FACTOR IN A BIOREACTOR

RELATED APPLICATION DATA

The present application claims priority from European Patent Application No.

EP14172338.7 filed June 13 2014, the entire contents of which are hereby incorporated by reference.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates to improved methods for the recombinant production of von Willebrand factor (vWF) in a bioreactor by separating different species of vWF multimers during fermentation.

BACKGROUND

Biotechnology offers the promise of producing low cost biopharmaceutical products, including recombinant proteins. Many therapeutic proteins, such as coagulation factors, have therapeutic potential if they can be produced recombinantly in a way that retains their biological activity, and produces sufficient yields to be commercially viable. For instance, recombinantly produced coagulation factors such as von Willebrand Factor (vWF) have the potential to treat a wide variety of bleeding disorders. However, this potential has not been adequately met partly because of the inherent complexity of naturally-occurring biological molecules and the variety of limitations associated with the synthesis of their recombinant protein counterparts in genetically engineered cells. Therefore, a strong need exists in the art for improved methods of producing recombinant, highly biologically active therapeutic proteins such as vWF.

Recombinant therapeutic proteins are usually expressed in eukaryotic cells which are cultivated in bioreactors of varying size. Several modes of operating such bioreactors are known in the field like fed-batch processes and perfusion processes. In such fermentation processes it is desirable to maintain high cell viability and cell density in order to obtain the desired recombinant protein in a concentrated form which facilitates subsequent purification. WO 2008/006494 discloses a perfusion process in which the accumulation of the desired therapeutic protein in the cell culture supernatant is achieved to high concentrations by feeding culture media components to the cell culture in a bioreactor and wherein the cell culture comprising the cells, the therapeutic protein and the cell culture medium is circulated over a separation system wherein the separation system separates the therapeutic protein from substances of lower molecular weight than the therapeutic protein like from metabolites. The molecular weight cut-off (MWCO) of the substances to be separated from the therapeutic protein of interest in WO 2008/006494 is between 5,000 Da to 500,000 Da preferably at most 100,000 Da.

EP2171034 discloses a method of producing a biopolymer in a continuous fermentation process wherein an impurity filter is used which allows impurities with a molecular weight below the molecular weight of the biopolymer of interest to be removed from the biopolymer of interest in the bioreactor. This patent also discloses a continuous fermentation process in which a combination of a filter unit equipped with an impurity filter is combined with a filter unit equipped with a product filter which will let the biopolymer of interest pass in order to harvest the biopolypeptide of interest. Impurity filter pore sizes of up to 80,000 Da are described whereby the preferred range is 2,000 Da to 15,000 Da. As biopolymers of interest only polypeptides with a given molecular weight are described. The largest biopolymer of interest disclosed is Factor VIII having a molecular weight of about 280,000 Da.

BRIEF DECRIPTION OF THE DRAWINGS

Figure 1 shows the ratio of vWF Ristocetin Cofactor activity over vWF antigen (vWF:RCoF/ vWF:Ag) in the bioreactor during the batch process.

Figure 2 shows the ratio of vWF Ristocetin Cofactor activity over vWF antigen (vWF:RCoF/ vWF:Ag) in the retentate (bioreactor) and in the permeate during the process of Example 2. Figure 3 shows the accumulation of HMW vWF in the retentate (bioreactor) and the depletion of HMW vWF in the permeate at day 4 in Example 2. The accumulated pixel intensities related to standard human plasma of bands 1-5, 6-10 and >11 were determined as described in Example 2. Figure 4 shows the ratio of vWF Ristocetin Cofactor activity over vWF antigen (vWF:RCoF/ vWF:Ag) in the retentate (bioreactor) and in the permeate during the process of Example 3. Figure 5 shows the accumulation of HMW vWF in the retentate (bioreactor) and the depletion of HMW vWF in the permeate at day 9 in Example 3. The accumulated pixel intensities related to standard human plasma of bands 1-5, 6-10 and >11 were determined as described in Example 2. Figure 6 shows a schematic drawing of the hypothetical process setup of Example 4.

Figure 7 shows a schematic drawing of the hypothetical process setup of Example 5.

Figure 8 shows the accumulation of HMW vWF in the retentate (bioreactor) and the depletion of HMW vWF in the permeate at day 7 of Example 6. The accumulated pixel intensities related to standard human plasma of bands 1-5, 6-10 and >11 were determined as described in Example 2.

Figure 9 shows the ratio of vWF Ristocetin Cofactor activity over vWF antigen (vWF:RCoF/ vWF:Ag) in the retentate (bioreactor) and in the permeate during the process of Example 6 as described in Example 2.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the surprising discovery that a separation system can be used in a bioreactor during a fermentation process to separate certain multimeric forms of vWF from other multimeric forms of vWF. Such different multimeric forms of vWF with different molecular weight do have different properties and/or different biological activities. Such separation already taking place during the up-stream fermentation step of manufacturing greatly reduces the purification effort in downstream processing.

Embodiments of the present invention therefore include methods for producing a recombinant von Willebrand factor (vWF).

One embodiment of the present invention is a process for manufacturing a recombinant von Willebrand factor (vWF) by culturing host cells in a bioreactor in a cell culture medium, wherein the host cells produce recombinant vWF which is secreted into the cell culture medium and wherein the vWF in the cell culture medium comprises vWF multimers of different size,

wherein at least one cell culture medium component is fed to the cell culture medium and wherein the cell culture comprising the cells, the recombinant vWF and the cell culture medium is pumped over a separation system and wherein the separation system separates the vWF multimers into at least

a permeate fraction which is enriched in low molecular weight (LMW) multimers of vWF and reduced in high molecular weight (HMW) multimers of vWF as compared to the vWF multimers in the cell culture supernatant before separation and

a retentate fraction which is reduced in low molecular weight (LMW) multimers of vWF and enriched in high molecular weight (HMW) multimers of vWF as compared to the vWF multimers in the cell culture supernatant before separation

In one embodiment of the invention the two fractions of the vWF do have different biological activities.

In another embodiment of the invention the desired fraction of the recombinant vWF intended to be purified further is the permeate.

In another embodiment the recombinant VWF in the permeate is enriched in low molecular weight (LMW) vWF multimers and reduced in high molecular weight (HMW) vWF multimers, wherein the LMW vWF multimers correspond to bands 1 to 5 and the HMW vWF multimers correspond to bands 11 and higher in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330).

In another embodiment of the invention the LMW vWF Multimer Ratio in the permeate is equal to or below 0.9, or in other words the ratio of the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 1 to 5 in the retentate divided by the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 1 to 5 in the permeate is below 0.9, wherein the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 1 to 5 HMW refers to the numerical value which is obtained when the amount of bands 1 to 5 as determined in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330) is divided by the amount of bands 1 to 5 of a standard human plasma as determined in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330). Preferentially standard human plasma (SHP) (Siemens, Standard Human Plasma, ORKL17) is used.

In another embodiment of the invention the desired fraction of the recombinant vWF intended to be purified further is the retentate. In another embodiment of the invention the HMW vWF Multimer Ratio in the retentate is equal to or above 1.1, or in other words the ratio of the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 11 and higher in the retentate divided by the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 11 and higher in the permeate is above 1.2, wherein the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 11 and higher refers to the numerical value which is obtained when the amount of bands 11 and higher as determined in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330) is divided by the amount of bands 11 and higher of a standard human plasma as determined in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330). Preferentially standard human plasma (SHP) (Siemens, Standard Human Plasma, ORKL17) is used.

In certain embodiments the molecular weight cut off size of the separation system is about 0.05 μιη to 1 μιη.

In other embodiments of the invention the molecular weight cut off size of the separation system is about 550,000 to 1,000,000 Da.

In another embodiment of the invention the molecular weight cut off size of the separation system is about 750,000 Da.

In another embodiment of the invention a second separation takes place wherein the retentate as obtained in any of the processes described above is subjected to a second separation wherein ultra-large vWF multimers are enriched in said retentate, providing a second permeate in which the proportion of ultra-large vWF multimers in the total vWF multimer amount is reduced as compared to the proportion of ultra-large vWF multimers in the total vWF multimer amount in the retentate before said second separation.

In one embodiment the second separation is done in parallel with the first separation and in another embodiment the second separation is done after the first separation. DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, length, or other unit described herein.

Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

By "biological activity" is meant a measurable function of vWF which vWF performs also in vivo when administered to a human being. As used herein, the terms "function" and "functional" and the like refer to a biological, enzymatic, or therapeutic function of vWF. The biological activity of vWF can for example be determined by the artisan using methods to determine the ristocetin co-factor activity (vWF:RCoF) (Federici AB et al. 2004. Haematologica 89:77-85), the binding of vWF to GP lb of the platelet glycoprotein complex Ib-V-IX (Sucker et al. 2006. Clin Appl Thromb Hemost. 12:305-310), a collagen binding assay (Kallas & Talpsep. 2001. Annals of Hematology 80:466-471) or a FVIII binding assay. FVIII binding may be determined for example by Biacore analysis.

By "bioreactor" is meant any manufactured or engineered device or system that provides and supports a biologically active environment for cell culture which allows the cells to grow and produce the product of interest. Bioreactors in general measure and regulate the physical and chemical parameters which are needed to generate this biological active environment. Bioreactors can be operated in different modes as batch, fed-batch or continuous. Bioreactors which are operated in continuous mode are additionally equipped with a media inlet and a device for separating the cells from the supernatant. Commonly bioreactors are cylindrical or cubic, ranging in size from milliliters to cubic meters, and are often made of stainless steel or plastic.

The term "host cell" includes an individual cell or cell culture that can be or has been a recipient of an introduced, recombinant vector, isolated polynucleotide, or polypeptide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide. A host cell which comprises a recombinant vector is a recombinant host cell. Specific host cells are defined below.

A "separation system" according to the invention means a device which can be used to retain host cells within a bioreactor during a fermentation process wherein at least part of the cell culture supernatant is separated from the host cells by means of a separation system comprising a filter which has a certain molecular weight cut off. Host cells and vWF multimers above a certain molecular weight are retained by said filter with a certain molecular weight cut off. Small molecules, nutrients and metabolites as well as vWF multimers below a certain molecular weight cut off can pass said filter with a certain molecular weight cut off. Cell retention within the culture vessel may be achieved using a number of cell retention devices. Different separation principles may be used for this process. The methods could be based on gravity settling, acoustic settling, centrifugal settling or filtration. The following sets of apparatus may all be used for this process: internal or external filters like internal or external spin filters (e.g. Spinfilter P from Sartorius), external cross flow filters, external or internal hollow fiber cartridge filters (e.g. alternating tangential flow (ATF) from Refine). The pore size of the filter with a certain molecular weight cut off (MWCO) is preferably between 0.05 μιη and 1.0 μιη. The MWCO is preferably equal or above 500,000 Da, or equal or above 550,000 Da, or equal or above 600,000 Da, or equal or above 650,000 Da, or equal or above 700,000 Da, or about 750,000 Da, or equal or below 1,000,000 Da, or equal or below 950,000 Da or equal or below 900,000 Da, or equal or below 850,000 Da, or equal or below 800,000 Da. It is to be understood that the above lower molecular weight cut off values can be combined with the above mentioned upper molecular weight cut offs to define ranges for the preferred parts with a certain molecular weight cut off like for example a range between equal or above 500,000 Da up to 1,000,000 Da or between 550,000 Da up to 1,000,000 Da etc.

The part of the cell culture that has passed the separation device is called the "permeate" whereas the part of the cell culture which is retained by the separation device is called the "retentate".

A MWCO of 500,000 used in a separation system means that proteins below 500,000 Da pass the separation and are enriched in the permeate, whereas proteins above 500,000 Da are retained by the filter and are therefore enriched in the retentate.

This separation can be used to enrich for certain fractions of vWF multimers with different molecular size which do have different biological activities.

"Substantially " or "essentially " means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

The term "von Willebrand Factor" or "vWF", as used herein, refers to any polypeptide having the biological activity of wild type vWF or at least a partial biological activity of vWF. The gene encoding wild type vWF is transcribed into a 9 kb mRNA which is translated into a pre-propolypeptide of 2813 amino acids with an estimated molecular weight of 310,000 Da. The pre-propolypeptide consists of 2813 amino acids and contains a 22 amino acids signal peptide, a 741 amino acid pro- polypeptide and the mature subunit. Cleavage of the 741 amino acids pro -polypeptide from the N-terminus results in mature vWF consisting of 2050 amino acids. The cDNA sequence of wild type pre-pro-vWF is shown in SEQ ID NO: l. The amino acid sequence of wild type pre-pro-vWF is shown in SEQ ID NO:2. The term "vWF" as used hererein refers to the mature form of vWF unless indicated otherwise.

The pro-polypeptide of wild type vWF comprises multiple domains which are arranged in the following order:

D1-D2-D'-D3-A1-A2-A3-D4-B 1-B2-B3-C1-C2-CK

The Dl and D2 domain represent the pro-peptide which is cleaved off to yield the mature vWF. The carboxy-terminal 90 residues comprise the "CK" domain that is homologous to the "cystine knot" superfamily of protein. These family members have a tendency to dimerise through disulfide bonds.

Preferably, wild type vWF comprises the amino acid sequence of wild type vWF as shown in SEQ ID NO:2. Also encompassed are additions, insertions, N-terminal, C- terminal or internal deletions of vWF as long as at least a partial biological activity of vWF is retained. The biological activity of wild-type vWF can be determined by the artisan using methods for ristocetin co-factor activity (Federici AB et al. 2004. Haematologica 89:77-85), binding of vWF to GP Iba of the platelet glycoprotein complex Ib-V-IX (Sucker et al. 2006. Clin Appl Thromb Hemost. 12:305-310), a collagen binding assay (Kallas & Talpsep. 2001. Annals of Hematology 80:466-471) or a FVIII binding assay

Certain embodiments may include the recombinant production of wild-type vWF as for example described in WO 2010/048275, or variants thereof, for example, in which one or more amino acid deletions, additions, and/or substitutions have been introduced to increase or decrease at least one biological activity of the protein.

Accordingly, certain embodiments may employ any one or more of these vWF related sequences, including combinations and variants thereof. Also included are vWF-related sequences from other organisms, such as other mammals described herein and known in the art.

In certain embodiments th e term "vWF " includes "fusion proteins" of vWF, preferably "fusion proteins" of a vWF protein and a "heterologous" fusion partner. Also included are fusion proteins or modified proteins that comprise a heterologous fusion partner or heterologous sequence and at least one minimal fragment or portion of a vWF protein.

As used herein, a "fusion protein" includes a vWF protein or fragment thereof linked to either another (e.g., different) vWF protein (e.g., to create multiple fragments), to a vWF protein, or to both. A "non- vWF protein" refers to a "heterologous polypeptide" having an amino acid sequence corresponding to a protein which is different from a vWF protein, and which can be derived from the same or a different organism. The vWF protein of the fusion protein can correspond to all or a portion of a biologically active vWF protein amino acid sequence. In certain embodiments, a vWF fusion protein includes at least one (or two, three, etc.) biologically active portion(s) of a vWF protein.

More generally, fusion to heterologous sequences, such as albumin or immunoglobulins or fragments derived from immunoglobulins without an antigen binding domain, such as the Fc fragment, may be utilized to remove unwanted characteristics or to improve the desired characteristics (e.g., pharmacokinetic properties) of a vWF. For example, fusion to a heterologous sequence may increase chemical stability, decrease immunogenicity, improve in vivo targeting, and/or increase half-life in circulation of a vWF protein.

"Albumin", as used herein, includes polypeptides of the albumin family of proteins such as human serum albumin and bovine serum albumin, including variants and derivatives thereof, such as genetically engineered or chemically modified albumin variants and fragments of albumin proteins. The albumin portion of a fusion protein may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin-linked polypeptide may be from a different animal than the vWF protein portion of the fusion protein.

The albumin family of proteins, included within the term "albumin" used herein, comprise evolutionarily related serum transport proteins, for example, albumin, alpha-fetoprotein (AFP; Beattie & Dugaiczyk, Gene. 20:415-422, 1982), afamin (AFM; Lichenstein et al., J. Biol. Chem. 269: 18149-18154, 1994), and vitamin D binding protein (DBP; Cooke & David, J. Clin. Invest. 76:2420-2424, 1985). Alpha- fetoprotein has been claimed to enhance the half-life of an attached therapeutic polypeptide (see WO 2005/024044). Their genes represent a multigene cluster with structural and functional similarities mapping to the same chromosomal region in humans, mice and rat. Some embodiments of the invention, therefore, may use such albumin family members, or fragments and variants thereof as defined herein, as part of a fusion protein. Albumin family members of the therapeutic fusion proteins of the invention may also include naturally-occurring polymorphic variants of AFP, AFM and DBP.

VWF protein, or a fragment or variant thereof, may be fused to a human serum albumin polypeptide, or a fragment or variant thereof (see, e.g. WO 2009/156137). Human serum albumin (HSA, or HA), is a protein of 585 amino acids in its mature form, and is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. Among other benefits, fusion to HSA or a fragment or variant thereof can increase the shelf-life, serum half-life, and/or therapeutic activity of the vWF proteins described herein.

Preferably a fusion protein comprises albumin as the C- terminal portion, and a vWF protein as the N-terminal portion. In other embodiments, the fusion protein has vWF proteins fused to both the N-terminus and the C-terminus of albumin.

A peptide linker sequence may be employed to separate the components of a fusion protein. For instance, peptide linkers can separate the components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence may be incorporated into the fusion protein using standard techniques described herein and well-known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39- 46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.

One or more of the non-peptide or peptide linkers are optional. For instance, linker sequences may not be required in a fusion protein where the first and second polypeptides have non-essential N-terminal and/or C-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

Certain embodiments of the present invention also contemplate the use of modified vWF proteins, including modifications that improved the desired characteristics of the protein, as described herein. Modifications of vWF proteins include chemical and/or enzymatic derivatizations at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C- terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like. Exemplary modifications also include PEGylation of a vWF protein (see, e.g., Veronese and Harris, Advanced Drug Delivery Reviews 54: 453-456, 2002, herein incorporated by reference). VWF variants which are chemically conjugated to biologically acceptable polymers are described for example in WO 2006/071801.

The invention may also be used with "variants" of vWF proteins. The term protein "variant" includes proteins that are distinguished from SEQ ID NO:2 by the addition, deletion, and/or substitution of at least one amino acid residue, and which typically retain one or more activities of the reference protein. It is within the skill of those in the art to identify amino acids suitable for substitution and to design variants with substantially unaltered, improved, or decreased activity, relative to a reference sequence.

A protein variant may be distinguished from a reference sequence by one or more substitutions, which may be conservative or non-conservative, as described herein and known in the art. In certain embodiments, the protein variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the protein.

The terms "sequence identity" or, for example, comprising a "sequence 50% identical to," as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity " may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

Terms used to describe sequence relationships between two or more polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity" and "substantial identity." A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polypeptides may each comprise (1) a sequence (i.e., only a portion of the complete polypeptides sequence) that is similar between the two polypeptides, and (2) a sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences of the two polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.

As noted above, biologically active variant proteins may contain conservative amino acid substitutions at various locations along their sequence, as compared to a reference residue. A "conservative amino acid substitution" includes one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

This description also characterizes certain amino acids as "small " since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small " amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the a-amino group, as well as the a- carbon. For the purposes of the present invention, proline is classified as a "small " amino acid.

The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behavior.

Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in Table 1 below.

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic - hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a biologically active protein can readily be determined by assaying its chromogenic and/or coagulation activity, as described herein.

The terms "Low molecular weight vWF multimers" or "LMW vWF multimers" or "LMW vWF" are used synonymously and are meant to correspond to bands 1 to 5 in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330).

The terms "High molecular weight vWF multimers" or "HMW vWF multimers" or "HMW vWF" are used synonymously and are meant to correspond to bands 11 and higher in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330), wherein "higher" means band 11 and all larger vWF multimers.

"Ultra-large molecular weight vWF multimers" or "ULMW vWF multimers" are used synonymously and are meant to correspond to bands 20 and higher in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330), wherein "higher" means band 20 and all larger vWF multimers.

The term "HMW Multimer Ratio" refers to the ratio of the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 11 and higher in the retentate divided by the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 11 and higher in the permeate.

The term "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 11 and higher refers to the numerical value which is obtained when the amount of bands 11 and higher as determined in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330) is divided by the amount of bands 11 and higher of a standard human plasma as determined in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322- 330). Preferentially standard human plasma (SHP) (Siemens, Standard Human Plasma, ORKL17) is used.

The term "LMW Multimer Ratio" refers to the ratio of the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 1 to 5 in the retentate divided by the "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 1 to 5 in the permeate.

The term "accumulated pixel intensities related to standard human plasma" of vWF multimer bands 1 to 5 HMW refers to the numerical value which is obtained when the amount of bands 1 to 5 as determined in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330) is divided by the amount of bands 1 to 5 of a standard human plasma as determined in a densidometric vWF analysis according to Ott et al. (Am J Clin Pathol 2010;133:322-330). Preferentially standard human plasma (SHP) (Siemens, Standard Human Plasma, ORKL17) is used.

An LMW vWF Multimer Ratio in the permeate "below" the LMW vWF

Multimer Ratio of the cell culture medium before separation is typically a "statistically significant" decreased ratio, and may include a decrease that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.3, 4.4, 4.6, 4.8, 5, 6, 7, 8, 9, 10 or more times (including all integers and decimal points and ranges in between and above 1, e.g., 5.5, 5.6, 5.7. 5.8, etc.) below the LMW vWF Multimer Ratio of the cell culture medium before separation.

An HMW vWF Multimer Ratio in the permeate "above" the HMW vWF Multimer Ratio of the cell culture medium before separation is typically a "statistically significant" increased ratio, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.3, 4.4, 4.6, 4.8, 5, 6, 7, 8, 9, 10 or more times (including all integers and decimal points and ranges in between and above 1, e.g., 5.5, 5.6, 5.7. 5.8, etc.) above the HMW vWF Multimer Ratio of the cell culture medium before separation. Expression and Purification of Proteins of the Invention

Embodiments of the present invention include methods and related compositions for expressing, collecting, and optionally purifying the vWF fractions described herein.

For instance, certain embodiments include methods for producing a vWF or variant thereof, comprising culturing a cell in a cell culture medium, optionally a mammalian cell, where the cell comprises at least one introduced polynucleotide that encodes the vWF, where said polynucleotide is operably linked to at least one regulatory element and expresses the protein and collecting the protein from the cell or cell culture medium.

Recombinant vWF or variants thereof can be conveniently prepared using standard protocols. As one general example, recombinant vWF may be prepared by a procedure including one or more of the steps of: (a) preparing a construct comprising a polynucleotide sequence that encodes a protein and that is operably linked to at least one regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the polypeptide and (d) collecting or isolating the polypeptide from the host cell. To express a vWF, a nucleotide sequence encoding the polypeptide, or a functional equivalent, may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding vWF and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination and are known in the art.

The "regulatory elements" or "regulatory sequences" present in an expression vector include those non-translated regions of the vector, e.g., enhancers, promoters, 5' and 3' un-translated regions, ribosome binding site(s), RNA splice sites (if intron-containing genomic DNA is present), polyadenylation site(s), transcriptional termination sequence(s), which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.

Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, in mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred.

Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences.

In cases where sequences encoding the vWF, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature.

In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells. In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer or the CMV promoter/enhancer, may be used to increase expression in mammalian host cells.

Suitable host cells are higher eukaryotic cells such as mammalian cells. Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (e.g., HEK293 cells, 293 cells sub- cloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10)); mouse Sertoli cells; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO cells, VERO-76, ATCC CRL-1587); COS cells, human cervical carcinoma cells (Hela cells, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO cells, including DHFR-CHO cells); and myeloma cell lines such as NSO and Sp2/0.

Other suitable cell lines include, without limitation, Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1); DUKX cells (CHO cell line) and DG44 (CHO cell line). Also useful are 3T3 cells, Namalwa cells, myelomas and fusions of myelomas with other cells.

Certain preferred mammalian cell expression systems include CHO and HEK293-cell based expression systems. Particular examples of CHO cells include CHO-Kl and CHO-S cells. Also included are host cells that can be grown on serum free medium, such as certain HEK293 cells and CHO cells.

In some embodiments, the host cells are capable of growing in suspension cultures. As used herein, suspension-competent cells are those that can grow in suspension without making large, firm aggregates, that is, cells that are monodisperse or grow in loose aggregates with only a few cells per aggregate. Suspension-competent cells include, without limitation, cells that grow in suspension without adaptation or manipulation (e.g., hematopoietic cells, lymphoid cells) and cells that have been made suspension-competent by gradual adaptation of attachment dependent cells (e.g., epithelial cells, fibroblast cells) to suspension growth. In some embodiments, the host cells may include mutant or recombinant cells, including cells that express a qualitatively or quantitatively different spectrum of proteins that catalyze post-translational modification of proteins (for example, glycosylation enzymes such as glycosyl transferases and/or glycosidases, processing enzymes such as propeptides, including enzymes which promote the formation of functional vWF), relative to the cell type from which they were derived.

As one example, the host cell comprises an introduced polynucleotide that encodes at least one "processing factor" protein, where said polynucleotide is operably linked to at least one regulatory element and expresses the processing factor protein. The term "processing factor" includes any protein, peptide, non-peptide cofactor, substrate or nucleic acid which promotes the formation of a functional protein. Examples of processing factors include, but are not limited to paired basic amino acid converting enzyme (PACE), vitamin K-dependent epoxide reductase (VKOR), vitamin K-dependent gamma-glutamyl carboxylase (VKGC), or a combination thereof. Particular embodiments utilize the human sequence(s) for PACE, VKOR, and/or VKGC.

PACE, originally isolated from a human liver cell line, is a subtilisin-like endopeptidase, that is, a propeptide-cleaving enzyme which exhibits specificity for cleavage at basic residues of a protein (e.g., - Lys-Arg-, -Arg-Arg-, or -Lys-Lys-). The co-expression of PACE and a proprotein which requires processing for production of the mature protein results in high level expression of the mature protein.

For long-term, high-yield production of vWF, stable expression is generally preferred. For example, cell lines which stably express vWF may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1-2 days in non-selective media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.

Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Any number of selectable markers or systems may be used to recover or identify transformed or transduced cell lines, whether transient or stable. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) confers resistance to methotrexate; npt confers resistance to the aminoglycosides, neomycin and G-418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.

Host cells transformed with a polynucleotide sequence coding for vWF may be cultured under conditions suitable for the expression and recovery of the vWF from the cell culture. Any chemically- defined cell culture medium may be used. Particular examples include serum-free culture media, protein- free media, chemically-defined culture media, and culture media that lack animal-derived components.

Exemplary cell culture media include, without limitation, Basal Medium Eagle (BME); CMRL-1066 Medium; Dulbecco's Modified Eagle's Medium (DMEM); Glasgow Minimum Essential Medium, GMEM; H-Y Medium (Hybri-Max®); Medium 199; Minimum Essential Medium Eagle (EMEM); NCTC Medium; Swim's S-77 Medium; and Williams Medium E. Click's Medium; MCDB media 131 and Iscove's Modified Dulbecco's Medium (IMDM). Also included is DMEM/Ham's Nutrient Mixture F-12 (50:50), which is frequently used as a base media for the development of proprietary and specialty media to culture CHO cells for biomanufacturing, and MCDB medium 302, which was developed for CHO cells.

In particular embodiments, the media is a serum-free DMEM/HAM's F12 based formulation supplemented with one or more of the following: (a) glutamine; for example, final concentration about 0.9 g/1, (b) ferric sulfate x7H20, for example, at about 0.0006 g/1, (c) putrescine, x 2HC1, for example, at about 0.0036 g/1, (d) Vitamin Kl, for example, at about 0.0025 g/1, (e) Synperonic; for example, at a final concentration of about 1 g/1, (f) Phenol red, for example, at about 0.008 g/1, (g) Ethanolamine, for example, at about 0.00153 g/1, and/or (h) Na-hydrogencarbonate, for example, at about 2 g/1.

In specific embodiments, the cell culture medium is a CD-CHO medium or a

CD-CHO AGT™ medium or a CD OptiCHO™ AGT™ medium, which are commercially available (INVITROGEN®) or Pro-CHO™ or Power-CHO™ media

(LONZA®). CH-CHO, CD-CHO AGT™, and CD OptiCHO™ AGT™ medium can be used as a chemically-defined medium, a protein-free medium, and/or a serum-free medium, and can be supplemented with L-glutamine, if desired. The Ca 2+ levels of IX CD-CHO AGT™ medium without calcium supplementation can range from about 0.25-0.35 mM (as determined by atomic absorption spectroscopy).

As noted above, certain embodiments utilize a serum-free cell culture medium. One e x amp le includes a defined serum-free medium that is capable of growing a wide range of suspension and monolayer cells, and which includes a serum substitute composed of fetuin, transferrin, phosphatidylcholine (e.g., l-oleoyl-2- palmitoyl-phosphatidylcholine), linoleic acid, and cholesterol. Another example includes a cell culture medium containing a sterol such as cholesterol, which is stabilized by a surfactant rather than serum products or phospholipid micelles, and optionally containing soluble carboxylic acids as fatty acid precursors to satisfy lipid requirements, and/or alcohols that are capable of promoting cell.

Certain embodiments employ a cell culture medium that lacks "animal-derived" components. As used herein "animal-derived" components may include any components that are produced in an intact animal (e.g., proteins isolated and purified from serum) or components produced by using components produced in an intact animal (e.g., an amino acid made by using an enzyme isolated and purified from an animal to hydrolyse a plant source material). By contrast, a protein which has the sequence of an animal protein (that is, the protein has a genomic origin in an animal) but which is produced in vitro in cell culture (e.g., in a recombinant yeast or bacterial cell, or in mammalian cell line, recombinant or not), in media lacking components that are produced in, and isolated and purified from an intact animal is not an "animal-derived" component. For example, a protein which has the sequence of an animal protein (that is, has a genomic origin in an animal) but which is produced in a recombinant cell in media lacking animal derived components is not an "animal- derived" component. Accordingly, a cell culture medium lacking animal-derived components is one that may contain animal proteins that are recombinantly produced; such medium, however, does not contain, for example, animal serum or proteins or other products purified from animal serum. Such medium may, for example, contain one or more components derived from plants.

Any cell culture medium that supports cell growth and maintenance under the conditions of the invention may be used. Typically, the medium contains water, an osmolality regulator, a buffer, an energy source, amino acids, an inorganic or recombinant iron source, one or more synthetic or recombinant growth factors, vitamins, and cofactors. Media lacking animal-derived components and/or proteins are available from commercial suppliers, such as, for example, SIGMA®, SAFC, INVITROGEN®, GIBCO® or LONZA®. For example, certain embodiments employ suspension cell culture techniques. Examples of suspension cell culture techniques include perfusion processes, batch processes (e.g., simple batch processes, fed-batch processes), and draw-fill processes.

In a suspension cell-perfusion process the cells are typically inoculated into a seed culture vessel containing a cell culture medium, which preferably lacks animal- derived components and is propagated until the cells reach a minimum density.

Subsequently, the propagated seed culture is then transferred to a large- scale culture vessel, which likewise contains a culture medium that preferably lacks animal-derived components. The cells are propagated until at least a predetermined cell density is reached. In this phase the cells are grown in suspension to allow the cell number within the culture vessel to increase to a predetermined or critical value.

Medium exchange can be performed by continuously perfusing the culture vessel with fresh medium while the recombinant cells are retained in the culture vessel. The amount of perfused medium may depend on the cell density and is typically from about 10-300%, preferably from about 10% to 95%, 25% to 80%, of the tank volume per day (24 hours).

In the growth phase the culture is propagated until the cells reach a certain density. Reaching this density, the culture enters the production phase.

When the cell density reaches the value suitable for initiation of production phase, about 60-95% of the tank medium in the tank is changed every 24 hours, preferably about 80%. An 80% medium exchange is also preferably used in the production phase. Set-points may also be changed at this point and set at values suitable for production of the respective protein. The medium perfusion is preferably performed continuously. The flow rate of medium can be expressed in terms of percentage tank volume of medium per defined period of time.

Medium perfusion may be from about 10-200% tank volume per 10-48 hours; preferably, the medium perfusion is about 90% per 10-48 hours, more preferably the medium perfusion is about 80% tank volume every 24 hours.

Cell retention within the culture vessel may be achieved using a number of cell retention devices. Different separation principles may be used for this process. The methods could be based on gravity settling, acoustic settling, centrifugal settling or filtration. The following sets of apparatus may all be used for this process: internal or external filters like internal or external spin filters (e.g. Spinfilter P from Sartorius), external cross flow filters, external or internal hollow fiber cartridge filters (e.g. alternating tangential flow (ATF) from Refine). VWF multimers can vary from a simple dimer up to multimers consisting of more than 16 covalently linked vWF monomers. The different proteins forms do have different biological activities. For example low molecular weight multimers of vWF do have a lower ristocetin co-factor activity (vWF:RCoF), whereas high molecular weight forms of vWF do have a higher vWF:RCoF activity. A high vWF:RCoF activity of a vWF preparation correlates with a high activity in primary hemostasis as vWF preparations with high vWF:RCoF more efficiently contribute to the agglutination of platelets and to the tethering of platelets to collagen exposed by an injury. The ratio of vWF:RCoF over the antigen content (vWF:Ag) in human plasma is by definition 1.0.

It is thus desirable to manufacture pharmaceutical vWF preparations with a ratio of vWF:RCoF to vWF:Ag of at least 0.5, or at least 0.55, or at least 0.6, or at least 0.65 or at least 0.7, or at least 0.75, or at least 0.8, or at least 0.85, or at least 0.9, or at least 0.95 or at least 1.0.

In certain therapeutic situation it is also be preferable to manufacture vWF preparations which do have an increased vWF:RCoF/vWF:Ag ratio higher than that present in human plasma, for example of at least 1.05, or at least 1.1, or at least 1.15, or at least 1.2 or at least 1.25, or at least 1.3, or at least 1.35, or at least 1.40, or at least 1.45 or at least 1.50, or at least 1.55 or at least 1.60, or at least 1.65 or at least 1.70 or at least 1.75, or at least 1.80, or at least 1.85 or at least 1.90 or at least 1.95 or at least 2.0 .

In order to manufacture pharmaceutical vWF preparations with increased vWF:RCoF/vWF:Ag ratios it is normally necessary to devise down-stream purification procedures which increase the amount of higher molecular weight multimers of vWF. This is often cumbersome and may result in decreased yield of vWF product as each down-stream purification step is usually associated with some product loss.

The inventors of the present invention have now surprisingly found that the enrichment of higher molecular weight multimers of vWF can already be achieved at the up-stream stage of manufacturing during cell culture by using a separation device which retains the desired high molecular weight multimeric forms of vWF in the bioreactor (retentate) whereas the less desired lower molecular weight forms of vWF pass the separation device (permeate). However, as the lower molecular weight forms of vWF still are capable of binding and of stabilizing Factor VIII, the fraction in the permeate may also be a product for certain specific uses like for the stabilization of Factor VIII. In the latter case the low molecular weight fraction of vWF multimers may be the desired vWF form.

Harvesting the fraction which is enriched in HMW vWF multimers can be either done i) at the end of the fermentation process or ii) concomitantly by using an additional separation device. This additional separation device in the case ii) could be any separation device separating the cells form the retentate und which does not further separate LMW vWF multimers from HMW vWF multimers. As result of such a fermentation process in case ii) a vWF preparation is obtained which is enriched in HMW vWF multimers and the residence time in the bioreactor is reduced in comparison to a fermentation process as in case i). Shortening the residence is beneficial in general in order to minimize degradation events.

However, if the ratio of vWF:RCoF/vWF:Ag is higher than 2.0 there is an increasing risk of thromboembolic complications when such preparations are administered to patients, as such ultra- large molecular weight multimers of vWF may lead to the agglutination of platelets in non-physiological situations.

If such ultra-large molecular weight multimers are present in the cell culture medium it may be beneficial to apply separation devices with a molecular weight cut off (MWCO) of 10,000,000 Da which retains the ultra- large multimers of vWF in the bioreactor (retentate) whereas the desired high-molecular weight forms of vWF are separated into the filtrate.

The inventors also have found a process for manufacturing a recombinant von Willebrand factor (vWF) by culturing host cells in a bioreactor in a cell culture medium, wherein the host cells produce recombinant vWF which is secreted into the cell culture medium and wherein the vWF in the cell culture medium comprises vWF multimers of different size, wherein at least one cell culture medium component is fed to the cell culture medium and wherein the cell culture comprising the cells, the recombinant vWF and the cell culture medium is pumped over a separation system and wherein the separation system separates the vWF multimers into at least

a permeate fraction which is enriched in low molecular weight (LMW) multimers of vWF and reduced in high molecular weight (HMW) multimers of vWF as compared to the vWF multimers in the cell culture supernatant before separation and

a retentate fraction which is reduced in low molecular weight (LMW) multimers of vWF and enriched in high molecular weight (HMW) multimers of vWF as compared to the vWF multimers in the cell culture supernatant before separation

The HMW vWF Multimer Ratio is preferably equal or above 1.1, or above 1.2, or above 1.25, or above 1.30, or above 1.35, or above 1.40 or above 1.45 or above 1.50 or above 1.60, or above 1.70, or above 1.80, or above 1.90.

In a preferred embodiment the invention encompasses also a bioreactor comprising a cell culture medium comprising vWF multimers in a retentate and a cell culture medium comprising vWF multimers in a permeate wherein the HMW vWF Multimer Ratio is equal to or above 1.1, or above 1.2, or above 1.25, or above 1.30, or above 1.35, or above 1.40 or above 1.45 or above 1.50 or above 1.60, or above 1.70, or above 1.80, or above 1.90.

The LMW vWF Multimer Ratio in is preferably equal to or below 0.90, or below 0.85, or below 0.80, or below 0.75, or below 0.70 or below 0.65 or below 0.60, or below 0.55, or below 0.50.

A preferred embodiment of the invention encompasses also a bioreactor comprising a cell culture medium comprising vWF multimers and a cell culture medium comprising vWF multimers in a permeate wherein the LMW vWF Multimer Ratio is below 0.90, or below 0.85, or below 0.80, or below 0.75, or below 0.70 or below 0.65 or below 0.60, or below 0.55, or below 0.50.

By choosing the MWCOs of the separation device the man skilled in the art is enabled by the present invention to choose the molecular size of the vWF product which is then subsequently purified according to techniques well-known in the art.

Such separation of vWF multimers of different molecular weight already at the fermentation stage, i.e. during upstream processing, greatly facilitates separation in a subsequent post-fermentation or down-stream purification, as the desired fractions of vWF multimer are already enriched in the desired vWF multimers. Therefore potentially less purification steps are required in the down-stream processing resulting in faster processing time and higher yields. The invention usually also leads to higher purity of the resulting vWF preparation as part of the purification starts already in the bioreactor and subsequent downstream processing then start with a vWF already enriched for the desired molecular- weight fraction.

Certain embodiments may include culture methods that monitor and control certain parameters, such as pH, dissolved oxygen tension (DOT), and temperature. As one example, the pH can be controlled by regulating the C02 concentration in the headspace gas and/or sparger, and by addition of base to the culture medium when required. Certain embodiments therefore include large-scale production of a recombinant polypeptide in a cell culture medium, where the concentration of dissolved C02 in the culture is monitored and constant or intermittent sparging of air through the culture medium is employed to maintain certain concentration ranges of C02- In some aspects, the predetermined range for the concentration of dissolved C02 is about 80-200 mmHg, preferably about 100-180 mmHg, e.g., about 140 mmHg.

In most instances, the sparging rate of the air is controlled in relation to the monitored concentration of dissolved C02 in the culture medium. For example, the sparging rate of air can be in the range of about 0.000-0.100 L/min per L of culture liquid, preferably about 0.005-0.020 L/min per L of culture liquid, particularly where the monitored concentration of dissolved C02 is equal to (pre-determined) set-point concentration. In some instances, the sparging rate of air is in the range of 0.000- 0.005, such as around 0.0 L/min per L of culture liquid when the monitored concentration of dissolved C02 is equal to the set-point concentration -5 mmHg; and in the range of 0.010-0.100 L/min per L of culture liquid when the monitored concentration of dissolved C02 is equal to the set-point concentration +5 mmHg (see

U.S. Application No. 2006/0216790). Such methods can be used to maintain a desired pH in the cell culture medium, for example, where any solid or liquid substances added to the culture medium do not give rise to a localized pH value of above about 7.5.

Dissolved oxygen tension may be maintained, for instance, by sparging with air or pure oxygen or mixtures thereof. The temperature-control medium is typically water or other liquid, heated or cooled as necessary. In certain aspects, the water may be passed through a jacket surrounding the vessel or through a piping coil immersed in the culture.

The vWF produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), hydrophobic interaction chromatography, and high-pressure liquid chromatography (HPLC). Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration, reverse-phase, ceramic HyperD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Also included are analytical methods such as SDS- PAGE (e.g., Coomassie, silver stain), preparative isoelectric focusing (IEF), immunoblot, Bradford, differential solubility (e. g., ammonium sulfate precipitation) and ELISA, which may be utilized during any step of the production or purification process, typically to measure the purity of the protein composition.

In certain aspects, the recombinant vWF can be subjected to multiple chromatographic purification steps, including any combination of affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, dye chromatography, hydroxyapatite chromatography, size exclusion chromatography and preferably immunoaffinity chromatography, mainly to concentrate the desired protein and to remove substances which may cause fragmentation, activation and/or degradation of the recombinant protein during manufacture, storage and/or use. Illustrative examples of such substances that are preferably removed by purification include other protein contaminants, such as modification enzymes like PACE/furin, VKOR, and VKGC; proteins, such as host cell proteins, which are released into the tissue culture media from the production cells during recombinant protein production; non-protein contaminants, such as lipids; and mixtures of protein and non-protein contaminants, such as lipoproteins. Purification procedures for vWF proteins are known in the art (see for example WO 2011/022657).

In order to minimize the theoretical risk of virus contaminations, additional steps may be included in the process that allow effective inactivation or elimination of viruses. Such steps include, for example, heat treatment in the liquid or solid state, treatment with solvents and/or detergents, radiation in the visible or UV spectrum, gamma-radiation, and nanofiltration.

Pharmaceutical Compositions

Embodiments of the present invention include proteins and preferably vWF proteins that are produced according to the methods provided herein, and formulated in pharmaceutically-acceptable or physiologically- acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the modulatory or other effects desired to be achieved.

In the pharmaceutical compositions of the invention, formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, subcutaneous, and intramuscular administration and formulation.

In certain applications, the pharmaceutical or therapeutic compositions of the invention do not stimulate an immune reaction. In some aspects, the pharmaceutical composition comprising the protein(s) may be formulated in stable liquid form. In other aspects, the pharmaceutical composition may be formulated in lyophilized form; protein(s) may be lyophilized by a variety of procedures known in the art. Lyophilized formulations are typically reconstituted prior to use by the addition of one or more pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.

In certain embodiments, the proteins have a solubility that is desirable for the particular mode of administration, such a s intravenous administration. Examples of desirable solubilities include at least about 1 mg/ml, at least about 10 mg/ml, at least about 25 mg/ml, and at least about 50 mg/ml.

In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, subcutaneously, intravenously, intramuscularly, intra-arterially, intrathecally, intraparenchymally, intraventricularly, intraurethrally, intrasternally, intracranially, intrasynovially, or even intraperitoneally). Suitable devices for parenteral administration include needle (including micro-needle) injectors, needle-free injectors, and infusion techniques.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art.

Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection can also be prepared. The preparation can also be emulsified.

The pharmaceutical compositions may be formulated to be immediate and / or sustained release. Sustained release compositions include delayed, modified, pulsed, controlled, targeted and programmed release.

Methods of formulation are well known in the art. The recombinant proteins provided herein may be administered accordingly to any therapeutically effective dosing regimen. The dosage amount and frequency can be selected to create an effective amount of the protein(s) and minimize harmful effects. The effective amount will depend on a variety of factors, including the route of administration, the type of subject being treated, and the physical characteristics of the subject under consideration, such as weight, diet, concurrent medication and other factors that persons skilled in the medical arts will recognize.

Formulations of vWF have been described for example in WO 2010/048275. All publications, patent applications, and issued patents cited in this specification are incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES

All fermentations were performed using a Sartorius Biostat B-DCU Controller to control the pH at 7.0, the DO at 30% air saturation, the temperature and a stirrer speed of 150 rpm. The process was executed in a Sartorius B vessel with a working volume of 5L. After the inoculation of the reactor a growth phase at 37°C was performed, followed by a production phase at 33°C. All processes were performed with the same CHO-cell line expressing a wild-type full length vWF fused at its C-terminal end to human wild-type full-length albumin protein. The cell line was obtained as described in WO 2009/156137.

Example 1 (Batch process, comparative example)

A batch-process running with CSL-DBM-PFCDM-media (SAFC) was started with a cell density of 4.4* 10 ⁵ cells/mL. After 7 days a cell density of 6.9* 10 ⁶ cells/mL was reached. A sample from the bioreactor was taken daily and the vWF activity (vWF:RCoF) and antigen (vWF:Ag) was determined. From these measurements the ratio vWF:RCoF/vWF:Ag was calculated. Fig. 1 shows that this ratio is slightly decreasing after reaching a peak level at about day 2.

Example 2 (Process of the invention)

The retention module, an ATF 4 (Refine Technology) was operated with an exhaust flow rate of 3 L/min and a pressure flow rate of 3 L/min. A 750 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane obtained from GE Healthcare Life Sciences operated in ATF flow mode was used to enrich the high molecular weight (HMW) vWF in the bioreactor and to reduce the low molecular weight (LMW) vWF in the bioreactor. A CD CHO (Gibco) based culture media was used during growth phase and also as feed media. The culture was started with 5* 10 ⁵ cells/mL. After a growth phase (data not shown) of 4 days the ATF was started with a harvest rate for permeate of about 100% of the tank volume per day. At this time the cell density in the bioreactor was 3.8* 10 ⁶ cells/mL. When the cell density reached 7* 10 ⁶ cells/mL the temperature was shifted from 37°C to 33°C for the production phase. A sample from the bioreactor and of the permeate was taken daily and the vWF ristocetin cofactor activity (vWF:RCoF) and vWF antigen (vWF:Ag) was determined. From these measurements the ratio vWF:RCoF/vWF:Ag was calculated. It was shown that due to the removal of the LMW vWF the ratio vWF:RCoF/vWF:Ag in the retentate (bioreactor) was rising, whereas the ratio vWF:RCoF/vWF:Ag in the permeate was decreasing due to the removal of the HMW vWF (Fig. 2).

A vWF multimer analysis (multimer electrophoresis) was performed at day 4 according to Ott et al. (Am J Clin Pathol 2010;133:322-330) to show the enrichment of high-molecular-weight vWF in the retentate (bioreactor) and the separation and removal of low-molecular-weight vWF in the permeate. In addition to the multimer analysis of samples of the retentate and of the permeate a multimer analysis of a sample of standard human plasma (SHP) (Siemens, Standard Human Plasma, ORKL17) was performed according to Ott et al. (Am J Clin Pathol 2010;133:322-330) and used as a reference in each individual blot. The "accumulated pixel intensity related to standard human plasma" was then calculated by dividing the pixel intensity separately for each for the band sections 1-5, 6-10 and >11 in the respective sample by the average pixel intensity of the corresponding band sections of standard human plasma, wherein both individual accumulated pixel intensities were determined according to Ott et al. (Am J Clin Pathol 2010;133:322-330) (Table 2, Fig. 3).

Table 2 (vWF multimer analysis)

Accumulated pixel Accumulated pixel Accumulated pixel intensity related to intensity related to intensity related to standard human standard human standard human plasma of bands plasma of bands plasma of bands >11 6-10 1-5

Retentate 148.1 89.3 93.7

Permeate 26.6 102.8 111.9

Ratio

Retentate/Permeate 5.568 0.869 0.837 Example 3 (Process of the invention)

The retention module, an ATF 4 (Refine Technology) was operated with an exhaust flow rate of 3L/min and a pressure flow rate of 3 L/min. A 750 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane obtained from GE Healthcare Life Sciences operated in ATF flow mode was used to enrich the HMW vWF in the bioreactor and reduce the LMW vFW in the bioreactor. A PowerCHO-3 based cell culture medium (Lonza) was used for the entire process. The culture was started with 4* 10 ⁵ cells/mL. After a growth phase (data not shown) of 3 days the ATF was started with a harvest rate for permeate of about 100% of the tank volume per day. At this time the cell density in the bioreactor was 1.4* 10 ⁶ cells/mL. When the cell density reached 1.7* 10 ⁶ cells/mL the temperature was shifted from 37°C to 33°C for the production phase.

A sample from the bioreactor and of the permeate was taken daily and the activity (vWF:RCoF) and antigen (vWF:Ag) were determined as described in Example 1. From these measurements the ratio vWF:RCoF/vWF:Ag was calculated. It was shown that due to the removal of the LMW vWF the ratio vWF:RCoF/vWF:Ag in the retentate (bioreactor) was rising, whereas the ratio vWF:RCoF/vWF:Ag in the permeate was decreasing due to the removal of the HMW vWF (Fig. 4).

At the end of the process a multimer electrophoresis was performed to show the accumulation of high-molecular-weight vWF in the bioreactor and the separation/removal of low-molecular-weight vWF with the permeate. The accumulated pixel intensity related to standard human plasma was calculated as above.

A vWF multimer analysis (multimer electrophoresis) was performed at day 9 according to Ott et al. (Am J Clin Pathol 2010;133:322-330) to show the enrichment of high-molecular-weight vWF in the retentate (bioreactor) and the separation and removal of low-molecular-weight vWF in the permeate. In addition to the multimer analysis of samples of the retentate and of the permeate a multimer analysis of a sample of standard human plasma (SHP) (Siemens, Standard Human Plasma, ORKL17) was performed according to Ott et al. (Am J Clin Pathol 2010;133:322-330) and used as a reference in each individual blot. The "accumulated pixel intensity related to standard human plasma" was then calculated as described in Example 2 (Table 3, Fig. 5). Table 3 (vWF multimer analysis):

Example 4

Another aspect of the inventions is a process wherein the retentate as obtained in any of Examples 2 and 3 is directly harvested from the bioreactor using an additional separation device. The separation device in this case could be any separation device separating the cells from the retentate and which does not further separate LMW vWF multimers from HMW vWF multimers. Bioreactor conditions and media are as described in previous examples.

As result of such a fermentation process a vWF preparation is obtained which is enriched in HMW vWF multimers and the residence time in the bioreactor is reduced. Furthermore this kind of process is not limited in process time because of no accumulation of vWF multimers occurs. Example 5

Another aspect of the inventions is a process wherein the retentate as obtained in any of Examples 2 and 3 is then additionally subjected to a second separation wherein now the ultra-large vWF multimers (ULMW vWF multimers) are enriched in the retentate, providing a second permeate in which the proportion of ultra-large vWF multimers in the total vWF multimer amount is reduced as compared to the proportion of ultra-large vWF multimers in the total vWF multimer amount in the retentate before said second separation. Bioreactor conditions and media are as described in previous examples. A second ATF system is connected to the bioreactor (Fig. 6) which is equipped with a hollow fiber membrane of MWCO of about 10,000,000 Da or Ο.ΐμιη. By harvesting the permeate of the second separation system with a flow rate of 20-200 % of the tank volume the ULMW vWF multimers is removed. As a result of such a fermentation comprising a first separation leading to an enrichment of the HMW multimers of vWF with a second separation leading to a depletion of the HMW multimers from ULMW vWF multimers generates a VWF preparation in the permeate of the second separation which is depleted both for LMW vWF multimers and for ULMW vWF multimers, then having both high activity in primary hemostasis but avoiding possible adverse effects by the presence of ULMW vWF multimers.

The two separations can be done in parallel or sequentially. Example 6 (Process of the invention)

The fermentation was performed using a Sartorius Biostat B-DCU Controller to control the pH at 7.0, the DO at 30% air saturation, the temperature and a stirrer speed of 150 rpm. The process was executed in a Sartorius B vessel with a working volume of 5L.

After the inoculation of the reactor a growth phase at 37°C was performed, followed by a production phase at 33,5°C. The process was performed with a CHO-cell line expressing a wild-type full length vWF (NCBI Sequence NP_000543). The cell line was obtained as described in WO 2009/156137.

The retention module, an ATF 4 (Refine Technology) was operated with an exhaust flow rate of 3 L/min and a pressure flow rate of 3 L/min. A 750 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane obtained from GE Healthcare Life Sciences operated in ATF flow mode was used to enrich the high molecular weight (HMW) vWF in the bioreactor and to reduce the low molecular weight (LMW) vWF in the bioreactor.

A CD CHO (Gibco) based culture media was used during growth phase and also as feed media. The culture was started with 7.6* 10 ⁵ cells/mL.

After a growth phase (data not shown) of 1 day the ATF was started with a harvest rate for permeate of about 100% of the tank volume per day. Additionally the temperature was shifted from 37°C to 33.5°C for the entire production phase. At this time the cell density in the bioreactor was 10.9* 10 ⁵ cells/mL. At day 7 the cell density reached 70.0* 10 ⁵ cells/mL

A sample from the bioreactor and of the permeate was taken daily and the vWF ristocetin cofactor activity (vWF:RCoF) and vWF antigen (vWF:Ag) was determined.

From these measurements the ratio vWF:RCoF/vWF:Ag was calculated. It was shown that due to the removal of the LMW vWF the ratio vWF:RCoF/vWF:Ag in the retentate (bioreactor) was rising, whereas the ratio vWF:RCoF/vWF:Ag in the permeate was decreasing due to the removal of the HMW vWF (Fig. 9).

A vWF multimer analysis (multimer electrophoresis) was performed at day 7 according to Ott et al. (Am J Clin Pathol 2010;133:322-330) to show the enrichment of high molecular-weight vWF in the retentate (bioreactor) and the separation and removal of low-molecular-weight vWF in the permeate. In addition to the multimer analysis of samples of the retentate and of the permeate a multimer analysis of a sample of standard human plasma (SHP) (Siemens, Standard Human Plasma, ORKL17) was performed according to Ott et al. (Am J Clin Pathol 2010;133:322-330) and used as a reference in each individual blot. The "accumulated pixel intensity related to standard human plasma" was then calculated by dividing the pixel intensity separately for each for the band sections 1-5, 6-10 and >11 in the respective sample by the average pixel intensity of the corresponding band sections of standard human plasma, wherein both individual accumulated pixel intensities were determined according to Ott et al. (Am J Clin Pathol 2010;133:322-330) (Table 4, Fig. 8).

Table 4 (vWF multimer analysis):

Accumulated pixel Accumulated pixel Accumulated pixel intensity related to intensity related to intensity related to standard human standard human standard human plasma plasma plasma

of bands >11 of bands 6-10 of bands 1-5

Retentate 111,54 92,2 97,9

Permeate 96,44 86,0 108,4

Ratio

Retentate/Permeate 1,16 1,07 0,90