG TGCTATGTGGCCGGGAAGCCTGGCGACTTTGGGTACTGCGACCTCAACTATTGTGAGGAG GCCGTGGAG GAGGAGACAGGAGA GGGCTGGATGAGGACTCAGACAGGGCCATCGAAGGGCGTACCGCCACCAG GAG TACCAGACTTTCTTCAATCCGAGGACCTTTGGCTCGGGAGAGGCAGACTGTGGGCTGCGA CCTCTGTTC GAGAAGAAGTCGCTGGAGGACAAAACCGAAAGAGAGCTCCTGGAATCCTACATCGACGGG CGCATTGTG GAGGGCTCGGATGCAGAGATCGGCATGTCACCTTGGCAGGTGATGCTTTTCCGGAAGAGT CCCCAGGAG CTGCTGTGTGGGGCCAGCCTCATCAGTGACCGCTGGGTCCTCACCGCCGCCCACTGCCTC CTGTACCCG CCCTGGGACAAGAACTTCACCGAGAATGACCTTCTGGTGCGCATTGGCAAGCACTCCCGC ACCAGGTAC GAGCGAAACATTGAAAAGATATCCATGTTGGAAAAGATCTACATCCACCCCAGGTACAAC TGGCGGGAG AACCTGGACCGGGACATTGCCCTGATGAAGCTGAAGAAGCCTGTTGCCTTCAGTGACTAC ATTCACCCT GTGTGTCTGCCCGACAGGGAGACGGCAGCCAGCTTGCTCCAGGCTGGATACAAGGGGCGG GTGACAGGC TGGGGCAACCTGAAGGAGACGTGGACAGCCAACGTTGGTAAGGGGCAGCCCAGTGTCCTG CAGGTGGTG AACCTGCCCATTGTGGAGCGGCCGGTCTGCAAGGACTCCACCCGGATCCGCATCACTGAC AACATGTTC TGTGCTGGTTACAAGCCTGATGAAGGGAAACGAGGGGATGCCTGTGAAGGTGACAGTGGG GGACCCTTT GTCA GAAGAGCCCC TAACAACCGC GGTATCAAA GGGCATCGTCTCATGGGG GAAGGCTG GAC CGGGATGGGAAATATGGCTTCTACACACATGTGTTCCGCCTGAAGAAGTGGATACAGAAG GTCATTGAT CAGTTTGGAGAGTAGTGA (SEQ ID NO: 55)

Protein Sequence:

ANTFLEEVRKGNLERECVEETCSYEEAFEALESSTATDVFWA Y ACETARTPRDKLAACLEGNCAEGL GTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTT DPTVRRQEC SIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQYQGRLAVTTHGLPCLAWASAQ AKALSKHQD FNSAVQLVENFCRNPDGDEEGAWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDRA IEGRTATSE YQTFFNPRTFGSGEADCGLRPLFEKKSLEDKTERELLESYIDGRIVEGSDAEIGMSPWQV MLFRKSPQE LLCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVRIGKHSR RYERNIEKISMLEKIYIHPRYNWRE NLDRDIALMKLKKPVAFSDYIHPVCLPDRETAASLLQAGYKGRVTGWGNLKETWTANVGK GQPSVLQW NLPIVERPVCKDSTRIRITDNMFCAGYKPDEGKRGDACEGDSGGPFVMKSPFNNRWYQMG IVSWGEGCD RDGKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 56) Restriction Map(Nde I & Hind III) (figure 9)

Process Flow Diagram for the activation of prothrombin and purification of thrombin on

AKTAxpress

Prothrombin from Prothrombin spent medium harvested at day 7 by

CAP or CAP-T centrifugation @ 10,000 x g for 20 min

Harvest &

Clarification Filter the supernatant through 0.2pm filter membrane and store at -20°C until performing activation and purification

Prothrombin Mix 200ml_ prothrombin conditioned medium and 200pg Activation to recombinant ecarin-6xHis in lOmM Tris, pH7.4/200mM NaCI Prothrombin

by swirling, alternatively activating using Calcium chloride sol.

Incubate them for 90min at room temp

Add 2M Imidazole (pH8) for final concentration of 20mM Imidazole

Ecarin- 6xHis Column : HiTrap 5ml GE Chelating (17-0749-03) charged with removc 1 step Cu ⁺⁺

Capacity: Flow through mode (5ml resin is sufficient for whole sample)

/ Flow rate: 4mL/min (2cm/min)

Equilibration buffer: lOmM Tris, pH7.4 + 150mM NaCI + 20mM Imidazole

Wash buffer: the equilibration buffer

Desalti rig Column : Sephadex G25 fine (GE, 17-0032-02)

Capacity: 1L G25 for upto 250ml_ sample (Column: XK50)

Flow rate: 20ml/min (1.5cm/min)

Equilibration and Wash Buffer: 20mM MES (pH6.0) + 200mM choline chloride

Intermediate Column : HiTrap 16/10 Q-FF (GE, 17-0709-01) linked to HiTrap step 5ml SP-FF (GE, 17-5171-01) (^Q-SP^)

Capacity: 350ml_ of desalted sample

Flow rate: 4mL/min (2cm/min) Equilibration buffer/Wash buffer: 20mM MES (pH6.0) + 200mM choline chloride

Column Remove HiTrap 16/10 Q-FF

Separation

Polishin g & Column : HiTrap 5ml SP-FF (GE, 17-5171-01)

Concen tration Capacity: thrombin bound onto the column

Flow rate: 4ml_/min (2cm/min)

Wash buffer: 20mM MES (pH6.0) + 200mM choline chloride Elution buffer: 20mM MES (pH6.0) + 500mM choline chloride

Final Formulation 20mM MES (pH6.0) + 500mM choline chloride

Filter sterilization : 0.2μιη filter membrane & lyohilization

A6. Purification of recombinant human prothrombin

(from recombinant Ecarin-6His activated wt prothrombin)

1. prothrombin activation

· 200ml_ prothrombin culture super + 200ug ecarin for 90min at rt

• Test prothrombin activity at 60min to ensure the reaction is working

• One program for three columns

5ml GE Chelating to remove ecarin-6His

1L G25: buffer exchange to 20mM MES/200mM Choline CI

- Q-SP: to separate prothrombin from prothrombin and others

Method for the Preparation of Recombinant Human thrombin

Methods are disclosed for producing thrombin. The protein is produced from host cells transformed or transfected with DNA construct(s) containing information necessary to direct the expression of thrombin precursors. The DNA constructs generally include the following operably linked elements: a transcriptional promoter, DNA sequence encoding a prothrombin, and a transcriptional terminator, however, as an integrate part of this invention a gla-domain containing prothrombin or rather, a recombinant human gla-domain containing prothrombin. Trombin precursors produced from transformed or transfected host cells are activated either in vivo or in vitro. There is therefore a need in the art for methods for producing prothrombin that is essentially free of contaminating proteins. The present invention fulfils this need and provides other related advantages. For all the types of proteins to be obtained from human placenta cell lines, amnion cell lines, immortalized amnion cell lines encompassed in this invention are cDNA copies of certain human variants of recombinant prothrombin.

Selection of optimal media is within the level of the skill in the art. Certain thrombin precursors may preferably be produced from these transfectants by the addition of heparin or thrombin. The activation of prothrombin precursors containing a thrombin cleavage site in place of the wild-type thrombin activation site (Arg-Ile) may be enhanced by heparin added to the medium. Preferably, in this scenario, between 0.5 and 5.0 U/ml of heparin is added to the serum-free medium, more preferably between 1 and 5 U/ml and most preferably 1 U/ml of heparin is added to the serum-free medium according to some authors. To activate the protein produced from human placenta cell lines or from amnion cells more preferably will have an anticipated activity between 1 and 2 mu.g/ml of thrombin, with 1 mu.g/ml of thrombin added to the serum-free medium as being particularly preferred.

Thrombin precursors may be purified by conventional chromatography, and the thrombin precursor may then be activated by for instance snake venom activator in a serial dilution related to the protein concentration. Alternatively, thrombin precursors may be purified by affinity chromatography using anti-prothrombin antibodies. Other methods of thrombin purification have been suggested as for instance in U.S. Pat. No. 4,965,203. According to the present invention the recombinant human thrombin will be activated during purification by proteases such as factor Xa.

Purified prothrombin precursors may also be activated using the proteolytical activation of prothrombin as described by Heldebrant et al. and others

(Heldebrant C M, Butkowski R J, Bajaj S P, Mann K G. J. Biol. Chem. (1973) 248: 7149-7163; Downing M R, Butkowski R J, Clark M M, Mann K G, J. Biol.

Chem. (1975), 250: 8897; Krishnaswamy S, Church W R, Nesheim M E, Mann K G. J. Biol. Chem. (1987) 262 : 3291). Thrombin precursors that contain a thrombin activation site may be activated by the addition of thrombin. The activated thrombin can then be purified using column chromatography with a salt gradient. Methods of protein purification are well known in the art, for general purposes, see Scopes R. (Scopes, R., Protein Purification, Springer-Verlag, NY (1982), the higher purity that can be obtained the more clear-cut reaction of the thrombin, so a purification between 70-90% would most probably be ideal.

One embodiment according to the present invention provides a polynucleotide encoding a recombinant human prothrombin precursor molecule as defined in SEQ ID NO: 63 the polynucleotide containing i) Gla domain as defined SEQ ID NO: 16 In another embodiment, the present invention further comprises ii) Kringle 2. In yet another embodiment, the present invention further comprises a polynucleotide as described herein, wherein the polynucleotide further comprises iii) HCP4 (protein C), and the recombinant human prothrombin precursor molecule encoded has HPC4 linked at the 5' end (SEQ ID NO: 19)

In an embodiment the recombinant human thrombin precursor molecule according to the present invention has autolytic activity.

One embodiment according to the present invention provides a vector comprising the polynucleotide as defined in the present invention. In a further embodiment the polynucleotide according to the present invention is further operably linked to control sequences recognised by a host cell transformed with said vector. In yet a further embodiment present invention the vector according the present invention is in the form of a plasmid vector.

One embodiment according to the present invention provides a method for the preparation of recombinant human prothrombin or thrombin using a human expression system. In a further embodiment the human expression system comprises a vector as defined herein. In yet a further embodiment the human expression system is a human host cell as defined herein. In yet a further embodiment the human expression system is cultured under serum-free conditions. In an embodiment according to the present invention the recombinant human prothrombin or thrombin prepared according to the present invention contains a gla domain (SEQ ID NO: 16). One embodiment according to the present invention provides the recombinant human prothrombin or thrombin has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, nucleic acid homology with the natural human prothrombin or prothrombin gene. One embodiment according to the present invention provides the recombinant human prothrombin or prothrombin has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, amino acid homology with the natural human prothrombin or thrombin. One embodiment according to the present invention provides at least 90%, such as at least 91%, at least 92%, least 93%, at least 94%, least 95%, at least 96%, least 97%, at least 98%, least 99%, or 100%, of the protein has a glycosylation pattern that results in an immunogenicity response substantially identical to that of the natural human prothrombin or thrombin.

In an embodiment according to the present invention the recombinant human thrombin clots fibrinogen at a faster rate than the natural human thrombin.

In an embodiment according to the present invention the recombinant human thrombin retains at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, or 100%, of the initial fibrinogen polymerization activity after one week of storage at 4-8. degree. C.

In an embodiment according to the present invention the recombinant human preprothrombin or prothrombin can be activated by any means that are known to a person skilled in the art. In a preferred embodiment of the present invention the recombinant human preprothrombin or prothrombin is activated to recombinant human thrombin by use of ecarin. Activation with ecarin has the further advantages of making immobilisation of the activator possible; ecarin therefore provides for a one step purification method of recombinant human thrombin. In an embodiment according to the present invention the recombinant human prothrombin has a significantly lower autolytic activity than human natural prothrombin.

In an embodiment according to the present invention the human recombinant prothrombin is as defined in SEQ ID NO: 13 or SEQ ID NO: 15. In a further embodiment the human recombinant prothrombin as defined herein is for use in medicine.

In an embodiment according to the present invention the human recombinant preprothrombin is as defined in SEQ ID NO: 40 or SEQ ID NO: 68. In a further embodiment the human recombinant prothrombin as defined herein is for use in medicine.

In an embodiment according to the present invention the human recombinant thrombin is as defined in SEQ ID NO: 15. In a further embodiment the human recombinant thrombin as defined herein is for use in medicine. Applications of Human Recombinant Proteins According to the Present Invention

A most probable approach within the scope of this invention is the usage of one single recombinant human protein such as for instance certain recombinant Human prothrombin analogue(s) and/or recombinant prothrombin mutant(s), produced in mammal cells intended to be either used individually as sole product(s) without other combinations for local hemostasis or in certain bleeding areas in the organism where the applied recombinant human thrombin can react with fibrinogen present in the bleeding area, and thus prevent or stop the bleeding.

In one embodiment the concentration of prothrombin according to the present invention, such as recombinant human natural prothrombin or recombinant human prothrombin with one or more point mutations, may be less than 20 NIH units/ml, such as 1-20 NIH units/ml, such as less than 15 NIH units/ml, less than 10 NIH units/ml, less than 5 NIH units/ml, or less than 1 NIH unit/ml.

In one embodiment the concentration of fibrinogen according to the present invention, such as recombinant human natural fibrinogen, may be less than around 50 mg/ml, such as 1-50 mg/ml, such as less than 40 mg/ml, less than 30 mg/ml, less than 20 mg/ml, less than 10 mg/ml, or less than 2 mg/ml.

Recombinant human thrombin has a wide range of uses ranging from the treatment of coagulation disorders in humans to act as enzymatic initiator of clotting, treatment of burns, of skin grafting, both in minor and in major surgery either alone or as part of a product, such as tissue sealants.

The formulation of various wound tissue adhesives is discussed in detail in U.S. Pat. Nos. 4,427,650, 4,442,655, and 4,655,211, each of which is incorporated herein by reference.

The effective doses of recombinant human prothrombin will vary considerably ranging from 100 to 15,000 units depending on the manner in which prothrombin is used (the specific activity of pure prothrombin is 3,000 units per .mu.g protein). If the prothrombin or the thrombin is used in scaffolds or microcarriers such as Coloplast A/S's Aseed (MPEG PLGA) membrane or in such as for instance Biatain Foam (Coloplast A/S), the units needed are in the area between 25 - 150 per cm2.

Quite another model of this invention could be the use of some or one of the proteins which are within the scope of this invention that conveniently could be incorporated in substances which again could react with substances added to provoke gelation, adherence or gluing effect, as well as hemostatic effects.

Recombinant human prothrombin or thrombin according to the present invention may be formulated with any known pharmaceutically acceptable excipients. Additional compositions, kits, ingredient assemblies are included within the scope of the present invention for use in connection with the methods herein described as the follows.

5 An overview of the sequence listings enclosed is shown in Table 1 below.

TABLE-US-00001 TABLE 1 Description 1 Polynucleotide sequence of BC051332 (Human Prothrombin) - M400A (Base* 1198, 1199, 1200; ATG .fwdarw. gcc) 2 Amino acid sequence of BC051332 (Human Prothrombin) - M400A (Base* 1198,

10 1199, 1200; ATG .fwdarw. gcc) 3 Polynucleotide sequence of BC051332 (Human Prothrombin) - wt 4 Amino acid sequence of BC051332 (Human Prothrombin) - wt 5 Polynucleotide sequence of BC051332 (Human Preprothrombin)- M256A (Base* 766, 767, 768; ATG .fwdarw. gcc) 6 Polynucleotide sequence of BC051332

(Human Preprothrombin)- M256A (Base* 766, 767, 768; ATG .fwdarw. gcc) with

15 HPC4 7 Amino acid sequence of BC051332 (Human Preprothrombin)- M256A

(Base* 766, 767, 768; ATG .fwdarw. gcc) with HPC4 8 Polynucleotide sequence of BC051332 (Human Preprothrombin)- wt 9 Amino acid sequence of BC051332 (Human Preprothrombin)-wt 10 16 GLA domain (where X is defined as gamma- carboxyglutamic acid (Gla)). 17 Sequence of prothrombin analogue M84A from

20 Seq. (calculated from prothrombin Seq . no. 285) comprising alpha-prothrombin light (A) chain and heavy (B) chain

EXAMPLES

Example 1

25

Method for the Preparation of Recombinant Human Prethrombin Via a Prethrombin with a HPC4 Domain

The human prothrombin gene was purchased from the Gene Bank (GB accession 30 No. BC051332). PCR Gla-domain containing prothrombin region was obtained and an HPC4 (protein C) epitope was added to the 5' end in the DNA. Protein C undergoes Ca.sup.2+-induced conformational changes required for activation by the prothrombin-thrombomodulin complex. A Ca.sup.2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational 35 changes near the activation site in protein C as shown by Stearns et al (Stearns D J, Kurosawa S, Sims P J, Esmon N L, Esmon C T, The interaction of a Ca.sup.2+- dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca.sup.2+ binding to both antigen and antibody, J Biol Chem. (1988) 263(2) : 826-32).

Point mutation on 84 was made from Methionine to Alanine (ATG.fwdarw.GCC), see sequence of human M84A (SEQ ID NO: 6 and SEQ ID NO: 17), where the sequence (SEQ) numbering on B 116, corresponding to chymotrypsin "84" or prothrombin "400" (or prothrombin analogue M400A) was site mutated as described here, as a point mutation.

Another method for the production of recombinant human prothrombin is to activate this prothrombin from cloned recombinant Human preprothrombin-l as for instance M84A (or Preprothrombin analogue M256A) was inserted into a pHZsec vector, which is a proprietary vector owned by HumanZyme Inc.

(HumanZyme Inc., Chicago, USA). The M84A gene was then amplified in E. coli. The amplified gene was then transfected into human amnion cell line in a monolayer cell culture in medium containing serum according to the method developed by HumanZyme Inc. (HumanZyme Inc., Chicago). The expression of the human recombinant prothrombin analogue (M84A) was verified by Western blot analysis using mAb-HPC4. Both wild type preprothrombin-l and M84A preprothrombin-l have been successfully expressed from amnion cells or immortalized amnion cells. Example 2

Recombinant human prothrombin analogue (M84A, M400A (or analogue M256A) was expressed in CAP and/or CAP-T cells. Twelve (12) clones were selected from 6 wells to larger plates. The two top clones based on Western blot analysis using monoclonal antibody against HPC4 (mAb-HPC4). The cells were adapted for serum free medium and processed as a suspension culture. The recombinant human preprothrombin-l M84A (or preprothrombin analogue M256A) was purified using a pilot size purification method. The yield from a serum-free suspension culture of recombinant HU thrombin analogue (M84A (or thrombin analogue M256A)) per Liter was satisfactory. Untransfected cells (controls) died out while M84A (or analogue M256A) transfected cell lines showed continuous growth in the presence of antibiotics such as zeocin.

Amino acid sequence of human thrombin analogue M84A from Seq. (calculated from prothrombin) is according to SEQ ID NO 17.

This shows that it is relatively easy to purify recombinant human thrombin analogue M84A, when the crude protein prior to the elution only contains relatively few bands of proteins, when compared to the amount of proteins present in cell culture containing serum (as for instance 10% serum).

Example 3

Method for Preparing Prothrombin with Intact Gla Domain Selection of stable cell line transfected with recombinant human gla-domain containing prothrombin, which can be activated to recombinant thrombin, recombinant human thrombins whereof one of the recombinant human thrombins is the recombinant human thrombin analogue, M84A or even recombinant wild type thrombin derived from activation of gla-domain containing prothrombin. The entire Gla-domain containing prothrombin gene is amplified in E. coli, and the amplified Gla-domain containing prothrombin is transfected into placenta derived cells and/or amnion cell lines and grown in monolayer culture containing serum in the medium. Several clones were harvested and the clones indicating the content of M84A (or analogue M400A) was isolated and these clones were adapted into suspension cell culture in serum free medium. The M84A gla-domain prothrombin was harvested from the suspension cell culture by separating the cells from the supernatant. The M84A (or analogue M400A was then precipitated and purified and activated by one step method using a proteinase. Amino acid sequence of human thrombin analogue M84A from Seq. (calculated from prothrombin Seq. no. 285) is according to SEQ ID NO: 17.

During the process, the recombinant Gla-domain prothrombin, a prothrombin containing signal peptide, propeptide, Gla domain, two kringle domains and protease domain (e.g ., trypsin) is retained . Example 5

The human prothrombin gene was purchased from the Gene Bank (GB accession No. BC051332). PCR Gla-domain containing prothrombin region aa#44-622 (579 aa) was subjected to point mutation on 84 (Chy) (or at prothrombin analogue M400A) from Met to Ala (ATG.fwdarw.GCC)

Point mutation on amino acid position 84 (or at prothrombin analogue position 400) was made from methionine to alanine (ATG.fwdarw.GCC);

TABLE-US-00002 M84A-mF: 5'-GAAAAGATATCCGCCTTGGAAAAGATC-3' (SEQ ID NO. : 57) M84a-mR: 5 ' - G ATCTTTTCC A AG G CG G AT ATCTTTTC- 3 ' (SEQ ID NO. : 58)

The recombinant Human Prothrombin M84A (or Human Prothrombin analogue M400A) was then inserted into the pHZsec vector, followed by a vector specifically used by Cevec for amnion cells and immortalized amnion cells, and amplification of the M84A (or analogue M400A) gene was done in E. coli. The amplified gene was then transfected into amnion cell line in monolayer in 10% serum containing medium . The expression was found and verified by Western blot analysis on the conditioned medium with Anti-Human prothrombin (Enzyme Research

Laboratories).

Example 6

Human prothrombin gene was purchased from the Gene Bank (GB accession no. BC051332). PCR preprothrombin region aa#206-622 (417 aa) and added HPC4 epitope (18 aa) at N-terminus (5' end in DNA). Reference for HPC4 Stearns (Stearns D J, Kurosawa S, Sims P J, Esmon N L, Esmon C T. 1 Biol. Chem .

(1988), 263: 826-832) as follows: TABLE-US-00003 gcagcaaagcttgaagaccaagtagatccgcggctcattgatgggaaggt (SEQ ID NO. : 59), A A K L E D Q V D P R L I D G K V (SEQ ID NO. : 60), cgacctgtca (SEQ ID NO. : 61), D L S

Point mutation M84A on amino acid position 84 (or at analogue position M256A) from Met to Ala (ATG.fwdarw.GCC) was done. TABLE-US-00004 M84A-mF: 5'-GAAAAGATATCCGCCTTGGAAAAGATC-3' 3' (SEQ ID NO. : 57) M84a-mR: 5'-GATCTTTTCCAAGGCGGATATCTTTTC-3' 3' (SEQ ID NO. : 58)

The cloned recombinant Human Prothrombin M84A (or prothrombin analogue M256A) was inserted into the pHZsec vector. The cloned gene was amplified in E. coli. The gene was transfected into amnion cell line in monolayer culture. The expression was verified by Western analysis on the conditioned medium with mAb-HPC4. Twelve clones were selected and cells from 6 wells were transferred to a larger plate. The top two clones based on Western analysis (mAB-FIPC4) were selected. These cells were adapted to serum-free medium.

Pilot Purification of Preprothrombin M84A (or Analogue M256A)

Preprothrombin M84A (or analogue M256A was observed using mAb Anti-Protein C (Roche). Preprothrombin is eluted from crude Preprothrombin from serum-free medium using Resin: anti-Protein C Affinity Matrix (Roche). Prothrombin M84A Activation by Ecarin

Ecarin was obtained from Sigma (Sigma E0504), and is Echis carinatus venom, it is independent of Ca++, phospholipids, and plasma clotting factors. It can be used as possible immobilization agent.

The activation reaction condition was as follows, 50 .mu. l of Ecarin (50 EU/ml) was added to 5 ml of M84A preprothrombin (or preprothrombin analogue M256A) (0.5 mg/ml) either in crude media or in TBS (after purification through HPC4 column). Determination of the activation was done by using the thrombin assay with a chromogenic substrate (FPRpNA from Midwest Bio-Tech # 710013) at room temperature, and the following was done: [0210]970 .mu. l of 1. times. TBS (Tris- based saline, pH 7.4) [0211]25 .mu.l 1.7 mM FPRpNA (final concentration 40 . mu. M) [0212]5 .mu.l reaction mixture at each time period. The purification and activation of Preprothrombin M84A (or preprothrombin analogue M256A) to thrombin (.alpha. -thrombin M84A).

A-Prothrombin Purification from Heparin Sepharose Chromatography

5

According to FIG. 49. Lane 1 shows the Ecarin activated .alpha. -thrombin mixture was loaded on a heparin column pre-equilibrated with 10 mM Tris (pH 7.4)/200 mM Choline chloride. Lane 2 shows the Flow--thru which was collected until the baseline was obtained. Lane 3 shows Non-specific contaminant(s) which was 10 washed with 10 mM Tris (pH 7.4)/500 mM Choline chloride. Lane 4 shows the .alpha. -thrombin M84A eluted with 10 mM Iris (pH 7.4)/800 mM Choline chloride (see FIG. 49).

Example 7

15

Sequence CWU 1

1711743DNA artificial Recombinant human thrombin precursor molecule lgccaacacct tcttggagga ggtgcgcaag ggcaacctgg agcgagagtg cgtggaggag

20 60acgtgcagct acgaggaggc cttcgaggct ctggagtcct ccacggctac ggatgtgttc

120tgggccaagt acacagcttg tgagacagcg aggacgcctc gagataagct tgctgcatgt

180ctggaaggta actgtgctga gggtctgggt acgaactacc gagggcatgt gaacatcacc

240cggtcaggca ttgagtgcca gctatggagg agtcgctacc cacataagcc tgaaatcaac

300tccactaccc atcctggggc cgacctacag gagaatttct gccgcaaccc cgacagcagc

25 360accacgggac cctggtgcta cactacagac cccaccgtga ggaggcagga atgcagcatc

420cctgtctgtg gccaggatca agtcactgta gcgatgactc cacgctccga aggctccagt

480gtgaatctgt cacctccatt ggagcagtgt gtccctgatc gggggcagca gtaccagggg

540cgcctggcgg tgaccacaca tgggctcccc tgcctggcct gggccagcgc acaggccaag

600gccctgagca agcaccagga cttcaactca gctgtgcagc tggtggagaa cttctgccgc

30 660aacccagacg gggatgagga gggcgcgtgg tgctatgtgg ccgggaagcc tggcgacttt

720gggtactgcg acctcaacta ttgtgaggag gccgtggagg aggagacagg agatgggctg 780gatgaggact cagacagggc catcgaaggg cgtaccgcca ccagtgagta ccagactttc

840ttcaatccga ggacctttgg ctcgggagag gcagactgtg ggctgcgacc tctgttcgag

900aagaagtcgc tggaggacaa aaccgaaaga gagctcctgg aatcctacat cgacgggcgc

35 960attgtggagg gctcggatgc agagatcggc atgtcacctt ggcaggtgat gcttttccgg 1020aagagtcccc aggagctgct gtgtggggcc agcctcatca gtgaccgctg ggtcctcacc

1080gccgcccact gcctcctgta cccgccctgg gacaagaact tcaccgagaa tgaccttctg

1140gtgcgcattg gcaagcactc ccgcaccagg tacgagcgaa acattgaaaa gatatccatg

1200ttggaaaaga tctacatcca ccccaggtac aactggcggg agaacctgga ccgggacatt 5 1260gccctgatga agctgaagaa gcctgttgcc ttcagtgact acattcaccc tgtgtgtctg

1320cccgacaggg agacggcagc cagcttgctc caggctggat acaaggggcg ggtgacaggc 1380tggggcaacc tgaaggagac gtggacagcc aacgttggta aggggcagcc cagtgtcctg 1440caggtggtga acctgcccat tgtggagcgg ccggtctgca aggactccac ccggatccgc

1500atcactgaca acatgttctg tgctggttac aagcctgatg aagggaaacg aggggatgcc

10 1560tgtgaaggtg acagtggggg accctttgtc atgaagagcc cctttaacaa ccgctggtat

1620caaatgggca tcgtctcatg gggtgaaggc tgtgaccggg atgggaaata tggcttctac

1680acacatgtgt tccgcctgaa gaagtggata cagaaggtca ttgatcagtt tggagagtag 1740tga (SEQ ID NO. : 62)17432579PRTartificialRecombinant human prothrombin precursor molecule 2Ala Asn Thr Phe Leu Glu Glu Val Arg Lys Gly Asn Leu Glu Arg

15 Glul 5 10 15Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu 20 25 30Ser Ser Thr Ala Thr Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu 35 40 45Thr Ala Arg Thr Pro Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn 50 55 60Cys Ala Glu Gly Leu Gly Thr Asn Tyr Arg Gly His Val Asn lie Thr65 70 75 80Arg Ser Gly He Glu Cys Gin Leu Trp Arg Ser Arg Tyr Pro His Lys 85 90 95Pro Glu He Asn Ser Thr Thr

20 His Pro Gly Ala Asp Leu Gin Glu Asn 100 105 llOPhe Cys Arg Asn Pro Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr 115 120 125Thr Asp Pro Thr Val Arg Arg Gin Glu Cys Ser He Pro Val Cys Gly 130 135 140Gln Asp Gin Val Thr Val Ala Met Thr Pro Arg Ser Glu Gly Ser Serl45 150 155 160Val Asn Leu Ser Pro Pro Leu Glu Gin Cys Val Pro Asp Arg Gly Gin 165 170 175Gln Tyr Gin Gly Arg Leu Ala Val Thr Thr His

25 Gly Leu Pro Cys Leu 180 185 190Ala Trp Ala Ser Ala Gin Ala Lys Ala Leu Ser Lys His Gin Asp Phe 195 200 205Asn Ser Ala Val Gin Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly 210 215 220Asp Glu Glu Gly Ala Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe225 230 235 240Gly Tyr Cys Asp Leu Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr 245 250 255Gly Asp Gly Leu Asp Glu Asp Ser Asp Arg Ala He Glu Gly Arg

30 Thr 260 265 270Ala Thr Ser Glu Tyr Gin Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser 275 280 285Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu 290 295 300Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr He Asp Gly Arg305 310 315 320Ile Val Glu Gly Ser Asp Ala Glu He Gly Met Ser Pro Trp Gin Val 325 330 335Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser Leu 340 345

35 350Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro 355 360 365Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg He Gly 370 375 380Lys His Ser Arg Thr Arg Tyr Glu Arg Asn He Glu Lys He Ser Ala385 390 395 400Leu Glu Lys He Tyr He His Pro Arg Tyr Asn Trp Arg Glu Asn Leu 405 410 415Asp Arg Asp He Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser 420 425 5 430Asp Tyr He His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser 435 440

445Leu Leu Gin Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu 450 455 460Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gin Pro Ser Val Leu465 470 475 480Gln Val Val Asn Leu Pro He Val Glu Arg Pro Val Cys Lys Asp Ser 485 490 495Thr Arg He Arg He Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro 500 505

10 510Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro 515 520 525Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gin Met Gly lie 530 535 540Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr545 550 555 560Thr His Val Phe Arg Leu Lys Lys Trp lie Gin Lys Val He Asp Gin 565 570 575Phe Gly Glu (SEQ ID NO. : 63) 31743DNAHomo sapiens 3gccaacacct

15 tcttggagga ggtgcgcaag ggcaacctgg agcgagagtg cgtggaggag 60acgtgcagct

acgaggaggc cttcgaggct ctggagtcct ccacggctac ggatgtgttc 120tgggccaagt acacagcttg tgagacagcg aggacgcctc gagataagct tgctgcatgt 180ctggaaggta actgtgctga gggtctgggt acgaactacc gagggcatgt gaacatcacc 240cggtcaggca ttgagtgcca gctatggagg agtcgctacc cacataagcc tgaaatcaac 300tccactaccc

20 atcctggggc cgacctacag gagaatttct gccgcaaccc cgacagcagc 360accacgggac

cctggtgcta cactacagac cccaccgtga ggaggcagga atgcagcatc 420cctgtctgtg gccaggatca agtcactgta gcgatgactc cacgctccga aggctccagt 480gtgaatctgt cacctccatt ggagcagtgt gtccctgatc gggggcagca gtaccagggg 540cgcctggcgg tgaccacaca tgggctcccc tgcctggcct gggccagcgc acaggccaag 600gccctgagca agcaccagga

25 cttcaactca gctgtgcagc tggtggagaa cttctgccgc 660aacccagacg gggatgagga

gggcgcgtgg tgctatgtgg ccgggaagcc tggcgacttt 720gggtactgcg acctcaacta ttgtgaggag gccgtggagg aggagacagg agatgggctg 780gatgaggact cagacagggc catcgaaggg cgtaccgcca ccagtgagta ccagactttc 840ttcaatccga ggacctttgg ctcgggagag gcagactgtg ggctgcgacc tctgttcgag 900aagaagtcgc tggaggacaa

30 aaccgaaaga gagctcctgg aatcctacat cgacgggcgc 960attgtggagg gctcggatgc

agagatcggc atgtcacctt ggcaggtgat gcttttccgg 1020aagagtcccc aggagctgct gtgtggggcc agcctcatca gtgaccgctg ggtcctcacc 1080gccgcccact gcctcctgta cccgccctgg gacaagaact tcaccgagaa tgaccttctg 1140gtgcgcattg gcaagcactc ccgcaccagg tacgagcgaa acattgaaaa gatatccatg 1200ttggaaaaga tctacatcca

35 ccccaggtac aactggcggg agaacctgga ccgggacatt 1260gccctgatga agctgaagaa gcctgttgcc ttcagtgact acattcaccc tgtgtgtctg 1320cccgacaggg agacggcagc cagcttgctc caggctggat acaaggggcg ggtgacaggc 1380tggggcaacc tgaaggagac gtggacagcc aacgttggta aggggcagcc cagtgtcctg 1440caggtggtga acctgcccat tgtggagcgg ccggtctgca aggactccac ccggatccgc 1500atcactgaca acatgttctg

5 tgctggttac aagcctgatg aagggaaacg aggggatgcc 1560tgtgaaggtg acagtggggg accctttgtc atgaagagcc cctttaacaa ccgctggtat 1620caaatgggca tcgtctcatg

gggtgaaggc tgtgaccggg atgggaaata tggcttctac 1680acacatgtgt tccgcctgaa gaagtggata cagaaggtca ttgatcagtt tggagagtag 1740tga (SEQ ID NO. : 64)

17434579PRThomo sapiens 4Ala Asn Thr Phe Leu Glu Glu Val Arg Lys Gly Asn Leu

10 Glu Arg Glu l 5 10 15Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu 20 25 30Ser Ser Thr Ala Thr Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu 35 40 45Thr Ala Arg Thr Pro Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn 50 55 60Cys Ala Glu Gly Leu Gly Thr Asn Tyr Arg Gly His Val Asn He Thr65 70 75 80Arg Ser Gly He Glu Cys Gin Leu Trp Arg Ser Arg Tyr Pro His Lys 85 90 95Pro Glu He

15 Asn Ser Thr Thr His Pro Gly Ala Asp Leu Gin Glu Asn 100 105 l lOPhe Cys Arg Asn Pro Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr 115 120 125Thr Asp Pro Thr Val Arg Arg Gin Glu Cys Ser He Pro Val Cys Gly 130 135 140Gln Asp Gin Val Thr Val Ala Met Thr Pro Arg Ser Glu Gly Ser Serl45 150 155 160Val Asn Leu Ser Pro Pro Leu Glu Gin Cys Val Pro Asp Arg Gly Gin 165 170 175Gln Tyr Gin Gly Arg Leu Ala

20 Val Thr Thr His Gly Leu Pro Cys Leu 180 185 190Ala Trp Ala Ser Ala Gin Ala Lys Ala Leu Ser Lys His Gin Asp Phe 195 200 205Asn Ser Ala Val Gin Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly 210 215 220Asp Glu Glu Gly Ala Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe225 230 235 240Gly Tyr Cys Asp Leu Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr 245 250 255Gly Asp Gly Leu Asp Glu Asp Ser Asp Arg Ala

25 He Glu Gly Arg Thr 260 265 270Ala Thr Ser Glu Tyr Gin Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser 275 280 285Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu 290 295 300Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr He Asp Gly Arg305 310 315 320Ile Val Glu Gly Ser Asp Ala Glu He Gly Met Ser Pro Trp Gin Val 325 330 335Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser Leu

30 340 345 350Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro 355 360 365Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg He Gly 370 375 380Lys His Ser Arg Thr Arg Tyr Glu Arg Asn He Glu Lys He Ser Met385 390 395 400Leu Glu Lys He Tyr He His Pro Arg Tyr Asn Trp Arg Glu Asn Leu 405 410 415Asp Arg Asp He Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser 420 425

35 430Asp Tyr He His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser 435 440 445Leu Leu Gin Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu 450 455 460Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gin Pro Ser Val Leu465 470 475 480Gln Val Val Asn Leu Pro lie Val Glu Arg Pro Val Cys Lys Asp Ser 485 490 495Thr Arg He Arg He Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro 500 505 5 510Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro 515 520 525Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gin Met Gly He 530 535 540Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr545 550 555 560Thr His Val Phe Arg Leu Lys Lys Trp He Gin Lys Val He Asp Gin 565 570 575Phe Gly Glu (SEQ ID NO. : 65) 51257DNAartificialrecombinant human

10 prothrombin precursor molecule 5ctgtcacctc cattggagca gtgtgtccct gatcgggggc agcagtacca ggggcgcctg 60gcggtgacca cacatgggct cccctgcctg gcctgggcca gcgcacaggc caaggccctg 120agcaagcacc aggacttcaa ctcagctgtg cagctggtgg agaacttctg ccgcaaccca 180gacggggatg aggagggcgc gtggtgctat gtggccggga agcctggcga ctttgggtac 240tgcgacctca actattgtga ggaggccgtg gaggaggaga

15 caggagatgg gctggatgag 300gactcagaca gggccatcga agggcgtacc gccaccagtg

agtaccagac tttcttcaat 360ccgaggacct ttggctcggg agaggcagac tgtgggctgc gacctctgtt cgagaagaag 420tcgctggagg acaaaaccga aagagagctc ctggaatcct acatcgacgg gcgcattgtg 480gagggctcgg atgcagagat cggcatgtca ccttggcagg tgatgctttt ccggaagagt 540ccccaggagc tgctgtgtgg ggccagcctc atcagtgacc gctgggtcct

20 caccgccgcc 600cactgcctcc tgtacccgcc ctgggacaag aacttcaccg agaatgacct tctggtgcgc 660attggcaagc actcccgcac caggtacgag cgaaacattg aaaagatatc cgccttggaa

720aagatctaca tccaccccag gtacaactgg cgggagaacc tggaccggga cattgccctg

780atgaagctga agaagcctgt tgccttcagt gactacattc accctgtgtg tctgcccgac

840agggagacgg cagccagctt gctccaggct ggatacaagg ggcgggtgac aggctggggc

25 900aacctgaagg agacgtggac agccaacgtt ggtaaggggc agcccagtgt cctgcaggtg

960gtgaacctgc ccattgtgga gcggccggtc tgcaaggact ccacccggat ccgcatcact

1020gacaacatgt tctgtgctgg ttacaagcct gatgaaggga aacgagggga tgcctgtgaa

1080ggtgacagtg ggggaccctt tgtcatgaag agccccttta acaaccgctg gtatcaaatg

1140ggcatcgtct catggggtga aggctgtgac cgggatggga aatatggctt ctacacacat

30 1200gtgttccgcc tgaagaagtg gatacagaag gtcattgatc agtttggaga gtagtga (SEQ ID NO. : 66) 125761311DNAartificialrecombinant human prothrombin precursor molecule 6gcagcaaagc ttgaagacca agtagatccg cggctcattg atgggaaggt cgacctgtca 60cctccattgg agcagtgtgt ccctgatcgg gggcagcagt accaggggcg cctggcggtg

120accacacatg ggctcccctg cctggcctgg gccagcgcac aggccaaggc cctgagcaag

35 180caccaggact tcaactcagc tgtgcagctg gtggagaact tctgccgcaa cccagacggg 240gatgaggagg gcgcgtggtg ctatgtggcc gggaagcctg gcgactttgg gtactgcgac

300ctcaactatt gtgaggaggc cgtggaggag gagacaggag atgggctgga tgaggactca

360gacagggcca tcgaagggcg taccgccacc agtgagtacc agactttctt caatccgagg

420acctttggct cgggagaggc agactgtggg ctgcgacctc tgttcgagaa gaagtcgctg

5 480gaggacaaaa ccgaaagaga gctcctggaa tcctacatcg acgggcgcat tgtggagggc

540tcggatgcag agatcggcat gtcaccttgg caggtgatgc ttttccggaa gagtccccag

eOOgagctgctgt gtggggccag cctcatcagt gaccgctggg tcctcaccgc cgcccactgc

660ctcctgtacc cgccctggga caagaacttc accgagaatg accttctggt gcgcattggc

720aagcactccc gcaccaggta cgagcgaaac attgaaaaga tatccgcctt ggaaaagatc

10 780tacatccacc ccaggtacaa ctggcgggag aacctggacc gggacattgc cctgatgaag

840ctgaagaagc ctgttgcctt cagtgactac attcaccctg tgtgtctgcc cgacagggag

900acggcagcca gcttgctcca ggctggatac aaggggcggg tgacaggctg gggcaacctg

960aaggagacgt ggacagccaa cgttggtaag gggcagccca gtgtcctgca ggtggtgaac

1020ctgcccattg tggagcggcc ggtctgcaag gactccaccc ggatccgcat cactgacaac

15 1080atgttctgtg ctggttacaa gcctgatgaa gggaaacgag gggatgcctg tgaaggtgac

1140agtgggggac cctttgtcat gaagagcccc tttaacaacc gctggtatca aatgggcatc

1200gtctcatggg gtgaaggctg tgaccgggat gggaaatatg gcttctacac acatgtgttc

1260cgcctgaaga agtggataca gaaggtcatt gatcagtttg gagagtagtg a (SEQ ID NO. : 67) 13117435PRX artificialhuman recombinant preprothrombin 7Ala Ala Lys Leu Glu

20 Asp Gin Val Asp Pro Arg Leu He Asp Gly Lysl 5 10 15Val Asp Leu Ser Pro Pro Leu Glu Gin Cys Val Pro Asp Arg Gly Gin 20 25 30Gln Tyr Gin Gly Arg Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu 35 40 45Ala Trp Ala Ser Ala Gin Ala Lys Ala Leu Ser Lys His Gin Asp Phe 50 55 60Asn Ser Ala Val Gin Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly65 70 75 80Asp Glu Glu Gly Ala Trp Cys Tyr Val Ala Gly Lys Pro Gly

25 Asp Phe 85 90 95Gly Tyr Cys Asp Leu Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr 100 105 HOGly Asp Gly Leu Asp Glu Asp Ser Asp Arg Ala He Glu Gly Arg Thr 115 120 125Ala Thr Ser Glu Tyr Gin Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser 130 135 140Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leul45 150 155 160Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr He Asp Gly Arg 165 170

30 175Ile Val Glu Gly Ser Asp Ala Glu He Gly Met Ser Pro Trp Gin Val 180 185 190Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser Leu 195 200 205Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro 210 215 220Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg He Gly225 230 235 240Lys His Ser Arg Thr Arg Tyr Glu Arg Asn He Glu Lys He Ser Ala 245 250 255Leu Glu Lys He Tyr

35 He His Pro Arg Tyr Asn Trp Arg Glu Asn Leu 260 265 270Asp Arg Asp lie Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser 275 280 285Asp Tyr He His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser 290 295 300Leu Leu Gin Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu305 310 315 320Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gin Pro Ser Val Leu 325 330 335Gln Val Val Asn Leu Pro He Val Glu 5 Arg Pro Val Cys Lys Asp Ser 340 345 350Thr Arg He Arg He Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro 355 360 365Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro 370 375 380Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gin Met Gly Ile385 390 395 400Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr 405 410 415Thr His Val Phe Arg Leu Lys Lys Trp He Gin Lys Val

10 He Asp Gin 420 425 430Phe Gly Glu (SEQ ID NO. : 68)

43581311DNAartificialHuman Preprothrombin wild type with HPC4 protein

8gcagcaaagc ttgaagacca agtagatccg cggctcattg atgggaaggt cgacctgtca

60cctccattgg agcagtgtgt ccctgatcgg gggcagcagt accaggggcg cctggcggtg

120accacacatg ggctcccctg cctggcctgg gccagcgcac aggccaaggc cctgagcaag

15 180caccaggact tcaactcagc tgtgcagctg gtggagaact tctgccgcaa cccagacggg

240gatgaggagg gcgcgtggtg ctatgtggcc gggaagcctg gcgactttgg gtactgcgac

300ctcaactatt gtgaggaggc cgtggaggag gagacaggag atgggctgga tgaggactca 360gacagggcca tcgaagggcg taccgccacc agtgagtacc agactttctt caatccgagg

420acctttggct cgggagaggc agactgtggg ctgcgacctc tgttcgagaa gaagtcgctg

20 480gaggacaaaa ccgaaagaga gctcctggaa tcctacatcg acgggcgcat tgtggagggc

540tcggatgcag agatcggcat gtcaccttgg caggtgatgc ttttccggaa gagtccccag

600gagctgctgt gtggggccag cctcatcagt gaccgctggg tcctcaccgc cgcccactgc

660ctcctgtacc cgccctggga caagaacttc accgagaatg accttctggt gcgcattggc

720aagcactccc gcaccaggta cgagcgaaac attgaaaaga tatccatgtt ggaaaagatc

25 780tacatccacc ccaggtacaa ctggcgggag aacctggacc gggacattgc cctgatgaag

840ctgaagaagc ctgttgcctt cagtgactac attcaccctg tgtgtctgcc cgacagggag

900acggcagcca gcttgctcca ggctggatac aaggggcggg tgacaggctg gggcaacctg

960aaggagacgt ggacagccaa cgttggtaag gggcagccca gtgtcctgca ggtggtgaac

1020ctgcccattg tggagcggcc ggtctgcaag gactccaccc ggatccgcat cactgacaac

30 1080atgttctgtg ctggttacaa gcctgatgaa gggaaacgag gggatgcctg tgaaggtgac

1140agtgggggac cctttgtcat gaagagcccc tttaacaacc gctggtatca aatgggcatc

1200gtctcatggg gtgaaggctg tgaccgggat gggaaatatg gcttctacac acatgtgttc

1260cgcctgaaga agtggataca gaaggtcatt gatcagtttg gagagtagtg a (SEQ ID NO. : 69) 13119435PRTartificialHuman preprothrombin wild type with HPC4 protein 9Ala Ala

35 Lys Leu Glu Asp Gin Val Asp Pro Arg Leu He Asp Gly Lysl 5 10 15Val Asp Leu Ser Pro Pro Leu Glu Gin Cys Val Pro Asp Arg Gly Gin 20 25 30Gln Tyr Gin Gly Arg Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu 35 40 45Ala Trp Ala Ser Ala Gin Ala Lys Ala Leu Ser Lys His Gin Asp Phe 50 55 60Asn Ser Ala Val Gin Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly65 70 75 80Asp Glu Glu Gly Ala Trp Cys Tyr Val Ala Gly 5 Lys Pro Gly Asp Phe 85 90 95Gly Tyr Cys Asp Leu Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr 100 105 HOGly Asp Gly Leu Asp Glu Asp Ser Asp Arg Ala He Glu Gly Arg Thr 115 120 125Ala Thr Ser Glu Tyr Gin Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser 130 135 140Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu l45 150 155 160Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr He Asp Gly

10 Arg 165 170 175Ile Val Glu Gly Ser Asp Ala Glu He Gly Met Ser Pro Trp Gin Val 180 185 190Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys Gly Ala Ser Leu 195 200 205Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro 210 215 220Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg He Gly225 230 235 240Lys His Ser Arg Thr Arg Tyr Glu Arg Asn He Glu Lys He Ser Met 245 250

15 255Leu Glu Lys He Tyr He His Pro Arg Tyr Asn Trp Arg Glu Asn Leu 260 265

270Asp Arg Asp He Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser 275 280 285Asp Tyr He His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser 290 295 300Leu Leu Gin Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu305 310 315 320Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gin Pro Ser Val Leu 325 330

20 335Gln Val Val Asn Leu Pro He Val Glu Arg Pro Val Cys Lys Asp Ser 340 345

350Thr Arg He Arg He Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro 355 360 365Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro 370 375 380Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gin Met Gly Ile385 390 395 400Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr 405 410

25 415Thr His Val Phe Arg Leu Lys Lys Trp He Gin Lys Val He Asp Gin 420 425

430Phe Gly Glu (SEQ ID NO. : 70) 435101911DNAhomo sapiens lOgtcctaagtg tggtgggcac agcatggact gcagatagtg gtgaaggtga ctttctagct 60gaaggaggag gcgtgcgtgg cccaagggtt gtggaaagac atcaatctgc ctgcaaagat 120tcagactggc ccttctgctc tgatgaagac tggaactaca aatgcccttc tggctgcagg 180atgaaagggt tgattgatga

30 agtcaatcaa gattttacaa acagaataaa taagctcaaa 240aattcactat ttgaatatca gaagaacaat aaggattctc attcgttgac cactaatata 300atggaaattt tgagaggcga tttttcctca gccaataacc gtgataatac ctacaaccga 360gtgtcagagg atctgagaag cagaattgaa gtcctgaagc gcaaagtcat agaaaaagta 420cagcatatcc agcttctgca aaaaaatgtt agagctcagt tggttgatat gaaacgactg 480gaggtggaca ttgatattaa gatccgatct tgtcgagggt catgcagtag

35 ggctttagct 540cgtgaagtag atctgaagga ctatgaagat cagcagaagc aacttgaaca ggtcattgcc 600aaagacttac ttccctctag agataggcaa cacttaccac tgatcaaaat gaaaccagtt 660ccagacttgg ttcccggaaa ttttaagagc cagcttcaga aggtaccccc agagtggaag

720gcattaacag acatgccgca gatgagaatg gagttagaga gacctggtgg aaatgagatt

780actcgaggag gctccacttc ttatggaacc ggatcagaga cggaaagccc aaggaaccct

5 840agcagtgctg gaagctggaa ctctgggagc tctggacctg gaagtactgg aaaccgaaac

900cctgggagct ctgggactgg agggactgca acctggaaac ctggaagctc tggacctgga

960agtactggaa gctggaactc tgggagctct ggaactggaa gtactggaaa ccaaaaccct

1020gggagcccta gacctggtag taccggaacc tggaatcctg gcagctctga acgcggaagt 1080gctggacact ggacttctga gagctctgta tctggtagta ctggacaatg gcactctgaa

10 1140tctggaagtt ttaggccaga tagcccaggc tctgggaacg cgaggcctaa caacccagac

1200tggggcacat ttgaagaggt gtcaggaaat gtaagtccag ggacaaggag agagtaccac 1260acagaaaaac tggtcacttc taaaggagat aaagagctca ggactggtaa agagaaggtc 1320acctctggta gcacaaccac cacgcgtcgt tcatgctcta aaaccgttac taagactgtt

1380attggtcctg atggtcacaa agaagttacc aaagaagtgg tgacctccga agatggttct

15 1440gactgtcccg aggcaatgga tttaggcaca ttgtctggca taggcaccct ggatgggttc

1500cgccataggc accctgatga agctgccttc ttcgacactg cctcaactgg aaaaacattc

1560ccaggtttct tctcacctat gttaggagag tttgtcagtg agactgagtc taggggctca

1620gaatctggca tcttcacaaa tacaaaggaa tccagttctc atcaccctgg gatagctgaa

1680ttcccttccc gtggtaaatc ttcaagttac agcaaacaat ttactagtag cacgagttac

20 1740aacagaggag actccacatt tgaaagcaag agctataaaa tggcagatga ggccggaagt

1800gaagccgatc atgaaggaac acatagcacc aagagaggcc atgctaaatc tcgccctgtc

1860agaggtatcc acacttctcc tttggggaag ccttccctgt ccccctagtg a (SEQ ID NO. : 71) 191111625PRThomo sapiens HAIa Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg l 5 10 15Gly Pro Arg Val Val Glu Arg His Gin Ser Ala Cys Lys Asp

25 Ser Asp 20 25 30Trp Pro Phe Cys Ser Asp Glu Asp Trp Asn Tyr Lys Cys Pro Ser Gly 35 40 45Cys Arg Met Lys Gly Leu He Asp Glu Val Asn Gin Asp Phe Thr Asn 50 55 60Arg He Asn Lys Leu Lys Asn Ser Leu Phe Glu Tyr Gin Lys Asn Asn65 70 75 80Lys Asp Ser His Ser Leu Thr Thr Asn He Met Glu He Leu Arg Gly 85 90 95Asp Phe Ser Ser Ala Asn Asn Arg Asp Asn Thr Tyr Asn Arg Val Ser 100 105 l lOGIu Asp

30 Leu Arg Ser Arg He Glu Val Leu Lys Arg Lys Val He Glu 115 120 125Lys Val Gin His He Gin Leu Leu Gin Lys Asn Val Arg Ala Gin Leu 130 135 140Val Asp Met Lys Arg Leu Glu Val Asp He Asp He Lys He Arg Serl45 150 155 160Cys Arg Gly Ser Cys Ser Arg Ala Leu Ala Arg Glu Val Asp Leu Lys 165 170 175Asp Tyr Glu Asp Gin Gin Lys Gin Leu Glu Gin Val He Ala Lys Asp 180 185 190Leu Leu Pro Ser Arg Asp Arg

35 Gin His Leu Pro Leu He Lys Met Lys 195 200 205Pro Val Pro Asp Leu Val Pro Gly Asn Phe Lys Ser Gin Leu Gin Lys 210 215 220Val Pro Pro Glu Trp Lys Ala Leu Thr Asp Met Pro Gin Met Arg Met225 230 235 240Glu Leu Glu Arg Pro Gly Gly Asn Glu He Thr Arg Gly Gly Ser Thr 245 250 255Ser Tyr Gly Thr Gly Ser Glu Thr Glu Ser Pro Arg Asn Pro Ser Ser 260 265 270Ala Gly Ser Trp Asn Ser Gly Ser Ser Gly Pro 5 Gly Ser Thr Gly Asn 275 280 285Arg Asn Pro Gly Ser Ser Gly Thr Gly Gly Thr Ala Thr Trp Lys Pro 290 295 300Gly Ser Ser Gly Pro Gly Ser Thr Gly Ser Trp Asn Ser Gly Ser Ser305 310 315 320Gly Thr Gly Ser Thr Gly Asn Gin Asn Pro Gly Ser Pro Arg Pro Gly 325 330 335Ser Thr Gly Thr Trp Asn Pro Gly Ser Ser Glu Arg Gly Ser Ala Gly 340 345 350His Trp Thr Ser Glu Ser Ser Val Ser Gly Ser Thr Gly Gin Trp

10 His 355 360 365Ser Glu Ser Gly Ser Phe Arg Pro Asp Ser Pro Gly Ser Gly Asn Ala 370 375 380Arg Pro Asn Asn Pro Asp Trp Gly Thr Phe Glu Glu Val Ser Gly Asn385 390 395 400Val Ser Pro Gly Thr Arg Arg Glu Tyr His Thr Glu Lys Leu Val Thr 405 410 415Ser Lys Gly Asp Lys Glu Leu Arg Thr Gly Lys Glu Lys Val Thr Ser 420 425 430Gly Ser Thr Thr Thr Thr Arg Arg Ser Cys Ser Lys Thr Val Thr Lys 435 440

15 445Thr Val He Gly Pro Asp Gly His Lys Glu Val Thr Lys Glu Val Val 450 455 460Thr Ser Glu Asp Gly Ser Asp Cys Pro Glu Ala Met Asp Leu Gly Thr465 470 475 480Leu Ser Gly He Gly Thr Leu Asp Gly Phe Arg His Arg His Pro Asp 485 490 495Glu Ala Ala Phe Phe Asp Thr Ala Ser Thr Gly Lys Thr Phe Pro Gly 500 505 510Phe Phe Ser Pro Met Leu Gly Glu Phe Val Ser Glu Thr Glu Ser Arg 515 520 525Gly Ser Glu Ser

20 Gly He Phe Thr Asn Thr Lys Glu Ser Ser Ser His 530 535 540His Pro Gly He Ala Glu Phe Pro Ser Arg Gly Lys Ser Ser Ser Tyr545 550 555 560Ser Lys Gin Phe Thr Ser Ser Thr Ser Tyr Asn Arg Gly Asp Ser Thr 565 570 575Phe Glu Ser Lys Ser Tyr Lys Met Ala Asp Glu Ala Gly Ser Glu Ala 580 585 590Asp His Glu Gly Thr His Ser Thr Lys Arg Gly His Ala Lys Ser Arg 595 600 605Pro Val Arg Gly He His Thr Ser Pro

25 Leu Gly Lys Pro Ser Leu Ser 610 615 620Pro (SEQ ID NO. : 72)

625121389DNAhomo sapiens 12caaggtgtca acgacaatga ggagggtttc ttcagtgccc gtggtcatcg accccttgac 60aagaagagag aagaggctcc cagcctgagg cctgccccac cgcccatcag tggaggtggc 120tatcgggctc gtccagccaa agcagctgcc actcaaaaga aagtagaaag aaaagcccct 180gatgctggag gctgtcttca cgctgaccca gacctggggg

30 tgttgtgtcc tacaggatgt 240cagttgcaag aggctttgct acaacaggaa aggccaatca

gaaatagtgt tgatgagtta 300aataacaatg tggaagctgt ttcccagacc tcctcttctt cctttcagta catgtatttg 360ctgaaagacc tgtggcaaaa gaggcagaag caagtaaaag ataatgaaaa tgtagtcaat 420gagtactcct cagaactgga aaagcaccaa ttatatatag atgagactgt gaatagcaat 480atcccaacta accttcgtgt gcttcgttca atcctggaaa acctgagaag caaaatacaa

35 540aagttagaat ctgatgtctc agctcaaatg gaatattgtc gcaccccatg cactgtcagt 600tgcaatattc ctgtggtgtc tggcaaagaa tgtgaggaaa ttatcaggaa aggaggtgaa

660acatctgaaa tgtatctcat tcaacctgac agttctgtca aaccgtatag agtatactgt

720gacatgaata cagaaaatgg aggatggaca gtgattcaga accgtcaaga cggtagtgtt

780gactttggca ggaaatggga tccatataaa cagggatttg gaaatgttgc aaccaacaca

5 840gatgggaaga attactgtgg cctaccaggt gaatattggc ttggaaatga taaaattagc

900cagcttacca ggatgggacc cacagaactt ttgatagaaa tggaggactg gaaaggagac 960aaagtaaagg ctcactatgg aggattcact gtacagaatg aagccaacaa ataccagatc

1020tcagtgaaca aatacagagg aacagccggt aatgccctca tggatggagc atctcagctg 1080atgggagaaa acaggaccat gaccattcac aacggcatgt tcttcagcac gtatgacaga

10 1140gacaatgacg gctggttaac atcagatccc agaaaacagt gttctaaaga agacggtggt

1200ggatggtggt ataatagatg tcatgcagcc aatccaaacg gcagatacta ctggggtgga 1260cagtacacct gggacatggc aaagcatggc acagatgatg gtgtagtatg gatgaattgg 1320aaggggtcat ggtactcaat gaggaagatg agtatgaaga tcaggccctt cttcccacag

1380caatagtga (SEQ ID NO. : 73) 138913461PRThomo sapiens 13Gln Gly Val Asn

15 Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg Gly Hisl 5 10 15Arg Pro Leu Asp Lys Lys Arg Glu Glu Ala Pro Ser Leu Arg Pro Ala 20 25 30Pro Pro Pro He Ser Gly Gly Gly Tyr Arg Ala Arg Pro Ala Lys Ala 35 40 45Ala Ala Thr Gin Lys Lys Val Glu Arg Lys Ala Pro Asp Ala Gly Gly 50 55 60Cys Leu His Ala Asp Pro Asp Leu Gly Val Leu Cys Pro Thr Gly Cys65 70 75 80Gln Leu Gin Glu Ala Leu Leu Gin Gin Glu Arg Pro He

20 Arg Asn Ser 85 90 95Val Asp Glu Leu Asn Asn Asn Val Glu Ala Val Ser Gin Thr Ser Ser 100 105 llOSer Ser Phe Gin Tyr Met Tyr Leu Leu Lys Asp Leu Trp Gin Lys Arg 115 120 125Gln Lys Gin Val Lys Asp Asn Glu Asn Val Val Asn Glu Tyr Ser Ser 130 135 140Glu Leu Glu Lys His Gin Leu Tyr He Asp Glu Thr Val Asn Ser Asnl45 150 155 160Ile Pro Thr Asn Leu Arg Val Leu Arg Ser He Leu Glu Asn Leu Arg 165 170

25 175Ser Lys He Gin Lys Leu Glu Ser Asp Val Ser Ala Gin Met Glu Tyr 180 185

190Cys Arg Thr Pro Cys Thr Val Ser Cys Asn He Pro Val Val Ser Gly 195 200 205Lys Glu Cys Glu Glu He He Arg Lys Gly Gly Glu Thr Ser Glu Met 210 215 220Tyr Leu He Gin Pro Asp Ser Ser Val Lys Pro Tyr Arg Val Tyr Cys225 230 235 240Asp Met Asn Thr Glu Asn Gly Gly Trp Thr Val He Gin Asn Arg Gin 245 250

30 255Asp Gly Ser Val Asp Phe Gly Arg Lys Trp Asp Pro Tyr Lys Gin Gly 260 265 270Phe Gly Asn Val Ala Thr Asn Thr Asp Gly Lys Asn Tyr Cys Gly Leu 275 280 285Pro Gly Glu Tyr Trp Leu Gly Asn Asp Lys He Ser Gin Leu Thr Arg 290 295 300Met Gly Pro Thr Glu Leu Leu He Glu Met Glu Asp Trp Lys Gly Asp305 310 315 320Lys Val Lys Ala His Tyr Gly Gly Phe Thr Val Gin Asn Glu Ala Asn 325 330

35 335Lys Tyr Gin He Ser Val Asn Lys Tyr Arg Gly Thr Ala Gly Asn Ala 340 345 350Leu Met Asp Gly Ala Ser Gin Leu Met Gly Glu Asn Arg Thr Met Thr 355 360 365Ile His Asn Gly Met Phe Phe Ser Thr Tyr Asp Arg Asp Asn Asp Gly 370 375 380Trp Leu Thr Ser Asp Pro Arg Lys Gin Cys Ser Lys Glu Asp Gly Gly385 390 395 400Gly Trp Trp Tyr Asn Arg Cys His Ala Ala Asn Pro Asn Gly Arg Tyr 405 410 5 415Tyr Trp Gly Gly Gin Tyr Thr Trp Asp Met Ala Lys His Gly Thr Asp 420

425 430Asp Gly Val Val Trp Met Asn Trp Lys Gly Ser Trp Tyr Ser Met Arg 435 440 445Lys Met Ser Met Lys He Arg Pro Phe Phe Pro Gin Gin 450 455 (SEQ ID NO. : 74) 460141272DNAhomo sapiens 14gctcttttat ttctctcttc aacatgtgta gcatatgttg ctaccagaga caactgctgc 60atcttagatg aaagattcgg tagttattgt ccaactacct gtggcattgc

10 agatttcctg 120tctacttatc aaaccaaagt agacaaggat ctacagtctt tggaagacat cttacatcaa 180gttgaaaaca aaacatcaga agtcaaacag ctgataaaag caatccaact cacttataat

240cctgatgaat catcaaaacc aaatatgata gacgctgcta ctttgaagtc caggaaaatg

300ttagaagaaa ttatgaaata tgaagcatcg attttaacac atgactcaag tattcgatat

360ttgcaggaaa tatataattc aaataatcaa aagattgtta acctgaaaga gaaggtagcc

15 420cagcttgaag cacagtgcca ggaaccttgc aaagacacgg tgcaaatcca tgatatcact

480gggaaagatt gtcaagacat tgccaataag ggagctaaac agagcgggct ttactttatt

540aaacctctga aagctaacca gcaattctta gtctactgtg aaatcgatgg gtctggaaat

600ggatggactg tgtttcagaa gagacttgat ggcagtgtag atttcaagaa aaactggatt

660caatataaag aaggatttgg acatctgtct cctactggca caacagaatt ttggctggga

20 720aatgagaaga ttcatttgat aagcacacag tctgccatcc catatgcatt aagagtggaa

780ctggaagact ggaatggcag aaccagtact gcagactatg ccatgttcaa ggtgggacct

840gaagctgaca agtaccgcct aacatatgcc tacttcgctg gtggggatgc tggagatgcc

900tttgatggct ttgattttgg cgatgatcct agtgacaagt ttttcacatc ccataatggc

960atgcagttca gtacctggga caatgacaat gataagtttg aaggcaactg tgctgaacag

25 1020gatggatctg gttggtggat gaacaagtgt cacgctggcc atctcaatgg agtttattac

1080caaggtggca cttactcaaa agcatctact cctaatggtt atgataatgg cattatttgg

1140gccacttgga aaacccggtg gtattccatg aagaaaacca ctatgaagat aatcccattc

1200aacagactca caattggaga aggacagcaa caccacctgg ggggagccaa acaggctgga 1260gacgtttaat ga (SEQ ID NO. : 75) 127215411PRThomo sapiens 15Tyr Val Ala

30 Thr Arg Asp Asn Cys Cys He Leu Asp Glu Arg Phe Glyl 5 10 15Ser Tyr Cys Pro Thr Thr Cys Gly He Ala Asp Phe Leu Ser Thr Tyr 20 25 30Gln Thr Lys Val Asp Lys Asp Leu Gin Ser Leu Glu Asp He Leu His 35 40 45Gln Val Glu Asn Lys Thr Ser Glu Val Lys Gin Leu He Lys Ala He 50 55 60Gln Leu Thr Tyr Asn Pro Asp Glu Ser Ser Lys Pro Asn Met He Asp65 70 75 80Ala Ala Thr Leu Lys Ser Arg Lys Met Leu Glu Glu

35 He Met Lys Tyr 85 90 95Glu Ala Ser He Leu Thr His Asp Ser Ser He Arg Tyr Leu Gin Glu 100 105 llOIle Tyr Asn Ser Asn Asn Gin Lys He Val Asn Leu Lys Glu Lys Val 115 120 125Ala Gin Leu Glu Ala Gin Cys Gin Glu Pro Cys Lys Asp Thr Val Gin 130 135 140Ile His Asp He Thr Gly Lys Asp Cys Gin Asp He Ala Asn Lys Glyl45 150 155 160Ala Lys Gin Ser Gly Leu Tyr Phe He Lys Pro Leu Lys Ala Asn Gin 165 5 170 175Gln Phe Leu Val Tyr Cys Glu He Asp Gly Ser Gly Asn Gly Trp Thr 180 185 190Val Phe Gin Lys Arg Leu Asp Gly Ser Val Asp Phe Lys Lys Asn Trp 195 200 205Ile Gin Tyr Lys Glu Gly Phe Gly His Leu Ser Pro Thr Gly Thr Thr 210 215 220Glu Phe Trp Leu Gly Asn Glu Lys He His Leu He Ser Thr Gin Ser225 230 235 240Ala He Pro Tyr Ala Leu Arg Val Glu Leu Glu Asp Trp Asn Gly Arg 245 250

10 255Thr Ser Thr Ala Asp Tyr Ala Met Phe Lys Val Gly Pro Glu Ala Asp 260 265

270Lys Tyr Arg Leu Thr Tyr Ala Tyr Phe Ala Gly Gly Asp Ala Gly Asp 275 280 285Ala Phe Asp Gly Phe Asp Phe Gly Asp Asp Pro Ser Asp Lys Phe Phe 290 295 300Thr Ser His Asn Gly Met Gin Phe Ser Thr Trp Asp Asn Asp Asn Asp305 310 315 320Lys Phe Glu Gly Asn Cys Ala Glu Gin Asp Gly Ser Gly Trp Trp Met 325 330

15 335Asn Lys Cys His Ala Gly His Leu Asn Gly Val Tyr Tyr Gin Gly Gly 340 345

350Thr Tyr Ser Lys Ala Ser Thr Pro Asn Gly Tyr Asp Asn Gly He He 355 360 365Trp Ala Thr Trp Lys Thr Arg Trp Tyr Ser Met Lys Lys Thr Thr Met 370 375 380Lys He He Pro Phe Asn Arg Leu Thr He Gly Glu Gly Gin Gin His385 390 395 400His Leu Gly Gly Ala Lys Gin Ala Gly Asp Val (SEQ ID NO. : 76) 405

20 4101639PRThomo sapiensmisc_ M84A 17Thr Phe Gly Ser Gly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu l 5 10 15Lys Lys Ser Leu Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr 20 25 30Ile Asp Gly Arg lie Val Glu Gly Ser Asp Ala Glu He Gly Met Ser 35 40 45Pro Trp Gin Val Met Leu Phe Arg Lys Ser Pro Gin Glu Leu Leu Cys 50 55 60Gly Ala Ser Leu He Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys65 70 75

25 80Leu Leu Tyr Pro Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu 85 90 95Val Arg He Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Asn He Glu 100 105 l lOLys He Ser Ala Leu Glu Lys He Tyr He His Pro Arg Tyr Asn Trp 115 120 125Arg Glu Asn Leu Asp Arg Asp He Ala Leu Met Lys Leu Lys Lys Pro 130 135 140Val Ala Phe Ser Asp Tyr He His Pro Val Cys Leu Pro Asp Arg Glul45 150 155 160Thr Ala Ala Ser Leu

30 Leu Gin Ala Gly Tyr Lys Gly Arg Val Thr Gly 165 170 175Trp Gly Asn Leu Lys Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gin 180 185 190Pro Ser Val Leu Gin Val Val Asn Leu Pro He Val Glu Arg Pro Val 195 200 205Cys Lys Asp Ser Thr Arg He Arg He Thr Asp Asn Met Phe Cys Ala 210 215 220Gly Tyr Lys Pro Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp225 230 235 240Ser Gly Gly Pro Phe Val Met Lys Ser

35 Pro Phe Asn Asn Arg Trp Tyr 245 250 255Gln Met Gly He Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys 260 265 270Tyr Gly Phe Tyr Thr His Val Phe Arg Leu Lys Lys Trp lie Gin Lys 275 280 285Val He Asp Gin Phe Gly Glu 290 295 (SEQ ID NO. : 77). Items

1. A method for preparing a recombinant human gla domain (SEQ ID NO: 16) containing prothrombin or prothrombin comprising using a human expression system in placenta cells and in human amnion cell lines as well as immortalized amnion cell lines .

2. A method for preparing a recombinant human gla domain containing prothrombin or thrombin comprising a human amplifying vector system used in CAP and/or CAP-T cells.

3. The method according to claim 1, wherein the human expression system comprises a human placenta derived cell or an amnion cell line, or immortalized amnion cell line.

4. The method according to claim 51, wherein the human expression system is cultured under serum-free conditions.

Fibrinogen

Use of Placenta derived cells for Recombinant Clotting Proteins including Factor XHIa

Abstract

The present invention discloses a novel method for the preparation of

recombinant human fibrinogen and/or human Factor XIII expressed in CAP or CAP-T cell system from CEVEC Pharmaceuticals AG, Cologne, Germany.

Specifically, the present invention relates to novel methods for the transfection of alpha, beta and gamma chain for fibrinogen using at least two vector system, where of CEVEC's vector system is added and is amplifying the entire vector system adhered to these plasmids which can be made in a novel system consisting of amnion cells, amniocytes, immortalized amniocytes together with comparable vectos systems, CAP or CAP-T cells, which are high yield producer cells originated from CEVEC in Germany.

Moreover, the method employs serum-free culturing conditions and therefore provides the resulting recombinant human fibrinogen and/or the recombinant factor XIII, individually, expressed in CAP and/or CAP-T cells of which has made it possible to produce recombinant fibrinogen with a yield of over 100, and even as high as 300 mg to 1000 mg recombinant human glycosylated fibrinogen/liter cells at a cell concentration of 5-10 millions per ml. The proteins exhibit an increased safety to the patient when used in human medical treatments. The human recombinant fibrinogen is expressed in high producer cells called CAP cells This CAP and/or the CAP-T systems which are not embryonic cells, is the important part of the invention as well as the method of freeze drying the fibrinogen produced by the CAP cell system, because other commonly known transfected cell systems cannot produce the necessary level of fibrinogen to be useable as an alternative to plasma fibrinogen such as for instance CSL Behring's Hemocomplettan. The vector used, is the one used in the CAP system and is unique for proteins such as complex proteins, e.g., fibrinogen. Human cell lines are the most innovative choice of host cell for production of biopharmaceuticals since they allow for authentic posttranslational modification of therapeutic proteins. We present a new method for generating high and stable protein expressing cell lines based on human amniocytes without the requirement of antibiotic selection.

The fibrinogen alpha, beta and gamma gene is transfected using a new vector, called 'pmEcSH A for the expression of recombinant fibrinogen and for the expression of Factor XHIa in order to obtain a significantly and accelerated increase in the resulting secretion of two important proteins resulting in a combination of fibrinogen, both transfected specifically into the HEK293TS cell line.

Human Amnion cells also called amniocytes functions extremely well with adenoviral vectors, and can, also when immortalized, be significantly higher producers, when transfected even with complex proteins such as wild type human fibrinogen, P-A human fibrinogen, which can change the previously anticipated titers obtained with HEK 293TS cells to unique significantly higher titers than have ever been thought of being possible. In fact these amnionic cell types can raise the production up into the gram area instead of into milligram area, which appeared to be a surprisingly, and not anticipated high titer, that brings the production of both rec. fibrinogen and rec. prothrombin up to production levels that enable these productions to surpass any previously anticipated production levels thus finally make it possible for especially recombinant fibrinogen to be produced in amounts that absolutelly make it possible to compete out high quality plasmafibrinogen, which will mean that said fibrinogen gene transfected amniocytes or immortalized aminocytes can produce recombinant fibrinogen which would most probably be the future basis for the fibrinogen production in the future. Schiedner G, Hertel S, Kochanek S. Efficient transformation of primary human amniocytes by El functions of Ad5: generation of new cell lines for adenoviral vector production. Hum Gene Ther. 2000; 11 : 2105-2116. [PubMed]

The cells according to the present invention can be cultured under usual conditions for the cultivation of eukaryotic cells at about 37. degree. C, 95% humidity and 8% C02. The cells according to the present invention can be cultured in serum containing or serum free medium, in adherent culture or in suspension culture. The cultivation in suspension can take place in diverse fermentation vessels, e.g . in stirred tank reactors, wave reactors, in shaker flasks or vessels or spinner vessels, or in so called roller bottles. Thus, the cells are suitable for a scale up process into the industrial scale. The transfection of the cells for transient expression can take place with the diverse transfection methods as mentioned above. Transfection and transient expression can also be performed in the high throughput format and screening, respectively, e.g. in a 96 or 384 well format.

Background

It has been hypothesized that amniocytes might be easier to directly reprogram to the iPS cell state than other available somatic cell types because of the likely similarity of their transcriptional and epigenetic states to those of early embryonic cell types (Li, C, Zhou, J., Shi, G., Ma, Y., Yang, Y., Gu, 1, Yu, H., Jin, S., Wei, Z., Chen, F. et al. (2009) Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells. Hum . Mol. Genet., 18, 4340-4349; Galende, E., Karakikes, I., Edelmann, L, Desnick, R.J., Kerenyi, T., Khoueiry, G., Lafferty, , McGinn, T., Brodman, M., Fuster, V. et al. (2010) Amniotic fluid cells are more efficiently reprogrammed to pluripotency than adult cells. Cell Reprogram, 12, 117-125).

Amniocytes are autologous to the fetus and semi-allogeneic to each parent, thereby expanding the potential utility of amniocyte-derived iPS (AdiPS) cells to other family members. Because of their

early embryonic origin, amniocytes may have accumulated less genetic damage due to replication errors or somatic mutation than older cell types, potentially allowing derivation of iPS cells with improved lifespan and reduced neoplastic tendency.

The infection protocol involved using subconfluent cultures of mouse or human amniocytes (70% confluency) that were infected with pluripotency-inducing retroviral vectors in viral media [DMEM/10% FBS for mouse cultures; for human amniocytes, hES media containing 20% Knock Out Serum Replacement (KOSR, Invitrogen, Inc.) instead of FBS] on day 3. The retroviral vectors for Oct4, Sox2, c-Myc and Klf4 or combinations thereof were added to the amniocyte cultures for 48 h. The media were then changed back to amniocyte media (see Amniocyte isolation and other cell sources section) with continued culture in a feederless system until the emergence of the first morphologically distinct cell colonies. By day 5 post-infection (four factors, with mouse or human amniocytes) or day 7 post-infection (two factors with mouse amniocytes) in culture, colonies resembling ES cell colonies began to appear according to Chin et al. (Chin, A.C.,

Padmanabhan, 1, Oh, S.K. and Choo, A. B. (2009) Defined and serum-free media support undifferentiated human embryonic stem cell growth. Stem Cells Dev., 19, 753-761., 12, 117-125; Anchan RM, Quaas P, Gerami-Naini B, Bartake H, Griffin A, et al. Amniocytes can serve a dual function as a source of iPS cells and feeder layers. Human Molecular Genetics, 2011, Vol. 20, No. 5 962-974). While amniocytes may hold promising therapeutic potential [5-10], the molecular mechanisms controlling their developmental status are not understood, and a comprehensive characterization of these cells is clearly required before

patientderived amniocyte stem cell therapy becomes a clinical reality. Human amniocytes are considered an embryonic or fetal multipotent stem cell due to expression of transcriptional regulators (Prusa AR, Marton E, Rosner M, Bernaschek G, Hengstschlager M (2003) Oct-4-expressing cells in human amniotic fluid : a new source for stem cell research? Human reproduction 18 : 1489-93; Tsai MS, Hwang SM, Tsai YL, Cheng FC, Lee JL, et al. (2006) Clonal amniotic fluid- derived stem cells express characteristics of both mesenchymal and neuralstem cells. Biology of Reproduction 74: 545-51. Jezierski A, Gruslin A, Tremblay R, Ly D, Smith C, et al. (2010) Probing sternness and neural commitment in human amniotic fluid cells. Stem Cell Rev6: 199-214. Woodbury D ₍ Kramer BC, Reynolds K, Marcus AJ, Coyne TM, et al. (2006) Long-term cryopreserved amniocytes retain proliferative capacity and differentiate to ectodermal and mesodermal derivatives in vitro. Molecular Reproduction and Development 73 : 1463-72) and cell surface antigens (Roubelakis MG, Pappa KI, Bitsika V, Zagoura D, Vlahou A, et al. (2007) Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid : comparison to bone marrow mesenchymal stem cells. Stem Cells Dev 16: 931-52. Grisafi D, Piccoli M, Pozzobon M, Ditadi A, Zaramella P, et al. (2008) High transduction efficiency of human amniotic fluid stem cells mediated by adenovirus vectors. Stem Cells Dev 17: 953-62. De Coppi P, Bartsch G, Jr., Siddiqui MM, Xu T, Santos CC, et al. (2007) Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 25 : 100-6. Moschidou D, Mukherjee S, Blundell MP, Drews K, Jones GN, et al. (2012) Valproic Acid Confers Functional Pluripotency to Human Amniotic Fluid Stem Cells in a Transgene-free Approach. Mol Ther 20(10) : 1953-67) characteristic of stem cells. Interestingly, amniocytes can be efficiently reprogrammed into a primitive pluripotent state by DNA-integrating and non-integrating methods [18], and subsequently

differentiated along multiple lineages (Li C, Zhou J, Shi G, Ma Y, Yang Y, et al. (2009) Pluripotency can be rapidly and efficiently induced in human amniotic fluid- derived cells. Hum Mol Genet 18 : 4340-9. Anchan RM, Quaas P, Gerami-Naini B, Bartake H, Griffin A, et al. (2010) Amniocytes can serve a dual function as a source of iPS cells and feeder layers. Human Molecular Genetics 20: 962-74. Galende E, Karakikes I, Edelmann L, Desnick RJ, Kerenyi T, et al. (2009) Amniotic Fluid Cells Are More Efficiently Reprogrammed to Pluripotency Than Adult Cells. Cloning Stem Cells 12(2) : 117-25. Ge X, Wang IN, Toma I, Sebastiano V, Liu J, et al. (2012) Human amniotic mesenchymal stem cell-derived induced pluripotent stem cells may generate a universal source of cardiac cells. Stem cells and development 21(15) : 2798-808. Wolfrum K, Wang Y, Prigione A, Sperling K, Lehrach H, et al. (2010) The LARGE principle of cellular reprogramming : lost, acquired and retained gene expression in foreskin and amniotic fluid-derived human iPS cells. PLoS One 5: el3703. Liu T, Zou G, Gao Y, Zhao X, Wang H, et al. (2012) High Efficiency of Reprogramming CD34(+) Cells Derived from Human Amniotic Fluid into Induced Pluripotent Stem Cells with Oct4. Stem cells and development 21(12) : 2322-32. Easley CAt, Miki T, Castro CA _; Ozolek JA,

Minervini CF, et al. (2012) Human Amniotic Epithelial Cells are Reprogrammed More Efficiently by Induced Pluripotency than Adult Fibroblasts. Cellular reprogramming 14: 193-203). Alternatively, they can be reprogrammed through direct methods, which are thought to bypass pluripotency altogether [33], or as our data suggests, use some of the innate pluripotency of amniocytes. Like human embryonic stem cells (hESCs), amniocytes are highly proliferative, but unlike ESCs, they do not produce tumors in vivo and are not immortal (De Coppi P, Bartsch G, Jr., Siddiqui MM, Xu T, Santos CC, et al. (2007) Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 25 : 100-6). Despite these important findings, the regulatory networks controlling the developmental status of amniocytes are still undefined. To better define the developmental status of amniocytes, we examined samples from a large number of patients by immunostaining, flow cytometry, clonal analysis, qPCR and RNA-seq whole- genome profiling. Our bioinformatic analyses of amniocyte, hESC and hIPSC transcriptomes reveal clear distinctions among these populations. Relevant to clinical applications, we asked whether amniotic stem cell dynamics are dependent on gestation, gender, or time in culture. Strikingly, amniocyte profiles resemble transitioning cell-types that co-express markers for both undifferentiated and differentiated derivatives. Clonal analysis indicates that amniocytes are capable of self-renewal and generating multiple distinct pluripotent lineages. Together, our findings suggest molecular mechanisms maintain amniocytes in a stem cell state while simultaneously activating and repressing diverse sets of signaling and differentiation programs. Another important feature when using amnion cells as producer cells, because it preliminarily appears that it is not needed to knock any non transfected cells out with antibiotics such as for instance zeocin, it actually appears not to be necessary, from the initial reports. Hence, it is documented in the literature that amnion cells do not need antibiotic knock-out additions such as for instance Zeocin is not needed in case of these cells. I have chosen to enclose the antibiotics, including Zeocin, even though the need for products such as zeocin really does not need to be added, actually it appears not to be useable. The reason is that it may in the future show that certain amnion cells might need this selection method.

As part of this invention CEVEC Cap cells from CEVEC would also be a novel high producer cell line that would be suited well to produce WT P-A recombinant fibrinogen. The system is as stated in figure 10.

Stable Expression

Stable expression describes the production of recombinant proteins due to integration of the respective gene expression cassette into the chromosome of the producer cell line. In most cases, the gene of interest is integrated into the chromosome via non-homologous recombination in varying copy numbers.

Using CEVEC ^' s CAP (immortalized amnionic cells or immortalized amniocytes) for stable expression, stable integration of your gene is achieved with :

• Optimized expression cassettes

· Optimized selection cassettes

• Fast selection procedure

• Efficient transfection

The process of developing a high yield producer CAP cell line includes several important steps:

· Development of stably expressing pools

• Efficient single cell cloning with a selected pool

• Volumetric upscaling and optimization of fermentation process parameters

• Cell line development in suspension and serum-free medium

With optimized protocols for high producer cell and vector systems as well as the immortalized human amnion cell system as well as other human immortalized amnion cells, which is one of the keys to achieve recombinant human glycosylated proteins such as P-A recombinant human fibrinogen in amounts far exceeding previous obtained amount, which in previous Osther patent application Inspicos No. 17499 and 18223, where the amount obtained routinely could be proven to produce around 15 to 30 mg recombinant human fibrinogen, - now with the novel system used after transfection of adequate alpha-, beta-, and gamma genes for fibrinogen with P-A correction in the beta chain, can now be produced in a highly unexpected and surprising amount exceeding 100 and even 300 mg/liter amnion cells (~5- 10 million cells/ml or higher) and actually up to ~ 1000 mg recombinant human glycosylated fibrinogen, which make this product in quantity sufficiently high so that it can replace the current production rate of high quality plasma fibrinogen, thus being able to compete with plasma fibrinogen. This can be achieved resulting in yields of grams per liter even for complex human proteins with authentic human glycosylation patterns. Such yield of recombinant fibrinogen will eventually be capable of competing strongly with the non-pure human plasma fibrinogen, making it questionable whether one should prepare plasma fibrinogen for intravenous or systemic use and preferably only use recombinant high yield produced human fibrinogen, because of its exensive purity and reproducibility, when compareed to relatively impure plasma fibrinogen.

Cologne, Germany, January 3, 2013 / B3C newswire / - CEVEC Pharmaceuticals, the developer of the CAP® Technology, a novel human expression system derived from amniocytes, and VPM - Vakzine Projekt Management GmbH, announced today the signing of an exclusive license agreement on VPM's proprietary human CMV dense body technology.

CAP® Technology is based on an immortalized human amnion cell line for vaccine and complex protein production developed by CEVEC for industrial use. The serum-free suspension culture of this proprietary cell line allows for high virus yields for a broad variety of human pathogenic viruses. CAP® cells are uniquely suited to produce vaccines against challenging viral targets like human CMV. The non-infectious dense bodies are considered to be one of the most promising new CMV vaccine approaches since they result in efficient and long-lasting immunity. The CMV dense body project is foreseen to enter clinical development within the next 18 months. CEVEC receives exclusive word-wide development and commercialization rights from VPM and VPM will substantially contribute by taking over development project management services for the project.

The inventor has requested to use this new system for the production of recombinant fibrinogen and/or recombinant factor XIII, as well as recombinant thrombin using non-embryonic stem cells such as amnion cells (any placentar derived non-embryonic cells), CAP and/or CAP-T cells for the unique production aspects that these systems offer which is significantly different from different systems, such as HEK cell or PERC6 cells, both embryonic cell lines that is not able of being used for larger or higher yield production (presently giving a maximum of 15-30 mg/liter of cells, which will never be more than of academic interest.

This production does not inhibit multiplication of the cell line, which is anticipated to double every 35 - 45 hours. The fibrinogen Aalpa, Bbeta and gamma chain is used preferably in the composition 1 : 2 : 1 to obtain satisfactory secretion of fibrinogen as well as a satisfactory secretion of factor Xlll/XIIIa.

For high level r-protein expression it is important to use vectors with promoters that are highly active in the host cell line, such as the human cytomegalovirus (CMV) or SV40 promoters. One non-viral promoter, the elongation factor (EF)- l promoter, has also been used because it appears to be as strong as some of the viral promoters (Wurm and Bernard 1999). The CMV promoter is particularly powerful in HEK293 cells, where it has been shown to be strongly transactivated by the constitutively expressed adenovirus Ela protein (Gorman et al. 1989) and/or Elb, thereafter transfecting the permanent human amniocytic cells with a nucleic acid molecule comprising a nucleic acid sequence encoding the SV40 large T-antigen.

Plasmids for human amniocytes are as follows:

Plasmid pGS116 contains the human cytomegalovirus (hCMV) promoter, followed by a Simian virus 40 (SV40) intron, the human alpha- 1 antitrypsin (hAAT) cDNA and the SV40 polyadenylation site. Plasmid pGS119 was used to transform primary human amniocytes and contains the El and pIX region of adenovirus type 5 (Ad5) from nt. 505 to 4079. E1A is under the control of the murine

phosphoglycerate kinase (pgk) promoter, while E1B and pIX expression is controlled from their natural promoters. The E1B downstream intron, splice acceptor and polyA signal were replaced by corresponding motifs from SV40.

The first Recombinant Fibrin kit consisting of recombinant prothrombin M400A or recombinant wild type prothrombin made in transfected amnion cells (e.g., CAP and CAP-T), activated during processing for immediate use together with recombinant fibrinogen made in transfected amnion cells (e.g., CAP and/or CAP-T cells) as a unique Tissue Sealant with no risk of infection from plasma proteins.

Another part of the present invention is creation of a recombinant fibrin kit product, the first recombinant human fibrin kit that has been developed. This product consists of recombinant prothrombin, to be activated for instance with Calcium chloride as described above, immediately prior to use and then mixed together with recombinant human fibrinogen produced in amnion cells (e.g., CAP and CAP-T cells) to obtain recombinant fibrin. It is emphasized that - unlike any other known recombinant prothrombin and recombinant fibrinogen that either are non-human cells or especially human embryonic transfected cells. One of the significant and important difference is that the recombinant proteins such as recombinant prothrombin and recombinant fibrinogen according to my invention is that it is made in placenta originated cells and especially amnion cells (e.g., CAP and/or CAP-T cells from CEVEC) or from stem cells made from umbilical cord blood or from umbilical cord in the area called Wharton's gel, non of these cells are of embryonic origin.

WT P-A Recombinant human fibringen

The wild type recombinant human fibrinogen and/ or the wild type (beta chain P- A) recombinant human fibrinogen would essentially - apart from the significantly higher secretion namely going from the current 30 mg level obtained in my prior invention regarding these complex proteins to surprisingly high levels of this complex proteins to up to 1 gram (1000 mg), but the biotehcmical appearance of the fibrinogen produced by the amnion cells will not differ from the old previously , method also described by Kurt Osther and will appear as follows:

NPSffi; : CLUSTALW ALIGNMENT htl : npsa-pbilifccp.fi7 ^l cgi-b algi¾_clustaw.p!

P iin-cciis .

Prira

t : I t ! I

p58 _5¾_A

Priiiuccns .

5 : 1 5 : 1

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NM

PrirQ.c ns .

ΐϊΜ_02 871 SPeSESgSSTaTBSPSSSERGSaCSWrSSSSYSSSTSQWH3ESSS¾Ρ ·33¾Κ»ΚΚ

Priin.cc-ns ,

5 ! I 5 ! I

ε

Priii ccns . ¥SC¾ 3PGIRFEl

: I 5 : [

p5S4_ :SD'C?SAKDLGTLSGIGTLDGFRH KPDSSllFFr:TaSrGK

2 of 9/28/20061:31PM PS<¾ : CLUSTALW ALIGNMENT tttp: npsa-pfcii^beplr cgi-bm aiiaii_c!ustai .pl

. coriS .

E'riiri . cons .

Alignment data :

Alignment lengtii : 6 1

Identity (*) : 642 is 97.13 %

Strongly similar (:) : i is 0.15 %

Weakly similar ( ^' .) : 0 is 0.00 ¾

Different : IS is 2.72 %

Sequence 000! : p584_Fb_A { 661 residues).

Sequence 0002 : NM_02iS7i_FbA ( 644 residues).

Sequence 0003 : J00127 ( 544 residues).

CLUSTALW options used :

eadgaps=l

gapdist=8

gapext=0.2

gapopen=10.0

hgapresidues=GP SNDQERK

kmple= 1

matFix=goiinet

maxdiv=30

outordei-aligned

pairgap=3

score=percent

tepdiags=S

type=PROTEI

wiado\v=5

Result files (text) :

CLUSTALW

3 of 4 9/28/2006 1 :31 PM

Multiple sequence alignment between commercially available fibrinogen alpha chain cDNAs (NM 021871 (SEQ ID NO: 4), J00127 (SEQ ID NO: 78)) and UNC alpha chain p584. PS<¾ : CLUSTALW .fr .pi

10 20 30 40 SO SO

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70 80 90 100 110 120

i \ t I ! t

BCi06750_Fb F SPOOi!S&GG "·¾¾¾·/

BCIC77¾6_Fb ^" &FSPOFi::;SG :¾ ^: ft: ⁱ 5A30:;;j¾ Y5: i!!iAFIiSiKl LiJ !ilJLGVLGPT CQLQiAijLiJO

p668_Fij_S p ^R St-PP 13SGGY A PAKaAAT !»<KCia

Pria.cc RP&PPP I SGGGYRAF PaKSSJl^Qia ^FJ'S naGGCLSarjPDLG LCF GCQLQEaLL

mpIBliSVBELN!e^tfEAVSaTSSS;iFQlS»lYLLKDLWQKSQKQ D&m ⁷ V®;EY3SELE H

190 200 210 220 230 240

Friiii, ons _* ECEEIIR GGErSEKYLlQPD3S¥ PYRY'YCQ^raTENGG VlQEiRQDGSVDFGF.KS¾bFY

310 320 330 340 350 360

3CiS6760_Fb

SC10776S_Fb ^" FGGF KYaTK E KKYGGLFGEYSl 'JFFilSGLY ^!^

6 S_FS_b

KQGF NVJT*7TD K*F CGLP ZYWFGMF>FIS^

370 3S0 390 400 410 420

i i i I

BC106760_Eb_b

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c668_Fb_b IVCEiS/^iEYQIiVEjE iRGTS t^lKD A^

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f

BC106760 Fb b i<3E5RiF03FF£0.G; - of 4 9/28/200612:17 PM PS<¾ : CLUSTALW http://iipsa-pbii. ibcp.fr/c2i-bn/aiia11_clus tai .pi

pS74_Fb_g

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BC021€74_OpenE ioSys

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70 SO 90 100 110 120

i ! ! i ! i

p674_FiD_G' SDi EiSNSL!iOKtfiJLYS- M/O o^

SCO 07044_OpenB ioSys MSWSIi!KOfLEiLY

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130 140 150 160 170 130

190 200 210 220 230 240

: : · ! : :

p674_Fb_g E:iMKi£¾3 LYHES£IFY 1QE1 S£K ?JLS ¾l£¾ QE C DTVQli[^lTGi;

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BC021fc74_CpenEicSys SISYLQEl YSS QKI VllL EKVagiSSQCQEPC D VQ HlilTG!'

cons .

250 260 270 260 290 300

Friiii, ons _*

310 320 330 340 350 360

p674_F6__

SCO 07044_CpenB i oSy s EGF HLSPY TTEFSLG-iEKIHLIi^

BC021674_CpenBioS_ s i!SGFGiiLSPTG TEFKLGWSECTiS

PrLia.con.s. p674_I¾_g

3CQ07044_OceiiEioSy≤

BC021 74_CpenE icSy

PriTri.co s. DKYRlYTYAYsAjGGDAGBSilJGFDFGDDPSDKFFTSH

430 440 4S0 460 470 4S0

p674_Fb_g

sco 07044_opeuB icsys ;vyYQ';i;':' 5 ??>GYESi ^f ; ^; ·ΐγεκ·:·ΐΥΥ5<α : f

EC02 l¾74_OpeiiBioSy3

Frim.cons- soo 530

LYIGSG¾G.::i5LGG/AK¾AGDV KTV3KEI III3HTTFVEV

2 of 4 9/28/200611:50 AM

Multiple sequence alignment between commercially available fibrinogen gamma chain cDNAs (BC007044, BC021674 (SEQ ID NO: 12)) and UNC alpha chain p674. NFS.® : CLUSTALW ALIGNMENT

BCO 07044_ppenBicSys

BC021 £ 74_opersEiosys

Prim. cons. L IGEG'Q^HHLGGA Q.^GD T SKEIY E GLYL^FSIWAArtlLIIISH T 'E^ ⁷

Alignment data :

Alignment length : 536

Identity (*) : 43? is 81.53 %

Strongly similar (:) : 0 is 0.00 %

Weakly similar (.) : 0 is 0.00 %

Different : 99 is 18.47 %

Sequence 0O0! p674_Fb_g { 536 residues).

Sequence 0002 BC007044_Open3ioSys ( ^' 437 residues).

Sequence 0003 BC021674_Opeii3ioSys ( 4 7 residues).

CLUSTALW options used :

endgaps=l

gapdist=8

gapext=0.2

gapop-eu=10.0

hgap£asidues=GPS DQERK

kmple= 1

matiix=gonnet

maxdiv=30

outordei'=aiigned

pairgap=3

score=percenf

topcliag.s=5

type=PROTEIN

window=5

Result files (text) :

CLUSTALW

Garnier parameters

« Auto calculated

Decision constants Ηείίχ ϊδ SheetpS Tuinp C ilS

Return

PREDATOR parameters

Secondly structure data j DSSP f§

Return

SOPM parameters

Number of conformational states | 4 (Helix, Sheet, Turn, Coil) ¾ 3 of 4 ?'28 2(>06 11:50 AM

Multiple sequence alignment between commercially available fibrinogen gamma chain cDNAs (BC007044, BC021674 (SEQ ID NO: 12)) and UNC alpha chain p674. Table 1. Fibrinogen alpha chain Gene Cloning Reports of HZsec-Fb a-21 cloned from NM_021871 and HZsec-FbaNC- lA cloned from Fb a-NC. Silent mutations are highlighted in green and a point mutation is in red for both DNA sequences and their deduced amino acid sequences that are compared to those of UNC reported fibrinogen alpha chain sequence p584.

Table 1

Gene cloning Report (NM 021871)

Clone Name : HZsec-Fb a-21, or with the CEVEC CAP cell vector system or with other amniocyte or immortalized amniocytes with respective vector or with Adenoviral vector, and would be exactly the same with the CEVEC CAP system, just with quite another level of production, namely increased by a factor around 33 times.

Vector : HZsec (come from Humanzyme), compared to the vector used in the CEVEC CAP system or in other amniocyte systems.

Cloning site : Srf I

Gene : Fibrinogen a (NM_021871)

Primer :

HZsec-Fibrinogen a-F: (SEQ ID NO: 1)

5'-TCCACTGGTGACGCGCCCGCAGATAGTGGTGAAGGTGACTTTCTA-3'

HZsec-Fibrinogen a-R: (SEQ ID NO: 2)

5'-GGCGCGCCTGGCCGGCCCTCACTAGGGGGACAGGGAAGGCT-3'

Sequencing Primer :

HZsec-seqF: 5'-ATGGAGACAGACACACTCCTGC-3' (SEQ ID NO: 52)

Fibrinogen a-wl : 5'-GTCATGCAGTAGGG CTTTAG - 3 ' (SEQ ID NO: 79)

Fibrinogen a-w2 : 5'-AGACTGGGGCACATTTGAAG-3' (SEQ ID NO: 80)

Gene Sequence: (SEQ ID NO: 4)

GCAGATAGTGGT GAAGGTGACTTTCTAGCTGAAGGAGGAGGCGTGCGTGGCCCAAGGGTTGTGGAAAGA CA CAATC GCCTGCAA GATTCAGAC GGCCC CTGCTCT GATGAAGACTGGAACTACAAA GCCCT TCT GGCTGCAGGATGAAAGGGTTGATTGATGAAGTCAATCAAGATTTTACAAACAGAATAAAT AAGCTC AAAAAT TCACTATTT GAAT AT C AGAAGAAC AAT AAGGAT T C T CAT T C GT T GAC C AC T AAT AT AAT GGAA ATTTTGAGAGGCGATTTTTCCTCAGCCAATAACCGTGATAATACCTACAACCGAGTGTCA GAGGATCTG AGAAGCAGAATT GAAGTCCTGAAGCGCAAAGTCATAGAAAAAGTACAGCATATCCAGCTTCTGCAAAAA AATGTTAG GCiCAGTTGGTTGATATGAAACGACTGGAGGTGGACATT GATATTAAGATCCGATCTTGT CGAGGGTCATGCAGTAGGGCTTTAGCTCGTGAAGTAGATCTGAAGGACTATGAAGATCAG CAGAAGCAA

CTTGAACAGGTCATTGCCAAAGACTTACTTCCCTCTAGAGATAGGCAACACTTACCA CTGAT§§AAAATG

AAACCAGTTCCAGACTTGGTTCCCGGAAATTTTAAGAGCCAGCTTCAGAAGGTACCC CCAGAGTGGAAG

GC ATT AAC AGAC AT GC CGC AGAT GAGAAT GGAGT T AGAGAGACCT GGT GGAAAT GAGAT T ACT C GAGGA

GGCTCCAC TCTTATGGAACCGGATCAGAGACGGAAAGCCC AGGAACCCTAGCAGTGCTGGAAGCTGG

AACTCTGGGAGCTCTGGACCTGGAAGTACTGGAAACCGAAACCCTGGGAGCTCTGGG ACTGGAGGGACT

GCAACCTGGAAACCTGG|AGCTCTGGACCTGGAAGT CTGGAAGCTGGAACTCTGGGAGCTCTGGAACT

GGAAGTACTGGAAACCAAAACCCTGGGAGCCCTAGACCTGGTAGTACCGGAACCTGG AATCCTGGCAGC

TCTGAACGCGGAAGTGCTGG|CACTGGAC|TCTGAGAGCTCTGTATCTGGTAGTACT GGACAATGGCAC

TCTGAATCTGGAAGTTTTAGGCCAGATAGCCCAGGCTCTGGGAACGCGAGGCCTAAC AACCCAGACTGG

GGCACATTTGAAGAGGTGTCAGGAAATGTAAGTCCAGGGACAAGGAGAGAGTACCAC ACAGAAAAACTG

GTCACTTCTAAAGGAGATAAAGAGCTCAGGACTGGTAAAGAGAAGGTCACCTCTGGT AGCACAACCACC

ACGCGTCGTTCATGCTCTAAAACCGTTACTAAGACTGTTATTGGTCCTGATGGTCAC AAAGAAGTTACC

AAAGAAGTGGTGACCTCCGAAGATGGTTCTGACTGTCCCGAGGCAATGGATTTAGGC ACATTGTCTGGC

ATAGG|AC CTGGATGGGTTCCGCCATAGGCACCCTGATGAAGCTGCCTTCTTCGACACTGCCTCAACT

GGAAAAACATTCCCAGGTTTCTTCTCACCTATGTTAGGAGAGTTTGTCAGTGAGACT GAGTCTAGGGGC

TCAGAATCTGGCATCTTCACAAATACAAAGGAATCCAGTTCTCATCACCCTGGGATA GCTGAATTCCCT

TCCCGTGGTAAATCTTCAAGTTACAGCAAACAATTTACTAGTAGCACGAGTTACAAC AGAGGAGACTCC

ACATTTGAAAGCAAGAGCTATAAAATGGCAGATGAGGCCGGAAGTGAAGCCGATCAT GAAGGAACACAT

AGCACCAAGAGAGGCCATGCTAAATCTCGCCCTGTCAGAGGTATCCACACTTCTCCT TTGGGGAAGCCT

TCCCTGTCCCCCTAGTGA

Protein Sequence:

ADSGEGDFLAEGGGVRGPRWERHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDEVNQD FTNRINKL KNSLFEYQKNNKDSHSLTTNIMEILRGDFSSANNRDNT YNRVSEDLRSRIEVLKRKVIEKVQHIQLLQK NVRAQLVDMKRLEVDIDIKIRSCRGSCSRALAREVDLKDYEDQQKQLEQVIAKDLLPSRD RQHLPLIKM KPVPDLVPGNFKSQLQKVPPEWKALTDMPQMRMELERPGGNEITRGGSTS YGTGSETESPRNPSSAGSW NSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGS|GSWNSGSSGTGSTGNQNPGSPRPG STGTWNPGS SERGSAGHWTSESSVSGSTGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEVSGNVSPGT RREYHTEKL VTSKGDKELRTGKEKVTSGSTTTTRRSCSKTVTKTVIGPDGHKEVTKEWTSEDGSDCPEA MDLGTLSG IGTLDGFRHRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETESRGSESGIFTNTKESSS HHPGIAEFP SRGKSSSYSKQFTSSTSYNRGDSTFESKS YKMADEAGSEADHEGTHSTKRGHAKSRPVRGIHTSPLGKP

SLSP ..

Restriction Map(Nde I & Hind III) (figure 11) Gene cloning Report (UNC alpha)

clone Name : HZsec-FbaNC-iA or with the CEVEC CAP cell vector system or with other amniocyte or immortalized amniocytes with respective vector or with Adenoviral vector.

Vector : HZsec (come from Humanzyme) or with CEVEC CAP cell/vector system

Cloning site : Srf I

Gene : Fb a-NC

Primer :

HZsec-Fibrinogen a-F:

5' -TCCACTGGTGACGCGCCCGCAGATAGTGGTGAAGGTGACTTTCTA-3 ' (SEQ ID NO: 1) HZsec-Fibrinogen a-R:

5' -GGCGCGCCTGGCCGGCCCTCACTAGGGGGACAGGGAAGGCT-3 ' (SEQ ID NO: 2)

Sequencing Primer :

HZsec-seqF: 5 ' -ATGGAGACAGACACACTCCTGC-3 ' (SEQ ID NO:

52)

HZsec-seqR2: 5 ' -TGGTCGACGGCGCTATTCAG-3 ' (SEQ ID NO: 53)

Fibrinogen a-wl : 5' -GTCATGCAGTAGGGCTTTAG-3 ' (SEQ ID NO: 79)

Gene Sequence: (SEQ ID NO: 81)

GCAGATAGTGGTGAAGGTGACTTTCTAGCTGAAGGAGGAGGCGTGCGTGGCCCAAGGGTT GTGGAAAGA CATCAATCTGCCTGCAAAGATTCAGACTGGCCCTTCTGCTCTGATGAAGACTGGAACTAC AAATGCCCT TCTGGCTGCAGGATGAAAGGGTTGATTGATGAAGTCAATCAAGATTTTACAAACAGAATA AATAAGCTC AAAAATTC CTATTTGAATATCAGAAGAACATAAGGATTCTCTTCGTTGACCACTAATATATGGAA ATTTTGAGAGGCGATTTTTCCTCAGCCAATAACCGTGATAATACCTACAACCGAGTGTCA GAGGATCTG AGAAGCAGAATTGAAGTCCTGAAGCGCAAAGTCATAGAAAAAGTACAGCATATCCAGCTT CTGCAAAAA AATGTTAG GC§CAGTTGGTTGATATGAAACGACTGGAGGTGGACATTGATATTAAGATCCGATCTTG T CGAGGGTCATGCAGTAGGGCTTTAGCTCGTGAAGTAGATCTGAAGGACTATGAAGATCAG CAGAAGCAA CTTGAACAGGTCATTGCCAAAGACTTACTTCCCTCTAGAGATAGGCAACACTTACCACTG AT||AAAATG AAACCAGTTCCAGACTTGGTTCCCGGAAATTTTAAGAGCCAGCTTCAGAAGGTACCCCCA GAGTGGAAG GCATTAAC GACATGCCGCAGATG GAATGGAGTTAGAGAGACCTGGTGGAAATGAGATTACTCGAGGA GGCTCCAC TCTTATGGAACCGGATCAGAGACGGAAAGCCC|!AGGAACCCTAGCAGTGCTGGAAGCTG G AACTCTGGGAGCTCTGGACCTGGAAGTACTGGAAACCGAAACCCTGGGAGCTCTGGGACT GGAGGGACT GCAACCTGGAAACCTGGIAGCTCTGGACCTGGAAGTICTGGAAGCTGGAACTCTGGGAGC TCTGGAACT GGAAGTACTGGAAACCAAAACCCTGGGAGCCCTAGACCTGGTAGTACCGGAACCTGGAAT CCTGGCAGC TCTGAACGCGGAAGTGCTGG|CACTGGAC|TCTGAGAGCTCTGTATCTGGTAGTACTGGA CAATGGCAC TCTGAATCTGGAAGTTTTAGGCCAGATAGCCCAGGCTCTGGGAACGCGAGGCCTAACAAC CCAGACTGG GGC AC AT TT GAAGAGGT GT CAGGAAAT GT AAGTC C AGGGAC AAGGAGAGAGT ACC AC AC GAAAAAC T G GTCACTTCTAAAGGAGATAAAGAGCTCAGGACTGGTAAAGAGAAGGTCACCTCTGGTAGC ACAACCACC ACGCGTCGTTCATGCTCTAAAACCGTTACTAAGACTGTTATTGGTCCTGATGGTCACAAA GAAGTTACC AAAGAAGTGGTGACCTCCGAAGATGGTTCTGACTGTCCCGAGGCAATGGATTTAGGCACA TTGTCTGGC ATAGG|AC CTGGATGGGTTCCGCCATAGGCACCCTGATGAAGCTGCCTTCTTCGACACTGCCTCAACT GGAAAAACATTCCCAGGTTTCTTCTCACCTATGTTAGGAGAGTTTGTCAGTGAGACTGAG TCTAGGGGC CAGAATCTGGCATCTTCACAAATACAAAGGAATCCAGTTCTCATCACCCTGGGATAGCTG AATTCCCT TCCCGTGGTAAATCTTCAAGTTACAGCAAACAATTTACTAGTAGCACGAGTTACAACAGA GGAGACTCC ACATTTGAAAGCAAGAGCTATAAAATGGCAGATGAGGCCGGAAGTGAAGCCGATCATGAA GGAACACAT AGCACCAAGAGAGGCCATGCTAAATCTCGCCCTGTCAGAGGTATCCACACTTCTCCTTTG GGGAAGCCT TCCCTGTCCCCCTAGTGA

Protein Sequence (SEQ ID NO: 82)

ADSGEGDFLAEGGGVRGPRWERHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDEVNQD FTNRINKL KNSLFEYQKNNKDSHSLTTNIMEILRGDFSSANNRDN YNRVSEDLRSRIEVLKRKVIEKVQHIQLLQK NVRAQLVDMKRLEVDI DIKIRSCRGSCSRALAREVDLKDYEDQQKQLEQVIAKDLLPSRDRQHLPLI KM KPVPDLVPGNFKSQLQKVPPEWKALTDMPQMRMELERPGGNEITRGGSTS YGTGSETESPRNPSSAGSW NSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGS|GSWNSGSSGTGSTGNQNPGSPRPG STGTWNPGS SERGSAGHWTSESSVSGSTGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEVSGNVSPGT RREYHTEKL VTSKGDKELRTGKEKVTSGSTTTTRRSCSKTVTKTVIGPDGHKEVTKEWTSEDGSDCPEA MDLGTLSG IGTLDGFRHRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETESRGSESGIFTNTKESSS HHPGIAEFP SRGKSSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPVRG IHTSPLGKP

SLSP ..

Figure 5. pHZsec vector map and Srf I site or, alternatively with the CEVEC CAP cell / vector system. Table 2. Gene Cloning Reports of fibrinogen beta and gamma chains: Gene sequence and deduced protein sequence. No mutation was found.

Gene cloning Report (beta chain)

Clone Name : HZsec-Fb b-21, or with the CEVEC CAP cell vector system or with other amniocyte or immortalized amniocytes with respective vector or with Adenoviral vector. Vector : pHZsec (come from Humanzyme)

Cloning site : Srf I

Gene : Fibrinogen b (BC 106760)

Primer :

HZsec- Fi brinogen b-F : (S EQ ID NO: 5)

5'-TCCACTGGTGACGCGCCCCAAGGTGTCAACGACAATGAGGAG-3'

HZsec- Fi brinogen b-R: (S EQ I D NO : 6)

5'-GGCGCGCCTGGCCGGCCCTCACTATTGCTGTGGGAAGAAGG- 3'

Sequencing Primer :

HZsec-seq F : 5'-ATGGAGACAGACACACTCCTGC- 3' (SEQ ID NO : 52)

HZsec-seq R2 : 5'-TGGTCGACGGCGCTATTCAG- 3' (SEQ I D NO : 53)

Gene Sequence: (SEQ ID NO : 83)

CAAGGTGTCAACGACAATGAGGAGGGTTTCTTCAGTGCCCGT GGTCATCGACCCCTTGACAAGAAGAGA GAAGAGGCTCCCAGCCTGAGGCCTGCCCCACCGCCCATCAGT GGAGGTGGCTATCGGGCTCGTCCAGCC AAAGCAGCTGCCACTCAAAAGAAAGTAGAAAGAAAAGCCCCTGAT GCTGGAGGCTGTCTTCACGCTGAC CCAGACCTGGGGGTGTTGTGTCCTACAGGATGTCAGTT GCAAGAGGCTTT GCTACAACAGGAAAGGCCA ATCAGAAATAGT GTTGATGAGTTAAATAACAATGTGGAAGCT GTTTCCCAGACCTCCTCTTCTTCCTTT CAG AC A GT AT T T GC T GAAAGACC T GT GGC AAAAGAGGCAGAAGC AAGT AAAAGAT AAT GAAAAT G A GTCAATGAGTACTCCTCAGAACT GGAAAAGCACCAATTATATATAGATGAGACTGTGAATAGCAATATC CCAACTAACCTTCGTGTGCTTCGTTCAATCCTGGAAAACCTGAGAAGCAAAATACAAAAG TTAGAATCT GAT GTCTCAGCTCAAATGGAATATT GTCGCACCCCATGCACT GTCAGTTGCAATATTCCTGTGGTGTCT GGCAAAGAATGT GAGGAAATTATCAGGAAAGGAGGTGAAACATCT GAAAT GTATCTCATTCAACCT GAC AGTTCTGTCAAACCGTATAGAGTATACTGTGACATGAATACAGAAAATGGAGGATGGACA GTGATTCAG AACCGTCAAGACGGTAGTGTTGACTTTGGCAGGAAATGGGATCCATATAAACAGGGATTT GGAAATGTT GCAACCAACACAGATGGGAAGAATTACTGTGGCCTACCAGGT GAATATTGGCTTGGAAATGATAAAATT AGCCAGCTTACCAGGATGGGACCCACAGAACTTTTGATAGAAATGGAGGACTGGAAAGGA GACAAAGTA AAGGCTCACTAT GGAGGATTCACTGTACAGAATGAAGCCAACAAATACCAGATCTCAGTGAACAAATAC AGAGGAACAGCCGGTAATGCCCTCATGGATGGAGCATCTCAGCTGATGGGAGAAAACAGG ACCATGACC ATTCACAACGGCATGTTCTTCAGCACGTATGACAGAGACAAT GACGGCTGGTTAACATCAGATCCCAGA AAACAG GT T CT AAAGAAGAC GGT GGT GGAT GGT GGT AT AAT AGAT GT CAT GC AGCC AAT CCAAAC GGC AGATACTACT GGGGT GGACAGT ACACCT GGGACAT GGC AAAGCAT GGCAC AGAT GAT GGT GTAGTAT GG ATGAATTGGAAGGGGTCATGGTACTCAAT GAGGAAGAT GAGTATGAAGATCAGGCCCTTCTTCCCACAG CAATAGT GA

Protein Sequence: (SEQ ID NO : 84) QGVNDNEEGFFSARGHRPLDKKREEAPSLRPAPPPISGGGYRARPAKAAATQKKVERKAP DAGGCLHAD PDLGVLCPTGCQLQEALLQQERPIRNSVDELNNNVEAVSQTSSSSFQYMYLLKDLWQKRQ KQVKDNENV VNEYSSELEKHQLYIDETVNSNIPTNLRVLRSILENLRSKIQKLESDVSAQMEYCRTPCT VSCNIPVVS GKECEEIIRKGGETSEMYLIQPDSSV PYRVYCDMNTENGGWTVIQNRQDGSVDFGRKWDPYKQGFGNV ATNTDGKNYCGLPGEYWLGNDKISQLTRMGP ELLIEMEDWKGDKVKAHYGGFTVQNEANKYQI SVNKY RGTAGNALMDGASQLMGENRTMTIHNGMFFSTYDRDNDGWLTSDPRKQCSKEDGGGWWYN RCHAANPNG RYYWGGQYTWDMAKHGTDDGWWMNWKGSWYSMRKMSMKIRPFFPQQ ..

Restriction Map(Nde I & Hind III) (Figure 12)

Gene cloning Report (gamma chain)

Clone Name : HZsec-Fb r-21 or with the CEVEC CAP cell vector system or with other amniocyte or immortalized amniocytes with respective vector or with Adenoviral vector.

Vector : pHZsec (come from Humanzyme) or with the CEVEC CAP cell system with another vector

Cloning site : Srf I

Gene : Fibrinogen r (BC021674)

Primer :

HZsec-Fibrinogen r-F: or with CEVEC CAP vector system r-F

5 ' -TCCACTGGTG ACGCGCCCTATGTTGCTACCAG AG ACAACTGCTG - 3 ' (SEQ ID NO: 9) HZsec-Fibrinogen r-R:

5'-GGCGCGCCTGGCCGGCCCTCATTAAACGTCTCCAGCCTGTTTG-3' (SEQ ID NO: 10) Sequencing Primer :

HZsec-seqF: 5'-ATGGAGACAGACACACTCCTGC-3' (SEQ ID NO: 52)

HZsec-seqR2 : 5'-TGGTCGACGGCGCTATTCAG-3' (SEQ ID NO: 53)

Gene Sequence: (SEQ ID NO: 12)

TA GT GCTACCAGAGACAAC GC GCATCTTAGATGAAAGATTCGGTAGTTA GTCCAACTACC GT GGCATTGCAGATTTCCTGTCTACTTATCAAACCAAAGTAGACAAGGATCTACAGTCTTTG GAAGACATC TTACATCAAGTTGAAAACAAAACATCAGAAGTCAAACAGCTGATAAAAGCAATCCAACTC ACTTATAAT CCTGATGAATCATCAAAACCAAATATGATAGACGCTGCTACTTTGAAGTCCAGGAAAATG TTAGAAGAA ATTATGAAATATGAAGCATCGATTTTAACACATGACTCAAGTATTCGATATTTGCAGGAA ATATATAAT TCAAATAATCAAAAGATTGTTAACCTGAAAGAGAAGGTAGCCCAGCTTGAAGCACAGTGC CAGGAACCT TGCAAAGACACGGTGCAAATCCATGATATCACTGGGAAAGATTGTCAAGACATTGCCAAT AAGGGAGCT AAACAGAGCGGGCTTTACTTTATTAAACCTCTGAAAGCTAACCAGCAATTCTTAGTCTAC TGTGAAATC GATGGGTCTGGAAATGGATGGACTGTGTTTCAGAAGAGACTTGATGGCAGTGTAGATTTC AAGAAAAAC TGGATTCAATATAAAGAAGGATTTGGACATCTGTCTCCTACTGGCACAACAGAATTTTGG CTGGGAAAT GAGAAGATTCATTTGATAAGCACACAGTCTGCCATCCCATATGCATTAAGAGTGGAACTG GAAGACTGG AATGGCAGAACCAGTACTGCAGACTATGCCATGTTCAAGGTGGGACCTGAAGCTGACAAG TACCGCCTA ACATA GCCTACTTCGC GGTGGGGATGCTGGAGA GCCTTTGATGGCTT GATTT GGCGA GATCCT AGTGACAAGTTTTTCACATCCCATAATGGCATGCAGTTCAGTACCTGGGACAATGACAAT GATAAGTTT GAAGGCAACTGTGC GAACAGGATGGATCTGGTTGGTGGATGAACAAGTGTCACGCTGGCCA CTCAAT GGAGTTTATTACCAAGG GGCACTTACTCAAAAGCATCTACTCCTAATGGTTA GA AA GGCAT ATT TGGGCCACT GGAAAACCCGG GGTATTCCA GAAGAAAACCACTA GA GA AATCCCATTCAACAGA CTCACAATTGGAGAAGGACAGCAACACCACCTGGGGGGAGCCAAACAGGCTGGAGACGTT TAATGA

Protein Sequence: (SEQ ID NO: 11)

YVATRDNCCILDERFGSYCPTTCGIADFLSTYQTKVDKDLQSLEDILHQVENKTSEVKQL IKAIQLTYN PDESSKPNMIDAATLKSRKMLEEIMKYEASILTHDSSIRYLQEIYNSNNQKIVNLKEKVA QLEAQCQEP CKDTVQIHDI GKDCQDIANKGAKQSGLYFIKPLKANQQFLVYCEIDGSGNGWTVFQKRLDGSVDFKKN WIQYKEGFGHLS P G EFWLGNEKIHLI S QSAI PYALRVELEDWNGRT STADYAMFKVGPEADKYRL TYAYFAGGDAGDAFDGFDFGDDPSDKFFTSHNGMQFSTWDNDNDKFEGNCAEQDGSGWWM NKCHAGHLN GVYYQGG YSKAS PNGYDNGI IWA WKTRWYSMKKTTMKI IPFNRLTIGEGQQHHLGGAKQAGDV ..

Restriction Map(Nde I & Hind III) (Figure 13)

Transfection and expression of fibrinogen

Transfection requires certain amount of transfectant versus amount of plasmid transfected (lOug plasmid : 30ug polyethyleneimine). Since fibrinogen requires three plasmids (HZsec Fb a-21, HZsec Fb b-21, HZsec Fb r-21) transfectant (polyethtleneimine at lmg/ml solution) was needed three times of single gene transfection. The amount of transfectant used for fibrinogen transfection of three chains was found to be toxic to the cells. Therefore the culture medium was changed with fresh medium after four hours of tranfection. At 1 : 1 : 1 ratio transfection we obtained cells expression about lOmg/L production in suspension. Later we learned from literature study that amount of fibrinogen beta chain is rate-limiting in fibrinogen complex assembly so we decided to study different ratio tranfections for alpha : beta :gamma as 1 : 1 : 1, 1 : 2: 1, and 1: 5 : 1. It turned out in our hand that equal mount increment of plasmids yielded high expression as 2: 2: 2. At the same time the condition was highly toxic therefore culture medium must be changed with fresh medium after four hours of tranfection.

Antibiotics such as zeocin is not needed in the amnion cell system (e.g., CAP and/or CAP-T, from CEVEC)

Figure 14. A point mutation (The312 to Ala312) in fibrinogen alpha chain. Both cloned fibrinogen alpha chains from NM_021871 (FbgAa_OBS) and UNC Fb a p584 (FbgAa_UNC) have Ala312 instead of Thr312 reported public database as highlighted in yellow in Ref22.

Purification

Not knowing proper characteristics of fibrinogen all the culture supernatant was kept at 4°C after removing the cells by centrifugation. In the process of learning purification some fractions were concentrated using ultrafiltration tool at room temperature and left at 4°C for a few days. When the fraction was analyzed on SDS-PAGE its molecular weight was noticeably lowed to around 190kDa from 340kDa (Figure 15).

At that time we contacted UNC and discussed the finding and learned that fibrinogen is very susceptible to proteases therefore UNC culture condition adds a cocktail of protease inhibitors and PMSF (Figure 16). Since then all the harvested culture supernatant was mixed with protease inhibitor cocktail and PMSF and stored at -20°C until purification. Also all the centrifuge bottle and storage bottles were used as autoclaved or obtained as sterile.

The purification method used in UNC is ammonium sulfate precipitation (overnight for liters! ) followed by huge centrifugation (liter bottles! and careful handling not to disturb) and immuno affinity chromatography ^14,1 . Final step involves dialysis against TBS or HEPES buffer for physiological condition for following studies. It will be very costly and not easily scalable.

Therefore we avoided the method and looked for alternative ways allowing scalability, which is indeed useable when the transfected cell line such as for instance the CAP or CAP-T cell line is capable of resulting in producing levels that then can be applied to a simple purification method described below. One was using heparin column knowing that many blood proteins bind to heparin. Indeed it was working, however still initial capture for liters of culture medium was problem to exchange the culture medium to the loading buffer condition, mostly much less salt, to bind the proteins to the column resins. During the literature study on fibrinogen purification we found some patent applications, from the same authors, showing that IMAC charged with copper ion (Cu ²⁺ ) columns were used for purification of plasma fibrinogen ^v . In the lab we had two types of immbolilized metal ion affinity chromatography (IMAC) columns (one from GE lifescience and the other from TOSHO) that used for purification of other proteins including His-tag proteins with different metal ion charges (Cu ²⁺ , Ni ²⁺ , Co ²⁺ , etc). Therefore we applied the protocol for rhFbg capture from the culture medium on TOSHO IMAC charged with Cu ²⁺ based on the literature with an alternative buffer system lOmM Tris-HCI, pH7.4, 50mM NaCI, instead of phosphate and citrate based buffers knowing that citrates chelating calcium ions that are involved in blood clotting. rhFbg was bound to the IMAC column equilibrated with lOmM Tris- HCI, pH7.4, 50mM NaCI and was eluted with lOmM Tris-HCI, pH7.4, 50mM NaCI, 50mM Arg (Figure 17). This method does not require any buffer exchange or treatment before loading the culture medium, just directly load on IMAC-Cu ²⁺ column equilibrated with lOmM Tris-HCI, pH7.4, 50mM NaCI. With the finding, rhFbg elution condition from IMAC-Cu ²⁺ was further studied with different Arg concentrations to improve the purity from the capture step and learned that rhFbg elution was in 20mM Arg fraction as well as 50mm Arg fraction. Since they eluted in different conditions they kept separately and individually loaded on heparin column directly.

The copper ion and other impurity proteins did not bind to heparin column were found in flow through fraction. rhFbg was then eluted with Tris-based saline (TBS or lOmM Tris-HCI, pH7.4, 150mM NaCI) and it was greater than 95% pure as it is, and not needed buffer exchange since it is already physiological buffer condition. Purified fibrinogen was analyzed on the gel under non-reducing and reducing conditions. Gel analysis showed that IMAC 20mM Arg fraction has more degraded rhFbg. Therefore purification was focused on 50mM Arg fraction since then (Figure 18). Purified rhFbg from IMAC and heparin was in TBS and filter sterilized through 0.22um and was placed in a deep freezer (-80°C) after quantification. Quantification and clottability Purified rhFbg was quantified using OD280nm = 1.51 for lmg/ml ^v . This fibrinogen was slow in polymerization so clottability was tested greater than 95% for quality control (Figure 19). According to discussion with UNC lab polymerization or clottability is used for quality control. After delivering 15mg and 50mg of rhFbg to HG, we learned than the rhFbg showed significant slower polymerization compared to hpFbg (Figures 20 and 21). Certificate of Analysis of rhFBG 15mg (CHI121407) and 50mg (CHI121807A and CHI121807B) are attached after Figures 20 and 21. Clotting time test performed in Coloplast showed that rhFbg showed a significant slower rate as being compared with hpFbg (Calbiochem, Cat# 341578) (Figure 22 from Coloplast report of June 26, 08). rhFbg Bb (Pro to Ala)

Slower clotting time with rhFbg was investigated with two possibilities including alpha chain instability and beta chain mutation of Alanine 162 (UNC) to Proline (HZ rhFbg). At the same time we looked at available crystal structure of fibrinogen (chicken fibrinogen, 1EI3) and learned that the Alanine to Proline mutation was close to one of bundling area and raised a concern that may cause misfolding of protein (Figure 1623). Therefore HG and HZ decided to mutate back the Proline to Alanine and hold the alpha chain instability study for the time being due to the complexity. Interestingly there was only one paper reporting the polymorphism^, and one paper reported beta chain sequence with Proline ^v " and one paper reported with Alanine ^vl ". Yet no functional comparison was reported. In May 2009 human plasma fibrinogen crystal structure was solved by Doolittle group with Prol62 ^lx .

Transfection and expression of wt rhFbg

HG and HZ mutually have called rhFbg with beta chain with Ala l62 as wildtype recombinant human fibrinogen or wt rhFbg. Transfection was performed in alpha : beta : gamma as 1 : 1 : 1, 1: 2 : 1, and 1 : 5: 1. Transfected cells were set for antibiotics selection zeocin 400ug/ml and 800ug/ml. Over two weeks time period, cells selected from zeocin 400ug/ml of 1 : 2: 1 transfection in well B3 (Fbg l21B3) were selected for expression because of higher expression than others, and set for serum-free suspension culture for large scale production. Expression level was however significantly lower than "old rh Fbg". Real expression level should be tested by ELISA in HG. However, Zeocin is not needed in producing recombinant fibrinogen or even factor XIII in gene transfected CAP and/or CAP-T cells.

Gene cloning Report (beta chain P162A)

Clone Name : HZsec- Fibrinogen b-P2A-3, or - preferably using amnion cells as host cells for the vector including the correct vector- system combined with fibrinogen b, fibrinogen a and fibrinogen gamma genes

Vector : pHZsec (come from Humanzyme)

Cloning site : Srf I

Gene : Fibrinogen b-P2A

Primer :

Fbb-P2A_F: AGACTGTGAATAGCAATATCgCAACTAACCTTCGTGTGCTT (SEQ ID NO: 85)

Fbb-P2A_R: AAGCACACGAAGGTTAGTTGcGATATTGCTATTCACAGTCT (SEQ ID NO: 85)

Sequencing Primer :

HZsec-seqF: 5'-ATGGAGACAGACACACTCCTGC-3' (SEQ ID NO: 52)

HZsec-seqR2 : 5'-TGGTCGACGGCGCTATTCAG-3' (SEQ ID NO: 53)

Gene Sequence: (SEQ ID NO. : 87)

CAAGGTGTCAACGACAATGAGGAGGGTTTCTTCAGTGCCCGTGGTCATCGACCCCTTGAC AAGAAGAGA GAAGAGGCTCCCAGCCTGAGGCCTGCCCCACCGCCCATCAGT GGAGGTGGCTATCGGGCTCGTCCAGCC AAAGCAGCTGCCACTCAAAAGAAAGTAGAAAGAAAAGCCCCT GATGCTGGAGGCTGTCTTCACGCTGAC CCAGACCTGGGGGTGTTGTGTCCTACAGGATGTCAGTT GCAAGAGGCTTT GCTACAACAGGAAAGGCCA ATCAGAAATAGT GTTGATGAGTTAAATAACAATGTGGAAGCTGTTTCCCAGACCTCCTCTTCTTCCTTT CAG AC AT GTA TTGC GAAAGACC T GT GGC AAAAGAGGCAGAAGC AAG AAAAGA AAT GAAAAT G A GTCAATGAGTACTCCTCAGAACTGGAAAAGCACCAATTATATATAGATGAGACTGTGAAT AGCAATATC

iCAACTAACCTTCGTGTGCTTCGTTCAATCCTGGAAAACCTGAGAAGCAAAATACAA AAGTTAGAATCT GAT GTCTCAGCTCAAATGGAATATTGTCGCACCCCATGCACTGT CAGT TGCAATATTCCT GTGGTGTCT GGCAAAGAATGT GAGGAAATTATCAGGAAAGGAGGTGAAACATCTGAAAT GTATCTCATTCAACCTGAC AGTTCTGTCAAACCGTATAGAGTATACTGTGACATGAATACAGAAAATGGAGGATGGACA GTGATTCAG AACCGTCAAGACGGTAGTGTTGACTTTGGCAGGAAATGGGATCCATATAAACAGGGATTT GGAAATGTT GCAACCAACACAGATGGGAAGAATTACTGTGGCCTACCAGGT GAATATTGGCTTGGAAATGATAAAATT AGCCAGCTTACCAGGATGGGACCCACAGAACTTTTGATAGAAATGGAGGACTGGAAAGGA GACAAAGTA AAGGCTCACTAT GGAGGATTCACTGTACAGAATGAAGCCAACAAATACCAGATCTCAGTGAACAAATAC AGAGGAACAGCCGGTAATGCCCTCATGGATGGAGCATCTCAGCTGATGGGAGAAAACAGG ACCATGACC ATTCACAACGGCATGTTCTTCAGCACGTATGACAGAGACAAT GACGGCTGGTTAACATCAGATCCCAGA AAACAGTGTTCTAAAGAAGACGGTGGTGGATGGTGGTATAATAGATGTCATGCAGCCAAT CCAAACGGC AGATACTACT GGGGT GGACAG ACACCT GGGACAT GGC AAAGCAT GGCAC AGAT GAT GGT GTAG AT GG ATGAATTGGAAGGGGTCAT GGTACTCAAT GAGGAAGAT GAGTATGAAGATCAGGCCCTTCTTCCCACAG CAATAGT GA

Purification

The wt rhFbg harvested culture supernatant was applied to the same purification scheme developed with old rhFbg . Again there were two fractions from IMAC in 20mM Arg elution and in 50mM Arg elution that are designated as <F5> and < F6>, respectively, as the collected fraction order. Both fractions were separately applied to heparin column and purified wt rhFbg in TBS. Their difference in heparin column elution profile was in the way that 20mM Arg fraction < F5> displayed sharp and narrower elution profile and that 50mM Arg fraction <F6> displayed broad and longer tail elution profile. There were also heterogeneity and inconsistence in wt rhFbg production as Dr. Lord's lab faced. We attempted to explain using 1970's papers, that high instability of fibrinogen alpha chain may cause the difference sizes of fibrinogens. At this time it is not clear what and how causes the alpha chain instability in the culture condition. Both fractions were separately filter sterilized and placed in -80°C freezer.

Quantification and clottability

Purified wt rhFbg was quantified using OD280nm . Both <F5> and < F6> wt rhFbg displayed good polymerization response, and <F6> polymerization was comparable to hpFbg purchased from Clabiochem (Cat# 341578).

Characteristics of rhFbg vs hpFbg

Carrying on the rhFbg production project has been faced a number of unexpected challenges due to the nature of fibrinogen.

1. Protein degradation : Fibrinogen is a structural protein in clotting system and is very fragile protein (very susceptible to proteases). First notice of fibrinogen degradation was molecular weight shift to 200kDa from 320kDa under non- reducing condition and disappearing alpha chain band under reducing condition. Therefore it is strongly recommended to add protease inhibitors into the culture supernatant before purification process. Degradation may come from two different stages: one is from culture stage and degraded before purification, and the other is from purification especially concentrating whole culture supernatant which increases concentration of proteases. According to Dr. Gorkun in Dr. Lord's lab 5 UNC, degree of alpha chain degradation and its mechanism are not fully

understood. Currently cocktail of protease inhibitors is added into the culture supernatant to prevent further degradation which is costly.

In late 1960's and early 1970's human plasma fibrinogen degradation and

10 heterogeneities were studied by two groups using plasmin as prime protease.

Their study clearly demonstrated higher susceptibility of alpha chain to the protease degradation that yielded different degrees of degradation. There has been no such study was performed with recombinant human fibrinogen produced in serum suspension culture. Dr. Lord's group in UNC has been studying certain 15 genetic variants of interest and their characteristics in polymerization.

2. Many natural variants: Besides the heterogeneity of degraded fibrinogen molecules, there are significantly large number of genetic variants have been reported in the literature (Table 1). Not doing amino acid sequence analysis there

20 is a good chance of batch to batch inconsistency in hpFbg depending on the

source. This inconsistency problem can be addressed only by recombinant human fibrinogen produced with known genetic materials. However study on fibrinogen functionality of each variant will be very lengthy because only mammalian cells can produce intact fibrinogen due to its complex nature.

25 Even in recombinant fibrinogen production attention must be paid to cDNA

sources of alpha, beta, and gamma chain. In this study we found there was wrong sequence information (Thr to Ala) in alpha chain and a critical polymorphism (Prol62Ala) in beta chain was for physiological function.

30 3. Handling issues with human plasma fibrinogen

To compare physical and biochemical properties human plasma fibrinogens were purchased from Sigma and Clabiochem. hpFbg is dried powder and is needed rehydrating the protein. According to manufacturer's manual rehydration should be done in 37oC water bath and the protein solution should be kept at -70oC or

35 37oC, not in ice. The reason is not clear. On the other hand rhFbg has been fine in ice during the purification and at the working bench for experiments. Although no through study was performed rhFbg seems much more stable than hpFbg in solution. Therapeutic usage of hpFbg requires heat treatment and/or Pasteurization that may cause its degradation , . However replacing with rhFbg may not need such harsh treatment because of its controlled production environment. Also homogeneity of rhFbg may give better stability for the treatment, although the supporting experiments have not been performed .

4. Polymerization requires a fine balance of thrombin, fibrinogen, and other chemicals in the reaction buffer

Fibrinogen polymerization is not only matter of fibrinogen, but also of thrombin and other ions in the buffer. Their concentration can influence polymerization rate and degree. Therefore accurate comparison only can be achieved with the same amount of thrombin in the same buffer system .

Recombinant human matrices towards human glycosylated recombinant proteins by using special selection methods present in culture medium.

The invention relates to recombinant human recombinant proteins such as factor XIII and fibrinogen. The present invention relates to a novel concept and method for preparing compositions of recombinant human proteins, each prepared in a novel manner that create authentic or natural human proteins produced in human cells, human cell lines, human stem cells, or human precursor cells. In this invention the cells consisting of human cells that are transfected with human genes expressing protein factors involved in the hemostasis and hemolytic process, such as a novel concept and method for preparing compositions such as transamidase XHIa, also called Factor XHIa which according to this invention now has been produced as a recombinant human protein resembling human authentic transamidase (also called fibrinoligase, plasma transglutaminase, fibrin stabilizing factor). The transamidase factor XHIa catalyzes the conversion of soluble fibrin to insoluble fibrin by crosslinking the lysine and glutamine side chains of the a- and γ- chains of fibrin to form homopolymers. Thus, factor XHIa also called factor XIII is considered a fibrin stabilizing factor. According to this invention the factor XHIa produced in CEVEC CAP cell lines. The present invention using amnionic cells or amniocytes also relates to novel methods for preparing compositions of kits containing recombinant human factor XHIa, and recombinant human fibrinogen, each prepared in a novel manner that create authentic, wild type human proteins with human glycosylation produced in human amnion cells, or immortalized human amnion cell lines, human stem cells, or human precursor cells. The results using the above described eel lines differs significantly from the results obtained when using embryonic cell lines such as in HEK or PERC6 cell lines.

The human amnionic cell types capable of being transfected with genes, and where the selection for all these proteins cloned and refined by being resistant against a certain antibiotic, Zeocin, which will weaken non - resistant cells including non- resistant human cells of any kind, in order to optimize the clones producing these particular proteins described and encompassed above.

Use of antibiotic selection in serum free medium used for transfected human cell lines

First of all, when using cells such as amniocytes transfected with the appropriate gene(s), it is certainly not necessary to utilize the zeocin knock out method or any other antibiotics for selection of transfected amnion cells versus non transfected amnion cells.

Stable, long-term expression of a gene of interest can be either achieved by eukaryotic vectors that harbor elements for episomal maintenance in the nucleus of a transfected cell or via direct integration of the transfected plasmid into the target cells genome. Episomal stability is often limited, resulting in gradual loss of transfected vectors that can only be prevented by sustained antibiotic selection eliminating cells that lost the plasmid. Furthermore, the functionality of episomal plasmid elements is often restricted to certain species. Although integration into the host cell chromosome is a rare event and, for most purposes, clonal events have to be isolated, stability of the intended genetic modification usually is much higher.

Since chromosomal integration into host chromosomes is a rare event, stably- transfected cells usually have to be selected and cultured in various ways. For the selection of stably-transfected cells, a selection marker is co-expressed on either the same construct or on a second, co-transfected vector. A variety of systems for selecting transfected cells exists, including resistance to antibiotics such as neomycin phosphotransferase, conferring resistance to G418, dihydrofolate reductase (DHFR), or glutamine synthetase (Southern and Berg, 1982). After gene transfer, cells are cultivated in medium containing the selective agent. Only those cells which have integrated the plasmid survive, containing

the drug resistant gene. Initially, the gene of interest has to be introduced into the cell, subsequently into the nucleus, and finally, it has to be integrated into chromosomal DNA. Since chromosomal integration into host chromosomes is a rare event, stably- transfected cells usually have to be selected and

cultured in various ways. For the selection of stably-transfected cells, a selection marker is co-expressed on either the same construct or on a second, co- transfected vector. A variety of systems for selecting transfected cells exists, including resistance to antibiotics such as neomycin phosphotransferase, conferring resistance to G418, dihydrofolate reductase (DHFR), or glutamine synthetase (Southern and Berg, 1982). After gene transfer, cells are cultivated in medium containing the selective agent. Only those cells which have integrated the plasmid survive, containing the drug resistant gene. Several options are used for the generation of a stable cell line, depending on the scope of the experiment. A mixed population of drug resistant cells can be used directly for experimental analysis (batch culture) with the advantage of generating fast results, but also the disadvantage of dealing with an undefi ned and genetically mixed cell population. To generate clonal cells, it is necessary to dilute the resistant cells in such a way that culture as single, isolated cells is achieved e.g., by plating in 96-well plates or by using other methods. Subsequently, the selection process is applied to the single cell cultures. The procedure of single cell cloning may be repeated several times to obtain 100% clonal purity. This culture method allows for conduction of the study or the screening using a defi ned and homogenous cell system. So far, generation of stable cell lines has been a major challenge for many cell types (e.g., Jurkat, MCF7 or U937) since overall transfection efficiencies and/or integration frequencies have been low. While common transfection methods, such as lipofection, can be used for the stable expression in easy-to-transfect cell lines (e.g., HeLa, COS-7 or CHO), Nucleofection® is the method of choice for stable expression in diffi cult-to-transfect cell types. The generation of stably-transfected cell lines is essential for a wide range of applications, such as gene function studies (Grimm, 2004), drug discovery assays or the production of recombinant proteins (Wurm, 2004). In contrast to transient expression, stable expression allows long term, as well as defined and reproducible, expression of the gene of interest.

Fibrinogen, produced in overexpressed Hep G2 cells an acute phase protein is increased in

Alternative cell systems for the production of fibrinogen

Earlier studies showed that overexpression of B beta fibrinogen chains, by transfection of Hep G2 cells with B beta cDNA, specifically enhanced the synthesis of all three fibrinogen chains (Roy, S. N., Mukhopadhyay, G., and Redman, C. M. (1990) J. Biol. Chem. 265, 6389-6393; Roy S, Overton O, Redman C.

Overexpression of Any Fibrinogen Chain by Hep G2 Cells specifically elevates the expression of the two Other Chains. The Journal of Biological Chemistry (1994), vol. 269, 691-695). To determine whether overexpression of any of the three component chains of fibrinogen affects the synthesis of the other two chains, we developed stable Hep G2 cell lines transfected with individual fibrinogen chain cDNAs.

As a control, cells were also transfected with expression vector, which did not contain fibrinogen cDNA. Transfection with any fibrinogen cDNA increased the synthesis of all three fibrinogen chains but not of other plasma proteins. Hep G2 cells transfected with B beta cDNA produced 3-4-fold more fibrinogen than control cells, and cells transfected with A alpha or gamma cDNA made about 2-fold more fibrinogen. Northern blot analyses showed that levels of all 3 fibrinogen mRNAs were increased and were highest in Hep G2-B beta cells. Nuclear run-on transcription assays demonstrated that increased expression of the chains was due to increased transcriptional activity. These studies show that transcription of the three fibrinogen chains is tightly linked, and increased expression of any chain specifically leads to increased synthesis of the other two chains. Factor XIIIa/Factor XHIb

The hemostatic system, consisting of blood vessels and blood, plays a crucial role in human survival. The importance of the plasma coagulation system in protecting life and preventing further blood loss following transection of a blood vessel has been understood for a long time. Blood normally is maintained in a fluid state, without evidence of bleeding or clotting. The presence of a bleeding diathesis in families with an X-linked pattern of inheritance of the disorder has been recognized for hundreds of years. Factor XIII is a transglutaminase that circulates in the plasma as a heterotetramer of two catalytic A subunits and two carrier B subunits. When thrombin has converted fibrinogen to fibrin, the latter forms a proteinaceous network in which every E-unit is crosslinked to only one D-unit. Factor XIII is activated by thrombin into factor XHIa; its activation into Factor XHIa requires calcium as a cofactor. A cleavage by thrombin between residue Arg37 and Gly38 on the N-terminus of the A subunit, leads to the release of the activation peptide (MW 4000 da).

Figure 24 is from Hematologic Technologies Inc. Overview of Factor XIII

Factor XIII is the zymogenic form of the glutaminyl-peptide g-glutamyl transferase factor XHIa (fibrinoligase, plasma transglutaminase, fibrin stabilizing factor, E.C. 2.3.2.13) (1-3). Factor XIII is unique among transamidases in that it is a zymogen in vivo (2). Factor XIII is found both extracellularly in plasma and intracellularly in platelets, megakaryocytes, monocytes, placenta, uterus, liver and prostrate tissues. Plasma factor XIII is synthesized in the liver and circulates as a tetramer (Mr= 320,000), composed of 2 pairs of nonidentical subunits (A2B2) (4). The intra-cellular forms are synthesized in the tissues where they reside as dimers (Mr= 146,000) of 2 identical A chains (A2) (7-11). The A subunits of plasma and intracellular forms of factor XIII are functionally identical. The A subunit contains 6 free sulfhydryl groups one of which is the active site (12).

Especially, due to the fact that Factor XIII is present in placenta, it would be obvioius, that the cell type such as amniocytes amnion cells, such as CEVEC CAP systems, would be perfect in the production of Factor XIII. In the presence of calcium the carrier subunits dissociate from the catalytic subunits, leading to a 3D change in conformation of factor XIII and hence the exposure of catalytic cysteine residue. Upon activation by thrombin, factor XHIa acts on fibrin to form y-glutamyl-C-lysyl amide cross links between fibrin molecules to form an insoluble clot (see Figure 25).

• Factor XIII is a transglutaminase that circulates as a zymogen comprised of 2 catalytic A subunits and 2 carrier B subunits

· The A subunit is synthesized in platelets, monocytes and macrophages while the B subunit is synthesized in the liver; the A and B dimers then assemble in the plasma to form a heterotetramer

• Factor XIII is activated by thrombin and is responsible for catalyzing the final step in the coagulation cascade by cross-linking fibrin (in the presence of calcium) · Deficiency is due to a defect in either the A gene (type 2) or B gene (type 1)

• Factor XIII deficiency is a congenital disorder that is inherited as an autosomal recessive trait and is associated with a variable bleeding tendency

• Acquired factor XIII deficiency is associated with liver failure, inflammatory bowel disease, leukemia, disseminated intravascular coagulation, Henoch- Schonlein purpura, systemic lupus erythematosus and exposure to certain drugs (phenytoin, isoniazid, valproate).

Activation of factor XIII (FXIII) by thrombin and calcium is a 2-step process (see Figure 26). Thrombin cleaves an arginine-lysine bond in the A subunit and calcium causes dissociation of the B subunit, exposing the active site on the A subunit (XHIa). Factor XHIa catalyzes the formation of covalent bonds between glutamine and lysine residues on the fibrin a and g chains, enhancing the mechanical strength of the fibrinpolymer. Recombinant Factor XIII

A new recombinant FXIII (rFXIII), originally developed by ZymoGenetics and later transferred to Novo Nordisk (Novo Nordisk A/S, Copenhagen, Denmark), has been manufactured in Saccharomyces cerevisiae (yeast) and contains no

human/mammalian products.7 The rFXIII associates in plasma with the endogenous FXIII-B subunit to form the stable FXIII heterotetramer. In a phase 1 clinical trial, rFXIII had a half-life similar to that of native FXIII. This new product was found to have a good safety profile and to be appropriate for development for monthly prophylactic administration in patients with FXIII-A subunit deficiency. 7 Therefore, this multinational, open-label, single-arm, multiple-dosing, phase 3 prophylaxis trial was undertaken to evaluate the efficacy and safety of rFXIII for the prevention of bleeding in congenital FXIII-A subunit deficiency.

BRIEF SUMMARY OF THE Factor XIII invention

The present invention provides methods for the isolation of highly purified factor XIII. In a particular embodiment, using the methods disclosed, a factor XIII composition that is at least 95% pure with respect to contaminating proteins can be obtained. Factor XIII is obtained by gene transfection into non-embryonic placentar cells, amnion cells (e.g., CAP or CAP-T cells) These methods are particularly adapted for the purification of recombinant factor XIII from a recombinant host cell. In a particular embodiment the recombinant host cell is a recombinant yeast cell and the methods provide for the purification of yeast- produced recombinant human factor XIII.

The present invention provides purification methods that do not require a precipitation and/or a crystallization step. The present methods also do not require the use of expensive antibodies or monoclonal antibodies specific for factor XIII for purification of the factor. Within one typical embodiment a biological fluid comprising recoverable amounts of factor XIII is fractionated by immobilized metal affinity chromatography to produce a highly concentrated factor XIII product that does not contain proteins of the biological fluid that have been found in factor XIII product purified by other typical purification methods. In a particular embodiment, the biological fluid is a lysate from a recombinant yeast cell transformed to produce factor XIII wherein the methods of the present invention remove yeast proteins that typically remain with prior purification methods.

In a particular embodiment of the present invention immobilized metal affinity chromatography is combined with other affinity purification techniques. Typically, immobilized metal affinity chromatography is combined in any order with anion exchange and hydrophobic interaction fractionation to purify the factor XIII. Specifically in this embodiment a biological fluid comprising recoverable amounts of factor XIII is first partially purified by anion exchange fractionation prior to using a combination of immobilized metal affinity chromatography, hydrophobic interaction fractionation and anion exchange chromatography to complete the purification process. In a particular embodiment the biological fluid is first partially purified by anion exchange fractionation to produce a first fraction enriched for factor XIII; this first fraction is further fractionated by hydrophobic interaction fractionation to produce a second fraction enriched for factor XIII; subsequently the second enriched fraction is further fractionated by immobilized metal affinity chromatography to produce a third fraction enriched for factor XIII. This third fraction can optionally be further fractionated by anion exchange fractionation to produce a highly purified factor Xlll-containing peak fraction.

The biological fluid comprising recoverable amounts of factor XIII can include cell culture supernatants, cell lysates, clarified cell lysates, cell extracts, tissue extracts, blood, plasma, and fractions thereof. In a typical embodiment of the present invention the biological fluid is a cell lysate, particularly a cell lysate of a recombinant cell that has been engineered to produce factor XIII. Factor XIII of the present invention comprises any factor XIII including but not limited to a mammalian factor XIII, including in particular human factor XIII. Factor XIII can include polyprotein comprising the a2 dimer, comprising the a2b2 tetramer, and the like. Typically, the factor XIII is the a2 subunit dimer produced by a recombinant cell. The recombinant cell of the present invention can include bacterial, yeast and cultured mammalian cells. Typically, the yeast cells used for recombinant expression of factor XIII include those of the genus Saccharomyces, including for example, Saccharomyces cerevisiase, and species of Pichia and Kluyveromyces. In one particular embodiment of the present invention the anion exchange fractionation of the biological sample comprising factor XIII is carried out on a substrate, solid phase, or media that has been derivatized with DEAE. Prior to elution of the sample from the anion exchange medium the sample can be washed with a buffer solution comprising a density modifier, such as a sugar, e.g., sucrose, or glycerol to remove cellular debris. Hydrophobic interaction fractionation as used in the present invention comprises use of a chromatography media that has been derivatized with a butyl, phenyl or octyl group. In a particularly preferred embodiment the hydrophobic interaction chromatography media is derivatized with a phenyl group. Use of the chromatography media derivatized with a phenyl group has been found to require a lower salt buffer to elute the fractions comprising the largest amounts of factor XIII.

Immobilized metal affinity chromatography as used in the processes of the present invention comprises the use of chromatographic media that is charged with, for example Cu2+, Zn2+, or Ni2+. In a particular embodiment the chromatographic media charged with Ni2+ has been found to give the highest yield of factor XIII. Further, the factor XIII enriched fraction collected at each step of the disclosed process can be filtered to remove any particulates or aggregates that may have formed during each step in the process.

One particularly preferred embodiment of the present invention comprises fractionating the biological fluid by anion exchange with an anion exchange media, e.g., a DEAE derivatized chromatography media, to produce a first fraction enriched for factor XIII; this first fraction is further fractionated by hydrophobic interaction employing a phenyl derivatized chromatography media to produce a second fraction enriched for factor XIII; subsequently the second enriched fraction is further purified by immobilized metal affinity chromatography comprising a Ni2+ charged chromatography media to produce a third fraction enriched for factor XIII; the third fraction is finally fractionated by anion exchange with a QAE derivatized chromatography media to produce a purified factor Xlll-containing peak fraction. In a typical embodiment the factor XIII product produced by this process is at least 95% pure with respect to contaminating proteins. In particular embodiments, the product can comprise less than 5% yeast proteins. Further the product can comprise less than 1% activated factor XIII, less than 2% aggregates and/or can also comprise less than 5% charge isomers of factor XIII. Yield for the process can be at least about 45%.

A further understanding of the nature and advantages of the invention will become apparent by reference to the remaining portions of the specification. About CSL Behring

CSL Behring is a leader in the plasma protein therapeutics industry. Committed to saving lives and improving the quality of life for people with rare and serious diseases, the company manufactures and markets a range of plasma-derived and recombinant therapies worldwide. CSL Behring therapies are indicated for the treatment of coagulation disorders including hemophilia and von Willebrand disease, primary immune deficiencies, hereditary angioedema and inherited respiratory disease. The company's products are also used in cardiac surgery, organ transplantation, burn treatment and to prevent hemolytic diseases in newborns. CSL Behring operates one of the world's largest plasma collection networks, CSL Plasma. CSL Behring is a subsidiary of CSL Limited (ASX: CSL), a biopharmaceutical company headquartered in Melbourne, Australia . For more information, visit www.cslbehring .com. Contact:

Greg Healy

Senior Manager, Public Relations and Communications

U.S. Commercial Operations

610-878-4841

greg. healy@cslbehring.com

The recognition of factor deficiencies as the cause of hemophilias spurred investigations into the causes of other bleeding disorders and led to progress in understanding normal hemostasis. Knowledge of the fact that blood clots that are formed in the presence of calcium are stronger, insoluble in alkali, and resistant to proteolytic degradation led to the concept of insoluble clots in the earlier part of the last century. In 1948, Laki and Lorand recognized that a serum factor, termed fibrin stabilizing factor, was responsible for the characteristics of insoluble fibrin clots. [ 1] In 1960, Duckert et al described the first case of an "undescribed congenital haemorrhagic diathesis probably due to fibrin stabilizing factor deficiency," which was a description of the consequences of severe factor XIII (FXIII) deficiency.

The importance of FXIII in the process of coagulation is underscored by symptoms borne by patients who are homozygously deficient in FXIII or who have an antibody that disrupts FXIII function. Paradoxically, alterations in FXIII may predispose patients to thrombosis. Based on all available data, FXIII is clearly involved in the clot preservation side of the delicate balance between clot formation and stability and clot degradation. FXIII participates in other physiologic processes, including wound repair and healing. The many functions of FXIII and the disruptions of those functions by mutations in the genes coding for FXIII are the subjects of on-going investigations.

Gene polymorphisms are being evaluated for their influence on susceptibility to venous and arterial thromboembolism. Variants of coagulation factors, including factor XIII Val34Leu, have been implicated in influencing susceptibility to thromboembolic diseases.

There is a question as to whether factor XIII Val34Leu polymorphism is protective against idiopathic venous thromboembolism . The substitution of leucine for valine at amino acid position 34 of the factor XIII gene, commonly referred to as FXIII Val34Leu polymorphism, has been reported to confer protection against venous thromboembolism . However, the results of a recent study of white Canadian study population do not support an independent association of the FXIII Val34Leu polymorphism with idiopathic venous thromboembolism .

An association may exist between the factor XIII Leu allele and a modest protective effect against AMI and may provide useful information in profiling susceptibility to myocardial infarction.

Factor XIII has a variety of uses, potential and real. Plasma levels of factor XIII were found decreased in children with Henoch-Schonlein purpura having severe abdominal symptoms. Thus, it has been suggested that measurement of factor XIII level may be of value to detect the vasculitic process of Henoch-Schonlein purpura before the rash occurs or long after it has disappeared in patients with isolated abdominal or scrotal problems.

Immunohistochemistry may show factor XHIa (FXIIIa). FXIIIa-positive dermal dendritic cells were increased in a variety of skin tumors, including

dermatofibromas. Severe factor XIII deficiency, a rare autosomal recessive coagulation disorder, is associated with a relatively common prevalence of F13B gene defects, at least within the German population. The regions in and around the cysteine disulphide bonds in the FXIII-B protein at the sites of frequent mutations. FXIIIs aids immobilization and killing of bacteria as well as

phagocytosis by macrophages, likely functioning as part of the innate immune system . LOCUS HUMFXIIIB 3821 bp mRNA linear PRI 08-

NOV-1994

DEFINITION Human factor XIII subunit a mRNA, 3' end.

ACCESSION M14539

VERSION M14539.1 GI:182836

KEYWORDS factor XIII; fibrin; fibrinoligase ; glycoprotein; plasma transglutaminase .

SOURCE Homo sapiens (human)

ORGANISM Homo sapiens

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;

Euteleostomi ;

Mammalia; Eutheria; Euarchontoglires ; Primates; Haplorrhini; Catarrhini; Hominidae; Homo.

REFERENCE 1 (bases 1 to 3821)

AUTHORS Ichinose,A., Hendrickson, L . E . , Fujikawa, K. and Davie, E.W. TITLE Amino acid sequence of the a subunit of human factor XIII

JOURNAL Biochemistry 25 (22), 6900-6906 (1986)

PUBMED 3026437

COMMENT Original source text: Human placenta, cDNA to mRNA, clones lambda-HFXIIIa [2.14, 3.82] .

Draft entry and computer-readable sequence for [1] kindly provided

by A.Ichinose, 23-FEB-1987. The mature factor XIII is cleaved by

thrombin at a point between positions 201 and 202 to produce active

factor XIII-a.

FEATURES Location/Qualifiers

source 1..3821

/organism="Homo sapiens"

/mol_type="mRNA"

/db xref="taxon: 9606"

/map="6p25-p24"

gene 1..3821

/gene="F13Al"

CDS <1..2286

/gene="F13Al"

/note=" factor XIII precursor"

/codon_start=l

/protein id="AAA52489.1"

/db_xref="GI : 182837"

/db_xref="GDB: GOO- 120-614"

/translation="REEVPEAHRASPREGTSGGERLQDLVKSKMSETSRTAFGGRRAV

PPNNSNAAEDDLPTVELQGWPRGVNLQEFLNVTSVHLFKERWDTNKVDHHTDKYENN

KLIVRRGQSFYVQIDFSRPYDPRRDLFRVEYVIGRYPQENKGTYIPVPIVSELQSGK W

GAKIVMREDRSVRLSIQSSPKCIVGKFRMYVAVWTPYGVLRTSRNPETDTYILFNPW C

EDDAVYLDNEKEREEYVLNDIGVI FYGEVNDIKTRSWSYGQFEDGILDTCLYVMDRAQ MDLSGRGNPIKVSRVGSAMV AKDDEGVLVGSWDNIYAYGVPPSAWTGSVDILLEYRS

SENPVRYGQCWVFAGVFNTFLRCLGIPARIVTNYFSAHDNDANLQMDI FLEEDGNV S

KLTKDSVWNYHCWNEAWMTRPDLPVGFGGWQAVDSTPQENSDGMYRCGPASVQAIKH G

HVCFQFDAPFVFAEVNSDLIYITAKKDGTHVVENVDATHIGKLIV KQIGGDGMMDIT

DTYKFQEGQEEERLALETALMYGAKKPLNTEGVMKSRSNVDMDFEVENAVLGKDFKL S

ITFRNNSHNRYTITAYLSANITFYTGVPKAEFKKETFDVTLEPLSFKKEAVLIQAGE Y

MGQLLEQASLHFFVTARINETRDVLAKQKSTVL I PEII IKVRGTQWGSDMTVTIQF

TNPLKETLRNVWVHLDGPGVTRPMKKMFREIRPNSTVQWEEVCRPWVSGHRKLIASM S

SDSLRHVYGELDVQIQRRPSM"

(SEQ ID NO: 88)

siq peptide <1..90

/gene="F13Al"

/note=" factor XIII signal peptide"

mat peptide 91..2283

/gene="F13Al"

/product="factor XIII"

ORIGIN 199 bp upstream of Smal site.

1 cgggaggaag tccccgaggc gcacagagca agcccacgcg agggcacctc tggaggggag

61 cgcctgcagg accttgtaaa gtcaaaaatg tcagaaactt ccaggaccgc ctttggaggc

121 agaagagcag ttccacccaa taactctaat gcagcggaag atgacctgcc cacagtggag

181 cttcagggcg tggtgccccg gggcgtcaac ctgcaagagt ttcttaatgt cacgagcgtt

241 cacctgttca aggagagatg ggacactaac aaggtggacc accacactga caagtatgaa

301 aacaacaagc tgattgtccg cagagggcag tctttctatg tgcagattga cttcagtcgt

361 ccatatgacc ccagaaggga tctcttcagg gtggaatacg tcattggtcg ctacccacag

421 gagaacaagg gaacctacat cccagtgcct atagtctcag agttacaaag tggaaagtgg

481 ggggccaaga ttgtcatgag agaggacagg tctgtgcggc tgtccatcca gtcttccccc

541 aaatgtattg tggggaaatt ccgcatgtat gttgctgtct ggactcccta tggcgtactt

601 cgaaccagtc gaaacccaga aacagacacg tacattctct tcaatccttg gtgtgaagat

661 gatgctgtgt atctggacaa tgagaaagaa agagaagagt atgtcctgaa tgacatcggg

721 gtaatttttt atggagaggt caatgacatc aagaccagaa gctggagcta tggtcagttt

781 gaagatggca tcctggacac ttgcctgtat gtgatggaca gagcacaaat ggacctctct

841 ggaagaggga atcccatcaa agtcagccgt gtggggtctg caatggtgaa tgccaaagat

901 gacgaaggtg tcctcgttgg atcctgggac aatatctatg cctatggcgt ccccccatcg 961 gcctggactg gaagcgttga cattctattg gaataccgga gctctgagaa tccagtccgg

1021 tatggccaat gctgggtttt tgctggtgtc tttaacacat ttttacgatg ccttggaata

1081 ccagcaagaa ttgttaccaa ttatttctct gcccatgata atgatgccaa tttgcaaatg

1141 gacatcttcc tggaagaaga tgggaacgtg aattccaaac tcaccaagga ttcagtgtgg

1201 aactaccact gctggaatga agcatggatg acaaggcctg accttcctgt tggatttgga

1261 ggctggcaag ctgtggacag caccccccag gaaaatagcg atggcatgta tcggtgtggc

1321 cccgcctcgg ttcaagccat caagcacggc catgtctgct tccaatttga tgcacctttt

1381 gtttttgcag aggtcaacag cgacctcatt tacattacag ctaagaaaga tggcactcat

1441 gtggtggaaa atgtggatgc cacccacatt gggaaattaa ttgtgaccaa acaaattgga

1501 ggagatggca tgatggatat tactgatact tacaaattcc aagaaggtca agaagaagag

1561 agattggccc tagaaactgc cctgatgtac ggagctaaaa agcccctcaa cacagaaggt

1621 gtcatgaaat caaggtccaa cgttgacatg gactttgaag tggaaaatgc tgtgctggga

1681 aaagacttca agctctccat caccttccgg aacaacagcc acaaccgtta caccatcaca

1741 gcttatctct cagccaacat caccttctac accggggtcc cgaaggcaga attcaagaag

1801 gagacgttcg acgtgacgct ggagcccttg tccttcaaga aagaggcggt gctgatccaa

1861 gccggcgagt acatgggtca gctgctggaa caagcgtccc tgcacttctt tgtcacagct

1921 cgcatcaatg agaccaggga tgttctggcc aagcaaaagt ccaccgtgct aaccatccct

1981 gagatcatca tcaaggtccg tggcactcag gtagttggtt ctgacatgac tgtgacaatt

2041 cagtttacca atcctttaaa agaaaccctg cgaaatgtct gggtacacct ggatggtcct

2101 ggagtaacaa gaccaatgaa gaagatgttc cgtgaaatcc ggcccaactc caccgtgcag

2161 tgggaagaag tgtgccggcc ctgggtctct gggcatcgga agctgatagc cagcatgagc

2221 agtgactccc tgagacatgt gtatggcgag ctggacgtgc agattcaaag acgaccttcc

2281 atgtgaatgc acaggaagct gagatgaacc ctggcatttg gcctcttgta gtcttggcta

2341 aggaaattct aacgcaaaaa tagctcttgc tttgacttag gtgtgaagac ccagacagga

2401 ctgcagaggg ccccagagtg gagatcccac atatttcaaa aacatgcttt tccaaaccca

2461 ggctattcgg caaggaagtt agtttttaat ctctccacct tccaaagagt gctaagcatt

2521 agctttaatt aagctctcat agctcataag agtaacagtc atcatttatc atcacaaatg

2581 gctacatctc caaatatcag tgggctctct taccagggag atttgctcaa tacctggcct

2641 catttaaaac aagacttcag attccccact cagccttttg ggaataatag cacatgattt 2701 gggctctaga attccagtcc cctttctcgg ggtcaggttc taccctccat gtgagaatat

2761 ttttcccagg actagagcac aacataattt ttatttttgg caaagccaga aaaagatctt

2821 tcattttgca cctgcagcca agcaaatgcc tgccaaattt tagatttacc ttgttagaag

2881 aggtggcccc atattaacaa attgcatttg tgggaaactt aaccacctac aaggagataa

2941 gaaagcaggt gcaacactca agtctattga ataatgtagt tttgtgatgc attttataga

3001 atgtgtcaca ctgtggcctg atcagcagga gccaatatcc cttactttaa ccctttctgg

3061 gatgcaatac taggaagtaa agtggaagaa tttatctctt tagttagtga ttatatttca

3121 cccatctctc aggaatcatc tcctttgcag aatgatgcag gttcaggtcc cctttcagag

3181 atataataag cccaacaagt tgaagaagct ggcggatcta gtgaccagat atatagaagg

3241 actgcagcca ctgattctct cttgtccttc acatcaccca tgttgagacc tcagcttggc

3301 actcaggtgc tgaagggtaa tatggactca gccttgcaaa tagccagtgc tagttctgac

3361 ccaaccacag aggatgctga catcatttgt attatgttcc aaggctacta cagagaaggc

3421 tgcctgctat gtatttgcaa ggctgattta tggtcagaat ttccctctga taagtctagg

3481 gtgtgattta ggtcagtaga ctgtgattct tagcaaaaaa tgaacagtga taagtatact

3541 gggggcaaaa tcagaatgga atgctctggt ctatataacc acatttctaa gcctttgaga

3601 ctgttcctga gccttcagca ctaacctatg agggtgagct ggtcccctct atatatacat

3661 catacttaac tttactaagt aatctcacag catttgccaa gtctcccaat atccaatttt

3721 aaaatgaaat gcattttgct agacagttaa actggcttaa cttagtatat tattattaat

3781 tacaatgtaa tagaagctta aaataaagtt aaactgatta t

(SEQ ID NO: 89)

Human Factor XIII b subunit gene, complete cds

LOCUS HUMBFXIII 33206 bp DNA linear PRI 31-OCT-1994

DEFINITION Human factor XIII b subunit gene, complete cds.

ACCESSION M64554 J05294

VERSION M64554.1 GI : 179416

KEYWORDS blood coagulation factor; factor XIII; factor XHIb; zymogen. SOURCE Homo sapiens (human)

ORGANISM Homo sapiens

Eukaryota; Metazoa; Chordata ; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini;

Catarrhini; Hominidae; Homo.

REFERENCE 1 (bases 1 to 33206)

AUTHORS Bottenus,R.E., Ichinose,A. and Davie,E.W. TITLE Nucleotide sequence of the gene for the b subunit of human factor XIII

JOURNAL Biochemistry 29 (51), 11195- 11209 (1990)

PUBMED 2271707

REFERENCE 2 (sites)

AUTHORS Nishimura ₍ D.Y., Leysens,NJ. and Murray,J.C.

TITLE A dinucleotide repeat for the D1S53 locus

JOURNAL Nucleic Acids Res. 20 (5), 1167 (1992)

PUBMED 1549502

FEATURES Location/Qualifiers

source 1..33206

/organism = "Homo sapiens"

/moLtype="genomic DNA"

/db_xref="taxon :9606"

/map= "6p25-p24"

/ce I Ltype = "fibroblast, leu kocyte "

/tissue_type="liver"

/dev_stage= "fetus"

gene join(<2931..2994,7093..7293,8181..8366,9073..9249,

9606..9782, 12684..12863, 12951..13136,14251..14433, 17308..17508,19264..19446,29399..29612,30723..30943) /gene= "F13Al"

mRNA join(<2931..2994,7093..7293,8181..8366,9073..9249,

9606..9782, 12684..12863, 12951..13136,14251..14433, 17308..17508,19264..19446,29399..29612,30723..30943) /gene="F13Al"

/product="coagulation factor XHIb"

/note="G00-120-614"

CDS join(2931..2994,7093..7293,8181..8366,9073..9249,

9606..9782, 12684..12863, 12951..13136, 14251..14433, 17308..17508,19264..19446,29399..29612,30723..30756) /gene="F13Al"

/codon_start= l

/product="coagulation factor XHIb"

/ p rote i n_i d = "AAA51821.1"

/db_xref="GI : 179417"

/db_xref="GDB: G00- 120-614"

/translation = "MRLKNLTFIIILIISGELYAEEKPCGFPHVENGRIAQYYYTFKS FYFPMSIDKKLSFFCLAGYTTESGRQEEQTTCTTEGWSPEPRCFKKCTKPDLSNGYIS

DVKLLYKIQENMHYGCASGYKTTGGKDEEVVQCLSDGWSSQPTCRKEHETCLAPELY N GNYSTTQKTFKVKDKVQYECATGYYTAGGKKTEEVECLTYGWSLTPKCTKLKCSSLRL IENGYFHPVKQTYEEGDVVQFFCHENYYLSGSDLIQCYNFGWYPESPVCEGRRNRCPP

PPLPINSKIQTHSTTYRHGEIVHIECELNFEIHGSAEIRCEDGKSTEPPKCIEGQEK V ACEEPPFIENGAANLHSKIYYNGDKVTYACKSGYLLHGSNEITCNRGKWTLPPECVEN NENCKHPPVVMNGAVADGILASYATGSSVEYRCNEYYLLRGSKISRCEQGKWSSPPVC LEPCTVNVDYMNRNNIEMKWKYEGKVLHGDLIDFVCKQGYDLSPLTPLSELSVQCNRG EVKYPLCTRKESKGMCTSPPLIKHGVIISSTVDTYENGSSVEYRCFDHHFLEGSREAY

CLDGMWTTPPLCLEPCTLSFTEMEKNNLLLKWDFDNRPHILHGEYIEFICRGDTYPA E

LYITGSILRMQCDRGQLKYPRCIPRQSTLSYQEPLRT"

(SEQ ID NO: 90) Items

1. recombinant fibrinogen made in high producer non-embryonic cell lines such as placentar cells, umbilical cord cells umbilical cord blood cells, amnion cells.

2. recombinant factor XIII made in high producer non-embryonic cell lines such as placentar cells, umbilical cord cells umbilical cord blood cells, amnion cells

Deposit of biological material

Transformed primary human amniocytes have been deposited in the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) Mascheroder Weg lb, D-38124 Braunschweig on 26 October 1999DSM under accession number DSM ACC2416.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety. The invention will now be described in further details in the non-limiting examples below.

Figures

Figure 1 show the PreProthrombin Structure (Degen & Davie (1987). Gla-domain containing human prothrombin as being transferred via vector system into placenta cell line(s) and into amnion cells.

Figure 2 shows an overview of the structure of prothrombin M256A including the domains HPC4, Kringle 2, and Peptidase S. In addition, the site of the point mutation M256A is indicated. Figure 3 shows an overview of the structure of prothrombin M400A including the domains Gla, Kringle 1 , Kringle 2, and Peptidase S. In addition, the site of the point mutation M256A is indicated .

Figure 4 shows an overview of the the individual expressions such as

prothrombins (either gla-domain (containing) prothrombin, gla-domainless prothrombin as used by Zymogenetics for their production of recombinant human thrombin, prethrombin, and prethrombin-1, Fig. 4 provides an overview over these individual terms. As illustrated in Fig. 4, prethrombin = prothrombin without gla domain and without kringle I domain (Gla-less domainless prothrombin). To facilitate purification HPC4 epitope was added on N-terminaf. So, the first part of Fig. 4 shows the prothrombin including the Gla domain, and the Kringle 1 and Kringle 2. Furthermore, Fig .4 illustrates the method of preparing recombinant human prethrombin starting with non-mutated prothrombin. Figure 5 shows pHZsec vector map and Srf I site.

Figure 6 Both prothrombin and preprothrombin of wild type and mutant

M400A/M84A were cloned into the pHZsec vector at Srf I site. Figure 7 shows Restriction Map (Nde I & Hind III).

Figure 8 shows Restriction Map (Nde I & Hind III).

Figure 9 shows Restriction Map (Nde I & Hind III) Figure 10 shows CEVEC ^' s CAP cell system

Figure 11-13 shows Restriction Map (Nde I & Hind III)

Figure 14 shows a point mutation (The312 to Ala312) in fibrinogen alpha chain. Both cloned fibrinogen alpha chains from NM_021871 (FbgAa_OBS) and UNC Fb a p584 (FbgAa_UNC) have Ala312 instead of Thr312 reported public database as highlighted in yellow in Ref22.

Figure 15 shows a recombinant crude fibrinogen (cell culture supernatant on the left and concentrate using ultrafiltration at room temperature and left at 4° C for a few days. A noticeable molecular weight shif was noted, indicating degradation Figure 16 shows cocktail of protease inhibitors added in CHO cell culture in UNC.

Figure 17 shows initial screening of rhFbg capture on IMAC column charged with Cu2+. rhFbg culture medium (1) was applied on the IMAC and flow through (2) and wash (3) fraction did not contain fibrinogen. rhFbg was found in the elution with 50mM Arg fraction (4) and the culture medium was concentrated.

Figure 18 shows rhFbg purification from the culture medium using IMAC and Heparin. 20mM Arg fraction contained degraded fibrinogens that are missing alpha chain under reducing condition. Detail structure was not further studied.

Figure 19 shows determination of fibrinogen clottability and an example.

Figure 20 shows the polymerization of wt. Rhfibrinogen fraction obtained from HEK cells, purified and eluated with 20 mM arginine (named Fbg (F5) with lower polymerization than the fraction Rhfibrinogen fraction eluated with 50 mM fibrinogen, indicating that one fraction of purified rh fibrinogen (F5) most probably is defect, when compared to fraction F6. As comparison Calbiochem's plasma fibrinogen is the curve showing the lowest polymerization curve, fraction F5 is located as the middle polymerization curve and F6 as the highest polymerisation curve. Figure 20. Polymerization of 3 different fibrinogens, where of fibrinogen F5 anf fibrinogen F6 is harvested and purified from HEK cells. Recombinant fibrinogen (F5) eluated with 20 mM arginine corres. ^{Flbrin09en F5} :rization effect compared to recombinant fibrinogen (F6) eluated with 50 mM arginine from the same batch of crude fibrinogen from HEK cells. Polymerisation of plasma fibrinogen (CalbioCh.) is showing almost the same polymerization as Fibrinogen F6.

Figure 21 shows SDS PAGE gel electrophoresis of purified recombinant fibrinogen F5 from HEK cells eluated with 20 mM arginine and recombinant fibrinogen F6 from HEK cells eluated with 50 mM arginine

Figure 21 Purification of recombinant fibrinogen from HEK cells showed that during the purification process, when eluating the part of the recombinant fibrinogen, using 20 mM arginine (left portion with biomarker and two rows of bands) for the eluation showed under non-reduced condition a diffuse fibrinogen band, whereas when the eluation wat done at 50mM Arginine (the left portion with biomarker and two rows of bands) a more distinct non-reduced fibrinogen band appeared., when viewing the reduced SDS Page electrophoresis it is obvious that the alpha band, the uppermost band eluated using 20 mM arginine was significantly more diffuse than the sharper alpha band in the reduced fibrinogen eluated by 50 mM. The first

21B.SDS PAGE gel electrophoresis Marker lane, and # 1. lane Plasma-Fbg, non reduced and reduced #2. CHO cell non-reduced and reduced, #3.F6 nonreduced and reduced, #4. F5 Fbg, nonreduced and reduced. 5. "Proline" Fbg nonreduced and reduced, the one that showed inferior polymerisation at least 1 log lower than the one we are working with at CEVEC and the one that probably in density corresponds to F6. The same pattern when comparing F6 to F5, F6 has a more dense alpha band than F5. CHO cell alpha band is also less significant. The alpha band in the ": proline" recombinant fibrinogen, which did show at least 1 log lower clotting than F6 alpha, which is due to an amino acid shift in the beta chain, has the same intensity in the alpha band as has F6.

Figure 21 B. 1. P-Fbg, non reduced and reduced 2. CHO cell unreduced and reduced, 3.F6 unreduced and reduced, 4. F5 Fbg, unreduced and reduced. 5. "Proline" Fbg unreduced and reduced The same pattern when comparing F6 to F5, F6 has a more dense alpha band than F5. CHO cell alpha band is also less significant. The alpha band in the ": proline" recombinant fibrinogen, which did show at least 1 log lower clotting than F6 alpha, which is due to an amino acid shift in the beta chain, has the same intensity in the alpha band as has F6.

Figure 22A shows that in the high concentration of fibrinogen (1 mg/ml), a comparable clotting time of CalbioChem hpFbg (human plasma fibrinogen when clotted with HCI and M400a at 12 U/ml but decreasing the concentration of the thrombins showed a little faster clotting time using the M400a thrombin from HumaGene. The rhFbg, which in this case corresponds to the fibrinogen with the error in prolin in the beta chain had a significantly slower clotting time compared to the hpFbg. Figure 22B. The lower bands in orange and blac, shown the clotting assay at a concentration of 0.38 mg fibrinogen shows the clotting effect actually being essentially equal to Calbiochem's plasma fibrinogen. This recombinant fibrinogen has been changed in the beta chain from proline to alanine . Figure 23 shows chicken fibrinogen structure (1EI3) with yellow highlighted beta chain. Prol62 site is close to beta and gamma bundle. Interestingly chicken fibrinogen beta chain has Proline too.

Figure 24 shows tabel from Hematologic Technologies Inc.

Figure 25 shows Factor XIII crosslinks fibrin

Figure 26 shows that activation of factor XIII (FXIII) by thrombin and calcium is a 2-step process.

Figure 27 shows plasmid maps of Fibrinogen alpha into pStbl-Neo-CMV-MCS (-) (G418iNeomycin Resistance), Fibrinogen beta into pStbl-Bsd-CNIV-MCS 1-) (Blasticidin Resistance), Fibrinogen gamma into pStbl-Hygro-CMV-MCS (-) (Hygromycin Resistance) Figure 28 shows an overview of the different generated CAP cell polls.

Figure 29 shows a Western Blot under non-reducing conditions with supernatants from 26/4-2013.

5

Figure 30 shows a Western Blot under reducing conditions with supernatants from 26/4-2013.

Figure 31 shows a Western Blot under reducing conditions with supernatants from 10 29/4-2013 and 3/5-2013.

Figure 32 shows the numer of viable cells and cell viability during the pool generation of pool 3.

15 Figure 33 shows single cell cloning.

Figure 34 shows fed-batch culture of selected fibrinogen pools and number of viable cells.

Figure 35 shows shows fed-batch culture of selected fibrinogen pools - viability

20

Figure 36 shows single cell cloning 19 days post limiting dilution

Figure 37 shows single cell cloning 27 days post limiting dilution

25 Figure 38 shows Western blot of individual fibrinogen expressing CAP single cell clones

Figure 39 shows single cell cloning : proposed work packages.

30 Figure 40 shows single cell cloning - fed batch culture in 24 deep well plates, pool 1 - Cv.

Figure 41 shows single cell cloning - fed batch culture in 24 deep well plates, pool 1 - productivity. Figure 42 shows single cell cloning - fed batch culture in 24 deep well plates, pool 5 - Cv. Figure 43 shows single cell cloning - fed batch culture in 24 deep well plates, pool 5 - Productivity.

Figure 44 shows single cell cloning - top 9 clones cell viability. Figure 45 shows single cell cloning - top 9 number of viable cells. Figure 46 shows single cell cloning - top 9 clones fibrinogen titer. Figure 47 shows single cell cloning - top 9 clones western blot analysis.

Figure 48 shows western analysis on the conditioned medium with mAb-HPC4.

Figure 49 shows lane 1 : Ecarin activated alpha thrombin mixture, Lane 2 shows the Flow— thru, Lane 3 shows Non-specific contaminant(s).

Examples

Example 1

Production of recombinant human fibrinogen

Amniocyte Production Technology is a transient and stable protein expression covering early development up to clinical production.

Efficient production of complex proteins and antibodies High and consistent product quality with authentic PTM Easy-to-handle optimized expression system Ethically obtained human cell line with fully documented history. CAP cells

- Human cell line, adapted to serum-free suspension culture

- Optimized for expression of secreted proteins

- Optimized for stable transfection and selection CAP-T cells

- Derived from CAP cells

- Express 5\M large T antigen

- Optimized for transient gene expression Generation of Fibrinogen Expressing CAP Cell Pools

- Cloning of the three different Fibrinogen chains (alpha, beta, gamma)

- Fibrinogen alpha into pStbl-Neo-CMV-MCS (-) (G418iNeomycin Resistance)

- Fibrinogen beta into pStbl-Bsd-CNIV-MCS (-) (Blasticidin Resistance)

- Fibrinogen gamma into pStbl-Hygro-CMV-MCS (-) (Hygromycin Resistance) - The correct sequences of the Fibrinogen inserts were verified by close- meshed sequencing of the resulting pStbl plasmids.

- The plasmid maps may be seen in figure 27.

Transfection of cloned Fibrinogen alpha, beta and gamma constructs into CAP cells by two different transfection methods Ceve's proprietary transfection method and nucleofection. Transfection of Fibrinogen alpha, beta and gamma constructs into CAP cells using different ratios between the three Fibrinogen chains:

1 : 1 : 1 - 1 : 2: 1

- 1 : 4: 1

- 2.4 x (l : l : l) An overview of the different generated CAP cell polls can be seen in figure 28. Line of action

Successfully transfected cells were first selected by blasticidin treatment - subsequently cells were treated either with Blasticidin and Neomycin (G418) or Blasticidin and Hygromycin -> finally, cells were be treated with all three selection reagents ->To monitor the expression of the different Fibrinogen proteins western blot analysis was performed - the used polyclonal antibody recognizes

Fibrinogen alpha, beta, and gamma - For each lane 12 pi cell free (150g for 6 min.) cell culture supernatant was used. In figure 29 a Western Blot under non- reducing conditions with supernatants from 26/4-2013 can be seen. In figure 30 a Western Blot under reducing conditions with supernatants from 26/4-2013 can be seen. In figure 31 shows a Western Blot under reducing conditions with supernatants from 29/4-2013 and 3/5-2013 can be seen. Single cell cloning

Two pools were chosen for single cell cloning

Pool 3 : treated with Blasticidin, G418, and Hygromycin

Pool 5: treated with Blasticidin and G418 Three pools were chosen for single cell cloning

Pool 3 : treated with Blasticidin, G418, and Hygromycin

Pool 5 : treated with Blasticidin and G418

In addition : Pool 1: treated with Blasticidin, G418, and Hygronnycin In Figure 32 the number of viable cells and cell viability during the pool generation of pool 3 can be seen.

Figure 33 shows single cell cloning.

Pool 1 : Cevec's proprietary transfection method, plasmid ration 1: 1 : 1 -> 8 plates. Pool 3: Cevec's proprietary transfection method, plasmis ration 1 : 4: 1 -> 4 plates. Pool 5 : Nucleofection, plasmis ration 2.4 x 1 : 1 : 1 -> 8 plates.

Fed-batch Culture of the Different Pools to Determine Productivity

- Fed-batch in CAP-CDM media and feed for 14 days

- Western blot analysis of the cell culture supernants collected during the batch/fed batch culture to determine Fibrinogen quality

- ELISA to determine product titers

Figure 34 shows fed-batch culture of selected fibrinogen pools and number of viable cells. Figure 35 shows shows fed-batch culture of selected fibrinogen pools

- viability, Figure 36 shows single cell cloning 19 days post limiting dilution, Figure 37 shows single cell cloning 27 days post limiting dilution and

Figure 38 shows Western blot of individual fibrinogen expressing CAP single cell clones. Figure 39 Single cell cloning : proposed work packages.

Single Cell Cloning by Limiting Dilution Fed-Batch Culture of 60 Clones in

24 Deep Well Plates. After limiting dilution of two stably expressing recombinant fibrinogen CAP cell pools, altogether 196 single cell clones (from both pools) were shifted from 96 well plates into 24 well plates and the fibrinogen titer for each clone was determined by ELISA (data not shown). The top 60 clones with the highest fibrinogen titers were selected for a HT S fed-batch culture in a 24 deep well plate with a volume of 2 ml at 185 rpm, 37°C and 5% CO ² . Culture were fed at day 4, 7, 9, and 11. Productivity during the fed-batch culture for each clone was determined by ELSIA.

Figure 40 shows single cell cloning - fed batch culture in 24 deep well plates, pool 1 - Cv, Figure 41 shows single cell cloning - fed batch culture in 24 deep well plates, pool 1 - productivity, Figure 42 shows single cell cloning - fed batch culture in 24 deep well plates, pool 5 - Cv and Figure 43 shows single cell cloning - fed batch culture in 24 deep well plates, pool 5 - Productivity.

Summary:

60 fibrinogen expressing CAP single cell clones were screened. 9 of these clones reached, in theis suboptimal HTS setting, fibrinogen titres above 15 mg/L.

Interestingly, most of these high expressing clones are slow growing, reaching highest cell number only after 120 h. Thus, we postulated that in a small scale production run in shaking flasks with a daily feeding regime cell numbers and therefore fibrinogen titers could be futher enhanced. For that reason we choose to perform the small scale production round with the 9 top clones with feed instead of 4 clones with and without feed.

Further analysis of the yop 9 clones, fed batch small scale run. Growth curve and analysis of productivity

9 top clones chosen from the 24 deep well plate analysis were further evaluated by a fed batch cultivation in a 125 shaking flask (30ml scale) at 185 rpm, 37 °C and 5% CO ² , culture were fed daily from day 3 to day 10. Cell viability and number of viable cells were determined daily from day 3 to day 14 in addition cell culture supernatants were collected for determination of fibrinogen titres. ELISA was performed using the fibrinogen Cencor sent as standard (Pool 2 PSM 1290). Figure 44 shows single cell cloning - top 9 clones cell viability, Figure 45 shows single cell cloning - top 9 number of viable cells and Figure 46 shows single cell cloning - top 9 clones fibrinogen titer.

Summary: Small Scale Production Run at a 30 ml Scale. Out of the 9 top clones. 1A5 is the top producer with nearly 100 mg/L in a fed-batch run over 14 days. The other top clones are 2D10, 16A5 and 16F6. As 1A5, the top clone, is still a more slow growing cell line, which just reach I x 10 ⁷ cells/rnl during the fed batch culture 4 it could be hypothesized that with proper upstream process development titers could be significantly increased .

Figure 47 shows single cell cloning - top 9 clones western blot analysis.

Purification of fibrinogen :

• The first step consists of loading the IMAC CU colum where recombinant Fibrinogen is bound to the IMAC CU column when equilibrated with lOmM

Tris-HCI, pH7.4, 50mM NaCI.

• The fibrinogen is eluted was eluted with lOmM Tris-HCI, pH7.4, 50mM

NaCI, 50mM arginine, range 20mM - 50 mM arginine.

• The eluted fibrinogen is then applied onto a Heparin FF column. • The fibrinogen is then eluted with Tris-based saline (TBS or lOmM Tris-HCI, pH7.4, 150mM NaCI) with a purity greater than 95% pure

• No other buffer exchange is needed since the elution buffer already is a physiological buffer condition

· The purified recombinant fibrinogen is then either frozen at -20°C for later procedures such as freeze drying .

• Freeze-drying is then performed in aliquots of 1 to 5 gram per final vial (bottle) and can then be stored at temperatures ranging from freezing to 25 °C.

References

Maurer et al. 2010, Thromn Res. 2010; 125(4) : 287-291, Emerging roles of fibronectin in thrombosis. Rahe-Meyer et al. 2013, Anesthesiology 2013, 118: 40-50, Effects of Fibrinogen Concentrate as First-line Therephy during Major Aortic Replacement Surgery.

US20070111312

US20120040400

Degen SJ, Davie EW. Nucleotide sequence of the gene for human prothrombin.

Biochemistry. 1987 Sep 22;26(19) :6165-77.

Steam s-Kurosowa DJ, Kurosowa S, Mollica JS et al. The endothelial cell protein C receptor augments protein C activation by the throm-hin-thrombomodulin complex. Proc. Natl. Acad Sci USA. 1996,93: 10212-10216.

Bernd Voedisch, Protein Congress 2012, London 02 April, 2012, Applicability of CAP-T cells to TGE for research purposes, in which it was described that CAP-T™ cells stably express intracellular T-Antigen of Simian Virus 40 (SV40). An optimized plasmid contains an expression casettet for the gene of interest and the SV40 origin of replication (S40ori).

Schiedner G, Hertel S, Biaiek C, Kewes H, Waschutza G and Volpers C, Efficient and reproducible generation of high-expressing, stable human cell lines without need for antibiotic selection. BMC Biotechnology 2008a, 8: 13. Doi: 10.1186/1472- 6750-8.13

Sequence listing

SEQ ID Description

NO

1 Primer: HZsec-Fibrinogen a-F

2 Primer: HZsec-Fibrinogen a-R

3 Fibrinogen Alpha chain (clone: NM_021871)

Amino acid sequence

4 Fibrinogen Alpha chain (clone: NM_021871)

Polynucleotide sequence

5 Primer: HZsec-Fibrinogen b-F

6 Primer: HZsec-Fibrinogen b-R

7 Fibrinogen beta chain (clone: NM 106760)

Amino acid sequence

8 Fibrinogen beta chain (clone: NM 106760)

Polynucleotide sequence

9 Primer: HZsec-Fibrinogen r-F

10 Primer: HZsec-Fibrinogen r-R

11 Fibrinogen gamma chain (clone: BC21674)

Amino acid sequence

12 Fibrinogen gamma chain (clone: BC21674)

Polynucleotide sequence

13 Amino acid sequence of BC051332 (human prothrombin) - M400A

14 Polynucleotide sequence of BC051332 (human prothrombin) - M400A

15 Amino acid sequence of BC051332 (human prothrombin) - WT

16 Polynucleotide sequence of BC051332 (human prothrombin) - WT

17 Amino acid sequence of BC051332 (human prethrombin) - M84A

18 Polynucleotide sequence of BC051332 (human prethrombin) - M84A

19 Amino acid sequence of BC051332 (human prethrombin) - M84A with

HPC4

20 Polynucleotide sequence of BC051332 (human prethrombin) - M84A with HPC4 Amino acid sequence of human tissue factor

Polynucleotide sequence of human tissue factor

Amino acid sequence of human labile factor

Polynucleotide sequence of human labile factor

Amino acid sequence of human fibrinogen stabilizing factor, Al polypeptide

Polynucleotide sequence of human fibrinogen stabilizing factor, Al polypeptide

Amino acid sequence of human fibrinogen stabilizing factor, B polypeptide

Polynucleotide sequence of human fibrinogen stabilizing factor, B polypeptide