MODIFIED GRANULOCYTE COLONY STIMULATING FACTOR (G-CSF)

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FIELD OF THE INVENTION

[0001] This invention relates to novel protein conjugates, particularly pegylated proteins, and their methods of making and use. One aspect of the present invention relates to pegylated-G-CSF having unexpected efficacy and stability.

BACKGROUND

[0002] One of the most serious potential side effects of many types of chemotherapy drugs is a low white blood cell count (neutropenia). Neutropenia can put some patients at risk for severe infections and may interrupt chemotherapy treatment. In fact, complications associated with a low white blood cell count are the most common causes of dose reductions or delays in chemotherapy (see Link, et al. (2001) Cancer.;92: 1354-1367; Lyman, et al. (2003) J Clin Oncol. 21 :4524-4531; and Lyman, et al. (2002) Am J Med. 112:406-411).

[0003] Granulocyte colony stimulating factor (G-CSF) is a major regulator of the development of antibacterial neutrophilic granulocytic leukocytes (neutrophils). The mouse version of G-CSF was purified from explanted tissues in 1983 and the human equivalent purified from a cancer cell line grown in culture inadveretently expressing G-CSF in high concentration sin 1985 (see e.g. Welte, et al. (1985) PNAS USA 82:1526-30). The human G- CSF was found to be a glycoprotein around 19kD which was variably acidic depending on the carbohydrate component. It was later found that the carbohydrate component was optional for biologic activity. The cloning and characterization of human recombinant G-CSF took place between 1984 and 1986 and led to its expression in E. coli cells and eventually to human clinical trials testing the compound in patients suffering from chemotherapy-induced neutropenia. In 1991, recombinant human G-CSF made in E. coli was approved by the U.S. FDA for this use (named Filgrastim, tradenamed Neupogen®) and in 1993 a related Chinese hamster ovary cell expressed form was approved in Europe (under the name lenograstim). It was found that the core protein included 174 amino acids, although multiple variants are known to exist (see e.g. Ngata, et al. (1986) Nature 319:415-18; Souza, et al. (1986) Science 232:61-5; U.S. Patent No. 4,999,291).

[0004] U.S. Patent Nos. 4,810,643, 4,999,291, 5,582,823 and 5,580,755, assigned to

Amgen, Inc. and claiming priority back to U.S. Patent Application 07/768,959, filed August 23, 1985, provide certain human pluripotent G-CSF molecules and methods of their production. These molecules form the basis for the approved Neupogen and Neulastin products. There is no discussion of potential pegylation of the molecule in these cases.

[0005] In recent years, non-antigenic water-soluble polymers, such as polyethylene glycol ("PEG"), have been used for the covalent modification of polypeptides of therapeutic and diagnostic importance. PEG is a polymer that is nontoxic, nonimmunogenic, highly water soluble, and readily cleared from the body. PEG has many applications and is commonly used in foods, cosmetics, beverages, and prescription medicines. Pharmaceutical grade PEGs are approved for use in the United States by the FDA and are widely used as

biopharmaceutical carriers, given their high degree of biocompatibility. PEGylation can modify certain characteristics of biopharmaceuticals without altering their function, thereby enhancing the therapeutic effect.

[0006] Generally, polyethylene glycol molecules are connected to the protein via a reactive group found on the protein. Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such attachment. PEG molecules have been attached through amino groups on polypeptides using methoxylated PEG ("mPEG") having different reactive moieties. Such polymers include mPEG-succinimidyl succinate, mPEG-succinimidyl carbonate, mPEGimidate, and mPEG-cyanuric chloride.

[0007] PEG can be coupled to active biopharmaceuticals through the hydroxyl groups at the ends of the polymer chain using a variety of chemical methods. For example, covalent attachment of PEG to therapeutic polypeptides such as interleukins (Knauf, M. J. et al., J. Biol. Chem. 1988, 263, 15,064; Tsutsumi, Y. et al, J. Controlled Release 1995, 33, 447), interferons (Kita, Y. et al, Drug Des. Delivery 1990, 6, 157), catalase (Abuchowski, A. et al, J. Biol. Chem. 1977, 252, 3, 582), superoxide dismutase (Beauchamp, C. O. et al, Anal. Biochem. 1983, 131, 25), and adenosine deaminase (Chen, R. et al., Biochim. Biophy. Acta 1981, 660, 293), has been reported to extend their half life in vivo, and/or reduce their immunogenicity and antigenicity.

[0008] U.S. Patent No. 4,002,531 describes the attachment of an aldehyde-linked

PEG to the enzyme tripsin. The attachment of the aldehyde is random via an acylation reaction, yielding in an amide linkage between the PEG and the enzyme. Similarly, reductive alkylation of lymphokines at neutral pH was described in European Patent Publication EP 0 154 316, which indicated that approximately 33% of the eleven lysine residues in the molecule were being modified. European Publication EP 0 335 423 describes the acylation of G-CSF using a PEG derivative.

[0009] To improve the stability and bioavailability, and ensure activity of peptides, site specific rather than random pegylation is desirable. Site-specific pegylation at the N- terminus, side chain and C-terminus of a potent analog of growth hormone-releasing factor has been performed through solid-phase synthesis (Felix, A. M. et al., Int. J. Peptide Protein Res. 1995, 46, 253).

[0010] PCT WO 2006/094530 to Siegried, Ltd., describes certain di-pegylated protein conjugates and processes for their preparation. Examples of dipegylated G-CSF are provided, in which the products are dipegylated at both the N-terminus and Lysl7 or at the N-terminus and Lys35.

[0011] Because Filgrastim was readily degraded in vivo, it was necessary to provide repeated daily injections during the course of chemotherapy. Therefore, there was a need to increase the pharmaceokinetic profile of Filgrastim. Using site specific pegylation at the N- terminus of G-CSF using aldehyde-activated PEG (see PCT Publication No. WO 96/11953 as well as U.S. Patent Nos. 5,824,784 and 7,090,835), PEG-Filgrastim was developed. This compound was approved by the U.S. FDA in 2002 under the tradename Neulasta®.

[0012] Although PEG-Filgrastim was developed using site-specific attachment through an amine bond to the N-terminal, such reactions require long reaction times and are heavily dependent on an acidid pH. WO 96/11953 describes that the reaction conditions are designed to permit the selective attachment of the polymer to the N-terminus of the protein. The characterization revealed that at the conditions described, in which the pH was adjusted to 4.0, the compositions contained primarily monopegylated products in which the PEG was attached to the N-terminus, Lys35 or Lys41. The composition of the pegylated G-CSF prepared using these processes has been confirmed, for example in Cindric, et al. (2007) J. Pharm. Biomed. Analys. 44:388-395.

[0013] Several alternate strategies for providing a stabilized G-CSF molecule have been proposed. Linking PEG to a cysteine residue has provided certain improvements in targeting. Thiol reactive PEGs (including PEG-maleimide) have been linked to G-CSF at its free cysteine residue. Veronese, et al. (2007) Bioconjugate Chem. 18: 1824-1830 described the pegylation of G-CSF at Cysl7, which was shown to increase aggregation although the aggregates were not covalently aggregated. Similarly, Hao, et al. (2006) Biodrugs 20:357-363 described the conjugation of PEG-maleimide to Cysl7, which was shown to increase the half life of the molecule.

[0014] Site-specific mutagenesis is a further approach which has been used to prepare polypeptides for site-specific polymer attachment. For example, U.S. Patent No. 6,646,110 describes polypeptide conjugates that exhibit G-CSF activity and have an amino acid residue that comprise and attachment group for a PEG or oligosaccharide moiety inserted. These can include lysine, glutamic acid, cysteine or aspartic acid.

[0015] As such, there remains a need for a pegylated composition, and/or method of production thereof, that produce a predictable and consistent product without the

complications of N-terminal targeting which retains the activity of the original molecule

SUMMARY OF THE INVENTION

[0016] The present invention is based on the production of novel forms of monopegylated proteins, e.g., granulocyte colony stimulating factor ("G-CSF") that are

unexpectedly active. The use of dimethyl sulfoxide ("DMSO") in the methods of producing PEG-linked proteins by reductive alkylation drives the PEG conjugation only to sites at or near the N-terminus. The process generally can drive the PEG to the lysine residue that is closest to the N-terminus. In particular, the invention provides a G-CSF molecule that includes a PEG-molecule linked to Lysl6 (Lys 17 if a terminal methionine is included). The invention also provides a method of linking an aldehyde-reactive PEG to a protein in the presence of DMSO.

[0017] In certain embodiments, the invention relates to a composition comprising at least one population of G-CSF proteins wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule and at least a 30% of the composition is not N- terminal pegylated. In a specific embodiment, each G-CSF molecule is covalently linked to a single polyethylene glycol molecule ("monopegylated G-CSF") and at least 30%> of the composition is not N-terminally pegylated. In another embodiment, the composition comprises at least 80%> monopegylated G-CSF wherein at least 30%> of the composition is not N-terminally pegylated. In further embodiments, the composition comprises at least 85%, at least 90%) or at least 95% monopegylated G-CSF molecules wherein at least 30%> of the composition is not N-terminally pegylated.

[0018] In specific embodiments of the composition, each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule through a particular lysine residue. In certain embodiments, the invention comprises a composition having at least one G- CSF molecule covalently linked to at least one polyethylene glycol molecule via the amino terminus of the GCSF protein through an amine linkage. In other embodiments, the invention comprises a composition having at least one G-CSF molecule covalently linked to at least one polyethylene glycol molecule through Lysl6 (Lysl7 if an N-terminal methionine residue is counted).

[0019] In some embodiments of the composition, the ratio of the N-terminally pegylated molecules to a second population, especially a second population at Lysl6 can range from less than about 1 to about 100, about 10 to about 90, about 20 to about 80, about 30 to about 70, about 40 to about 60, about 50 to about 50, about 60 to about 40, about 70 to about 30, wherein less than about 1 includes an amount undetectable using standard methods known in the art.

[0020] In certain embodiments, a substantially homogeneous composition is provided comprising a monopegylated protein wherein at least 30% of the protein molecules are not pegylated at the N-terminal. In certain specific embodiments, the composition comprises at least 30% monopegylated proteins which are pegylated at a lysine residue within 100 amino acids of the N-terminal. In specific embodiments, the lysine is within 80, within 70, within 60, within 50, within 40, within 30, within 20, within 19, within 18, within 17, within 16, within 15, within 14, within 13, within 12, within 11 , within 10, within 9, within 8, within 7, within 6, within 5, within 4, within 3 or within 2 amino acid residues from the N-terminal.

[0021] In certain embodiments, the invention relates to a pharmaceutical formulation comprising at least one population of G-CSF proteins wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule, optionally in a

pharmaceutically acceptable carrier. In certain embodiments, the carrier is substantially protein free. In certain embodiments, the pharmaceutical formulation comprises at least one population of G-CSF proteins wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule and at least a 30% of the composition is not N-terminal pegylated. In a specific embodiment, each G-CSF molecule is covalently linked to a single polyethylene glycol molecule ("monopegylated G-CSF") and at least 30%> of the composition is not N-terminally pegylated. In another embodiment, the formulation comprises at least 80% monopegylated G-CSF wherein at least 30% of the composition is not N-terminally pegylated. In further embodiments, the formulation comprises at least 85%, at least 90% or at least 95% monopegylated G-CSF molecules wherein at least 30%> of the composition is not N-terminally pegylated.

[0022] In specific embodiments of the pharmaceutical formulation, each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule through a particular lysine residue. In certain embodiments, the formulation includes at least one G- CSF molecule covalently linked to at least one polyethylene glycol molecule via the amino terminus of the GCSF protein through an amine linkage. In other embodiments, the formulation comprises at least one G-CSF molecule covalently linked to at least one polyethylene glycol molecule through Lysl6 (Lysl7 if an N-terminal methionine residue is counted).

[0023] In some embodiments of the formulation, the ratio of the N-terminally pegylated molecules to a second population, especially a second population at Lysl6 can range from less than about 1 to about 100, about 10 to about 90, about 20 to about 80, about 30 to about 70, about 40 to about 60, about 50 to about 50, about 60 to about 40, about 70 to about 30, wherein less than about 1 includes an amount undetectable using standard methods known in the art.

[0024] The G-CSF molecules and compositions of the invention are expected to exhibit prolonged stability under standard storage conditions, i.e., storage at standard temperature, e.g., about 25°C for at least three months. In certain embodiments, the protein- free carrier is serum-free, albumin-free or human serum albumin-free ("hsa-free")). In certain embodiments, the pharmaceutical formulations may be stored for extended period of time without substantial and/or detectable degradation of G-CSF as determined by methods described herein and/or known in the art. In certain embodiments, the pharmaceutical formulations of the invention are stable (i.e., do not exhibit detectable and/or do not exhibit substantial degradation) as determined at least 15 months after storage at about -20°C or 4°C. In other embodiments, the pharmaceutical formulations of the invention are stable (i.e., do not exhibit detectable and/or do not exhibit substantial degradation) as determined at least 10 months after storage at about 25°C or about 37°C. The stability of pharmaceutical

formulations of the invention may be assessed by any method known in the art. In certain embodiments, the stability of the pharmaceutical formulations of the invention is assessed by monitoring alteration in the protein concentration over time as determined by a bicinchoninic acid ("BCA") protein assay. In other embodiments, the stability of the pharmaceutical formulations of the invention is assessed by indication of protein degradation (i.e., G-CSF conjugate degradation) over time as determined by SDS PAGE analysis. In certain other embodiments, the stability can be measured by analyzing breakdown of the product by an HPLC. In still other embodiments, the stability of the pharmaceutical formulations of the invention is assessed by monitoring the activity of said formulation over time, wherein said activity is determined by any in vitro or in vivo method known in the art for determination of activity of said formulation {e.g., G-CSF activity). In a specific example in accordance with this embodiment, the activity of a pharmaceutical formulation of the invention comprising a plurality of G-CSF conjugates is evaluated by the ability of said pharmaceutical formulation in an invitro bioassay utilizing a G-CSF dependent clone of murine 32D cells. In other embodiments, the acitivyt of the formulation is measured in vivo in its capacity to alter white blood cell count in experimental animals, such as hamsters.

[0025] In certain embodiments, a pharmaceutical formulation is provided comprising a monopegylated protein wherein at least 30% of the protein molecules are not pegylated at the N-terminal. In certain specific embodiments, the formulation comprises at least 30% monopegylated proteins which are pegylated at a lysine residue within 100 amino acids of the N-terminal. In specific embodiments, the lysine is within 80, within 70, within 60, within 50, within 40, within 30, within 20, within 19, within 18, within 17, within 16, within 15, within 14, within 13, within 12, within 11, within 10, within 9, within 8, within 7, within 6, within 5, within 4, within 3 or within 2 amino acid residues from the N-terminal.

[0026] In another aspect of the invention, a method of increasing white cell count in a host is provided comprising administering a pharmaceutical formulation of the invention to a host in need thereof. In certain embodiments, the host is human. In certain embodiments, the hosts are at risk of or suffering from neutropenia. In certain other embodiments, the hosts are being treated with an agent that decreases their white blood cell count. In certain

embodiments, the hosts have decreased endogenous levels of G-CSF. In certain other embodiments, the hosts are undergoing radiation treatment. The hosts can be suffering from lung cancer, lymphoma, breast cancer, bone marrow transplantation, testicular cancer, AIDS- related malignancies, myleodysplasitc disorders, acute leukemia, congenital and cyclic neutropenias or aplastic anemia (see Mortsyn, et al.(1998) Filgrastim (r-metHuG-CSF). In Clinical Practice, 2 ^nd Ed., Marcel Dekker, Inc., New York, NY). In certain embodiments, the formulation is administered to a patient at risk of infection.

[0027] In certain embodiments, the formulation is provided in a single dose during a course of chemotherapy. In some embodiments, the formulation is provided as multiple doses over the course of chemotherapy. In certain embodiments, the formulation is administered once daily, once weekly, once every two weeks or once a month. The formulation can be administered within twenty four hours of a dose of chemotherapy. In certain embodiments, the formulation is administered at least 14 days before a dose of chemotherapy. [0028] In certain embodiments, the formulation is administered as an injection. In some embodiments, the formulation is suitable for subcutaneous administration. In other embodiments, the formulation is suitable for intravenous administration. The formulation can also be provided as an orally available form, receives a dose at least about once a week. In other embodiments, the patient receives a dose at least about once every two weeks, at least about once every three weeks, or at least about once every month.

[0029] Another aspect of the invention relates to a process of making a protein conjugate and a conjugate made by the process comprising, reacting a protein with an activated polyethylene glycol-aldehyde in a reaction buffer comprising DMSO to covalently link the protein with the activated water-soluble polymer. The process can also include removing substantially all unlinked water-soluble polymer to obtain said protein conjugate. In certain embodiments, the polyethylene glycol-aldehyde is:

. In certain embodiments, the

PEG is at least lOkD. In certain other embodiments, the PEG is at least 20kD. In specific embodiments, the PEG is 30kD or 40kD. The PEG can be linked to the protein using any linker, including an alkyl linkage of from 1 to 30, 1 to 20, 1 to 15, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 carbon atoms.

[0030] The process of the invention may allow the use of a reaction at a higher pH than other methods known in the art, such as substantially at neutral pH (about pH 7.0) (e.g., pegylation of a protein using aldehyde PEG (see, e.g., U.S. Patent No. 5,824,784). Such modifications relative to methods known in that art may provide manufacturing advantages in terms of costs, manufacturing efficiency, and/or ease of process.

[0031] In certain embodiments, the reaction is carried out at a pH of between 3 and 8.

In certain other embodiements, the reaction is carried out at a pH of between about 4 and about 7, or about 4.5 and about 6 or about 5. In certain other embodiments, the reaction is carried out at a pH about neutral. In certain other embodiments, the reaction is carried out at a slightly acidic pH, such as, for example, about 5 or about 6.

[0032] In yet a further embodiment, the reaction buffer comprises a molar ratio of protein to activated water-soluble polymer of about 1 to about 3 to about 1 to about 60. In other embodiments, the reaction buffer comprises a molar ratio of protein to activated water- soluble polymer of about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 30, about 1 to about 35, about 1 to about 40, about 1 to about 45, about 1 to about 50, about 1 to about 55, about 1 to about 60. In certain embodiments, the reaction buffer comprises a molar ratio of protein to activated water- soluble polymer of about 1 to about 7. In still a further embodiment, the removing of substantially all unreacted water-soluble polymer can be accomplished by methods known in the art such as, for example, dialysis or chromatography.

[0033] Additional advantages of the present invention will become readily apparent to those skilled in this art from the following detailed description, wherein only certain embodiments of the invention is shown and described. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of routine modifications in various respects, all without departing from the invention. The present invention may be practiced without some or all of these specific details. Accordingly, the description ise to be regarded as illustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS:

[0034] FIG. 1. Amino acid sequence of the predominant, fully processed human granulocyte colony stimulating factor ("G-CSF") (SEQ ID NO:l). DNA sequence disclosed as SEQ ID NO: 2.

[0035] FIG. 2. Sequencing data for G-CSF protein used in Examples. FIG. 2 discloses SEQ ID NOS 3 and 4, respectively, in order of appearance.

[0036] FIG. 3. Polyacrylamide Gel Electrophoresis of purified d-peg-GCSF produced according to the inventions. Lane 1 : Molecular weight size marker (in kD, 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31 21.5, 14.4); Lane 2: Purified d-peg-GCSF; Lane 3: Unmodified GCSF

[0037] FIG. 4. Polyacrylamide Gel Electrophoresis of purified peg-GCSF produced by existing method without DMSO, but with the sample PEG and reaction conditions. Lane 1: Molecular weight size marker (in kD, 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31 21.5, 14.4), Lane 2: Purified peg-GCSF

[0038] FIG. 5. Proliferative Effect of d-PEG-GCSF vs Reference (Neulasta) on

NFS60 cells.

[0039] FIG. 6. Proliferative Effect of d-PEG-GCSF vs Reference (Neulasta) on

NFS60 cells

[0040] FIG. 7. Proliferative Effect of d-PEG-GCSF vs Neulasta on NFS60 cells [0041] FIG. 8. In vivo assay of ANC over 5 day period after injection of the test compounds(d-PEG-GCSF vs Reference).

DETAILED DESCRIPTION

[0042] The present invention is based on the production of novel forms of mono- pegylated proteins, e.g., granulocyte colony stimulating factor ("G-CSF") that are

unexpectedly active. The use of dimethyl sulfoxide ("DMSO") in the methods of producing PEG-linked proteins by reductive alkylation drives the PEG conjugation only to sites at or near the N-terminus. In particular, the invention provides a G-CSF molecule that includes a PEG-molecule linked to Lysl6 (Lys 17 if a terminal methionine is included). The invention also provides a method of linking an aldehyde-reactive PEG to a protein in the presence of DMSO. The differentiated modification is such that the activity of the original molecule is preserved and the pharmacological profile of the molecule is improved.

DEFINITIONS

[0043] As used herein, the term "N-terminus," "amino-terminus," or analogous terms when used in the context of a covalent linkage of a protein to another molecule refer to a covalent linkage via the amino-terminal a-amino group of the protein.

[0044] As used herein, the term "wild type" or "native" refers to a protein or polypeptide in its operative or functional form, typically as it is found naturally functioning in the body. These terms also refer to the protein in a form in which it has not been artificially modified or altered. The terms can thus relate to recombinant proteins. Accordingly, the terms can refer to a protein with an altered glycosylation pattern, including lack of glycosylation, relative to that as produced in the animal from which the nucleic acid and/or amino acid sequence of the protein was originally derived.

[0045] As used herein, the term "G-CSF" or granulocyte colony stimulating factor, unless otherwise specified, refers to a protein having the amino acid sequence set out in SEQ ID NO: 1 (FIG. 1) or an amino acid sequence substantially homologous thereto, whose biological properties relate to the stimulation of white blood cell production. As used herein, these terms include such proteins modified deliberately, as for example, by site directed mutagenesis or accidentally through mutations; such that they have additions, deletions, or substitutions of amino acid residues with respect to native G-CSF. These terms include both natural and recombinantly produced human G-CSF. G-CSF refers to both the naturally occurring or recombinant protein, typically human, as obtained from any conventional source such as tissues, protein synthesis, cell culture with natural or recombinant cells.

[0046] "Substantially homologous" in reference to an amino acid sequence is defined herein as a sequence with at least 70%, typically at least about 80%, and more typically at least about 90% identity to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448

(1988).

[0047] As used herein, the term "conjugate" in reference to a protein or polypeptide is a protein or polypeptide or population thereof, that functions in interaction with one or more other chemical groups attached by covalent bonds.

G-CSF MOLECULES

[0048] Proteins which can be conjugated with polymer units according to the present invention include non-mutated and mutated proteins such as but not limited to growth factors, antibodies, hormones, in particular therapeutically active proteins such as but not limited to erythropoietin, interferon alpha, interferon beta, interferon gamma, consensus interferon, G- CSF, GM-CSF, hemoglobin, interleukins such as interleukin-2 and interleukin-6, tumour necrosis factor, various cytokines, growth factors such as human growth factor and epidermal growth factor, immuno-globulins such as IgG, IgE, IgM, IgA, IgD and/or structural and/or functional variants and/or fragments thereof as well as their proteinoids or synthetic proteinlike forms.

[0049] In general, protein such as G-CSF useful in the practice of this invention may be of any form isolated from mammalian organisms, a product of prokaryotic or eukaryotic host expression of exogenous DNA sequences obtained by genomic or cDNA cloning or by DNA synthesis or alternatively a product of chemical synthetic procedures or by endogenous gene activation. Thus, the protein can be of a natural or recombinant source obtained from tissue, mammalian- microbial cell cultures, plant cell cultures, transgenic animals, yeasts, fungi and/or transgenic plants. Suitable prokaryotic hosts include various bacteria such as E. coli; suitable eukaryotic hosts include yeasts such as S. cerevisiae or Pichia pastoris, mammalian cells such as Chinese hamster ovary cells or monkey cells, transgenic animals such as mice, rabbit, goat, sheep, plant cell culture and transgenic plants such as

Physcomitrellapatens (a moss). Depending upon the host employed, the protein expression product may be glycosylated with mammalian, plant or other eukaryotic carbohydrates, or it may be non-glycosylated. [0050] If the protein is G-CSF, the G-CSF expression product may also include an initial methionine amino acid residue at position 1. The present invention contemplates the use of any and all such forms of G-CSF, although recombinant G-CSF, especially E.coli- derived, is typical. Certain G-CSF analogues have been reported to be biologically functional, and these may also be conjugated according to the present invention. These G-CSF analogues may include those having amino acid additions, deletions and/or substitutions as compared to the G-CSF amino acid sequence according to SEQ ID No. 1. In certain embodiments, the sequence includes an insertion of amino acids as compared to SEQ ID No. 1, such as, for example, an insertion of VSE at positions 36, 37 and 38 of SEQ ID No. 1. In certain embodiments, the sequence is as in SEQ ID No. 2 (Figure 2).

[0051] Typically, the protein is one having the activity of G-CSF, including mutants of G-CSF, glycosylated G-CSF, non-glycosylated G-CSF and/or otherwise modified structural andlor functional variants of G-CSF. In a further embodiment, the protein has the amino acid sequence of G-CSF identified in SEQ ID NO. 1 which corresponds to

recombinant G-CSF produced in bacteria, having 174 amino acids and an extra N-terminal methionyl residue. Amino acid sequences of biological active G-CSF, which differ from SEQ ID NO. 1 in that they do not contain a methionyl residue at position 1, are also included.

G-CSF CONJUGATES

[0052] In certain embodiments, the invention relates to a composition comprising at least one population of G-CSF proteins wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule and at least a 30% of the composition is not N- terminal pegylated. In a specific embodiment, each G-CSF molecule is covalently linked to a single polyethylene glycol molecule ("monopegylated G-CSF") and at least 30% of the composition is not N-terminally pegylated. In another embodiment, the composition comprises at least 80%> monopegylated G-CSF wherein at least 30%> of the composition is not N-terminally pegylated. In further embodiments, the composition comprises at least 85%, at least 90%> or at least 95% monopegylated G-CSF molecules wherein at least 30% of the composition is not N-terminally pegylated.

[0053] In specific embodiments of the composition, each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule through a particular lysine residue. In certain embodiments, the invention comprises a composition having at least one G- CSF molecule covalently linked to at least one polyethylene glycol molecule via the amino terminus of the GCSF protein through an amine linkage. In other embodiments, the invention comprises a composition having at least one G-CSF molecule covalently linked to at least one polyethylene glycol molecule through Lysl6 (Lysl7 if an N-terminal methionine residue is counted).

[0054] In some embodiments of the composition, the ratio of the N-terminally pegylated molecules to a second population, especially a second population at Lysl6 can range from less than about 1 to about 100, about 10 to about 90, about 20 to about 80, about 30 to about 70, about 40 to about 60, about 50 to about 50, about 60 to about 40, about 70 to about 30, wherein less than about 1 includes an amount undetectable using standard methods known in the art.

[0055] In certain embodiments, a substantially homogeneous composition is provided comprising a monopegylated protein wherein at least 30% of the protein molecules are not pegylated at the N-terminal. In certain specific embodiments, the composition comprises at least

30% monopegylated proteins which are pegylated at a lysine residue within 100 amino acids of the N-terminal. In specific embodiments, the lysine is within 80, within 70, within 60, within 50, within 40, within 30, within 20, within 19, within 18, within 17, within 16, within 15, within 14, within 13, within 12, within 11, within 10, within 9, within 8, within 7, within 6, within 5, within 4, within 3 or within 2 amino acid residues from the N-terminal.

[0056] Most polypeptides have a plurality of potential PEG linkage sites. Therefore, although a homogenous population of IPEG-protein conjugates has one PEG molecule linked to each protein molecule, that linkage may not necessarily be in the same location on each protein in the population. Similarly, although a homogenous population of dipegylated protein conjugates has two PEG molecules linked to each protein molecule, those linkages may not necessarily be in the same locations on each protein in the population.

[0057] Several studies have indicated that pegylation at Lysl7 may be detrimental to the activity of recombinant G-CSF. For instance, scanning mutagenesis described by

Reidhaar-Olson et al. ((1996) Biochemistry 35:9034-9041) revealed that lysine 35 and lysine 41 are critical residues for biological activity, and structural simulation studies revealed that the pegylation of G-CSF at lysine 17 and lysine 24 might potentially result in a protein of lower biological activity due to their location within the region of protein receptor interaction. An attachment of a polymer moiety such as polyethylene glycol at these sites could therefore sterically hinder the receptor binding. In addition, Aritomi, et al. (1999) Nature 401 :713-717 assumed a negative impact of the Lysl7 modification on the biological activity of the protein. PEG MOLECULES

[0058] For purposes of this invention, the polymer-protein conjugates are typically prepared by reductive alkylation. Thus, the present invention provides polymer-protein conjugates, wherein at least one nitrogen atoms of amino groups of the protein are each conjugated with a polymer unit via an amine linkage. In the context of the present invention, the term amino group includes primary and secondary amino groups and in particular NH- or NH ₂ - groups in side-chains of amino acids such as NH ₂ -groups in the side-chain of lysine, NH- or NH ₂ -groups in the guanidino group of arginine or NH-groups in the imidazole side- chain of histidine.

[0059] The polymer unit generally comprises at least one polymer moiety and a linker moiety, which is between at least the one polymer moiety and the amine linkage. The linker moiety may be linear or branched. If the linker moiety is branched, a polymer unit may comprise more than one polymer moiety. The linker moiety is typically an aliphatic linker moiety. Suitable aliphatic linker moieties also include substituted alkyl diamines and triamines, lysine esters and malonic ester derivatives. The linker moieties are usually non- planar, so that the polymer chains are not rigidly fixed. Often, the linker moiety includes a multiple-functionalized alkyl group containing up to 18, and more typically from 1 to 10 carbon atoms. A hetero-atom such as nitrogen, oxygen or sulfur may also be included within the alkyl chain. The linker moiety may be branched, for example at a carbon or nitrogen atom. Examples for branched linker moieties and the resulting branched polymer units as well as methods for their preparation are described in WO 95/11 1924 and WO 03/1049699.

[0060] In one embodiment of the present invention, the linker moiety comprises at least one methylene group attached to the nitrogen atom of the amine linkage, e.g. from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, or less, such as 4, 3, 2 or 1 and most typically two methylene groups which are directly attached to the nitrogen atom of the amine linkage.

[0061] The polymers encompassed by instant invention include, but are not limited to, polyalkylene glycol and derivatives thereof, including PEG, methoxylated PEG

("mPEG"), PEG homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group. In certain embodiments, the polymer is mPEG and most often mono-methoxylated PEG. The water soluble polymers can be linear, branched, or star- shaped with a wide range of molecular weights. The size of the PEG can range from 10 to about 100 kD. In specific embodiments, the size of the PEG is about 10 to about 50 kD. In some embodiments, the molecular weight of a polyethylene glycol moiety attached to a amino group is from 2 to 100 kDa, more typically from 5 to 60 kDa and most typically from 10 to 30 kDa. In a further embodiment of the present invention, the number n of ethylene oxide residues in a polyethylene glycol moiety is from about 40 to about 2270, more typically from about 110 to about 1370 and most typically from about 225 to about 680.

[0062] The polymer moiety is usually a substantially non-antigenic or non- immunogenic polymer chain. Further, the polymer moieties used are typically selected from among water-soluble polymer moieties. This has the advantage that the protein to which the water-soluble polymer moieties are attached or conjugated does not precipitate in an aqueous environment such as a physiological environment. For reductive alkylation, the polymer selected should further have a single reactive aldehyde, so that the degree of polymerization may be controlled as provided for in the present processes. The polymer unit as well as the polymer moiety may be branched or unbranched. Typically, for therapeutic use of the end- product preparation, the polymer will be pharmaceutically acceptable. One skilled in the art will be able to select the desired polymer moiety based on considerations such as whether the polymer-protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis and other considerations.

[0063] Typically, the polymer moiety may be selected from the group consisting of polyalkylene glycol moieties, polysaccharide moieties such as dextran and its derivatives, polysaccharide and its derivatives, pyrrolidone moieties such as polyvinyl pyrolidone, cellulose moieties such as carboxymethyl cellulose, polyvinyl alcohol, poly- 1,3-dioxolan, poly- 1,3,6- trioxane, ethylene-maleic anhydride copolymer, polyaminoacid moieties and/or polyacrylamide moieties and/or other similar non-immunogenic polymer moieties (either homopolymers or random copolymers) and/or derivatives thereof. Such polymers are also capable of being functionalized or activated for inclusion in the present invention.

[0064] In particular, the polymer moiety is a polyalkylene glycol moiety. The term polyalkylene glycol designates polyalkylene glycol radicals or polyalkylene glycol moieties, where the alkylene radical is a straight or branched chain radical. The term polyalkylene glycol also comprises polyalkylene glycols formed from mixed alkylene glycols such as polymers containing a mixture of polyethylene and polypropylene radicals and polymers containing a mixture of polyisopropylene, polyethylene and polyisobutylene radicals. A polyalkylene glycol moiety in the polymer protein conjugates according to the present invention is usually a polyethylene glycol moiety or polyethylene glycol residue formed by removal of the two terminal hydroxyl groups.

[0065] Within this group are a-substituted polyalkylene oxide derivatives such as methoxy polyethylene glycols (mPEG) or other suitable alkyl substituted polyalkylene oxide derivatives such as those containing mono- or bis-terminal C ₁ -C4 groups. Straight-chained non-antigenic polymers such as monomethyl polyethylene glycol homopolymers are typical. Alternative polyalkylene oxides such as other polyethylene glycol homopolymers,

polyethylene glycol heteropolyrners, other alkyl polyalkylene oxide block copolymers and copolymers of block copolymers of polyalkylene oxides are also useful.

[0066] To effect covalent attachment of polyethylene glycol (PEG) and similar poly

(alkylene oxides) to a molecule, in particular, a protein, the hydroxyl end groups of the polymer must first be converted into reactive functional groups. This process is referred to herein as "activation" and the product is called "activated PEG." For example, methoxylated PEG ("mPEG") can be activated for subsequent covalent attachment to amino groups by methods well known in the art, i.e., mPEG can be modified to contain varying reactive moieties suitable for subsequent attachment to proteins via amino acid residues containing available amino residues, e.g., lysinyl residues.

[0067] The PEG reagent used in the process according to the present invention is usually a reagent having the formula: [R-L ¹ -(CH ₂ -CH ₂ -0), _n ]y-L ² -(CH ₂ ) _m -i-CFiO, wherein R is H, a lower alkyl, aryl or any suitable protecting group; n is an integer representing the number of ethylene oxide residues in a polyethylene glycol moiety; m is an integer representing the number of methylene groups; L ¹ is 0 , N, S andor a branched or non-branched linker moiety which can be absent or present; L ² is a branched or non-branched linker moiety which can be absent or present; and y is an integer with the proviso that y is 1 in the absence of L ² and y is at least 1 in the presence of L ² . In specific embodiments, the PEG is PEG acetaldehyde, most typically a methox -PEG acetaldehyde. The structure of the polyethylene gly col-aldehyde is:

PROCESS OF CONJUGATION [0068] A process of making a protein conjugate and a conjugate made by the process are provided comprising, reacting a protein with an activated polyethylene glycol-aldehyde in a reaction buffer comprising DMSO to covalently link the protein with the activated water- soluble polymer. The process can also include removing substantially all unlinked water- soluble polymer to obtain said protein conjugate. In certain embodiments, the polyethylene glycol-aldehyde is

. In certain embodiments, the PEG is

at least lOkD. In certain other embodiments, the PEG is at least 20kD. In specific

embodiments, the PEG is 30kD or 40kD.

[0069] The reducing agent used in the reactive alkylation is usually selected from, but not limited to, NaCNBH ₄ or NaBH ₄ .

[0070] Typically, the reaction is performed at a protein concentration from 0.5 to 100 mg/ml, more typically from 1 to 10 mg/ml and most typically from 3 to 7 mg/ml. The reaction can also be performed at a protein -to-polymer molar ratio of from 1 : 1 to 1 :400, and typically from 1 :5 to 1 :30 and most typically from 1 : 15 to 1 :30. In yet a further embodiment, the reaction buffer comprises a molar ratio of protein to activated water-soluble polymer of about 1 to about 3 to about 1 to about 60. In other embodiments, the reaction buffer comprises a molar ratio of protein to activated water-soluble polymer of about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 30, about 1 to about 35, about 1 to about 40, about 1 to about 45, about 1 to about 50, about 1 to about 55, about 1 to about 60. In certain embodiments, the reaction buffer comprises a molar ratio of protein to activated water-soluble polymer of about 1 to about 7. In still a further embodiment, the removing of substantially all unreacted water-soluble polymer can be accomplished by methods known in the art such as, for example, dialysis or chromatography.

[0071] Although any protein may be pegylated according to the methods described herein, the invention, in particular, encompasses the pegylation of therapeutic polypeptides. In certain embodiments, the therapeutic protein for use in accordance with the methods of the invention may be, e.g., a protease, pituitary hormone protease inhibitor, poietin, colony stimulating factor, hormone, clotting factor, anti-clotting factor, neurotropic factor, rheumatoid factor, CD protein, osteoinductive factor, interleukin, growth factor, interferon, cytokine, somatomedian, chemokine, immunoglobulin, gonadotrophin, interleukin, chemotactin, interferon, lipid-binding protein allergen, or a combination of the foregoing. Specific nonlimiting examples of such therapeutic proteins include, interferon-a2A, interferon-a2B, interferon β, interferon-γ, insulin- like growth factor- 1 (IGF-1), insulin- like growth factor-2 (IGF2), insulin, human growth hormone (hGH), transforming growth factor (TGF), erythropoietin (EPO), ciliary neurite transforming factor (CNTF), thrombopoietin (TPO), brain-derived neurite factor (BDNF), IL-1, insulintropin, IL-2, glial-derived neurite factor (GDNF), IL-1 RA, tissue plasminogen activator (tPA), superoxide dismutase (SOD), urokinase, catalase, streptokinase, fibroblast growth factor (FGF), hemoglobin, neurite growth factor, adenosine deamidase (NGF), granulocyte macrophage colony stimulating factor (GM-CSF), bovine growth hormone (BGH), granulocyte colony stimulating factor (G- CSF), calcitonin, platelet derived growth factor (PDGF), bactericidal/permeability increasing protein (BPI), L-asparaginase, arginase, uricase, γ-interferon, phenylalanine ammonia lyase, follicle stimulating hormone, proinsulin, epidermal growth factor, fibroblast growth factors, nerve growth factor (NGF), tumor necrosis factor, calcitonin, parathyroid hormone (PTH, including human PTH), bone morphogenic protein, hemopoietic growth factors, luteinizing hormone, glucagon, glucagon like peptide- 1 (GLP-1), peptide YY (PYY), factor VIIIC, factor IX, tissue factor, and von Willebrand factor, Protein C, atrial natriuretic factor, lung surfactant, bombesin, thrombin, enkephalinase, mullerian-inhibiting agent, relaxin A-chain, relaxin B-chain, prorelaxin, Dnase, inhibin, activin, vascular endothelial growth factor, integrin, protein A or D, bone-derived neurotrophic factor (BDNF), neurotrophin3, -4, -5, or - 6 (NT-3, NT-4, NT-5, or NT-6), CD-3, CD-4, CD-8, CD-19, M-CSF, GM-CSF, GCSF, biologically active fragments of any of the foregoing, or combinations of the foregoing.

[0072] In order to maintain the pH in the preferred range, the reaction may be performed in the presence of a buffer. For example, the buffer may be selected from a phosphate, acetate, HEPES, MES, or other similar buffers. The reaction buffer can generally be a standard buffer free of amine components, e.g., phosphate buffered saline (PBS). The reaction buffer usually includes a salt, e.g., Na, concentration of about 0.1 mM to about lOOmM, most often about lmM to about 50mM, and even more often about lOmM to about 20mM. In certain embodiments, the polypeptide is mixed with the dry activated water-soluble polymer under stirring. The reaction buffer usually has a pH of about 6.5 to about 8.5 or about 6.6 to about 7.5. In certain embodiments, the reaction buffer has a neutral pH of about 7.0.

[0073] The reaction buffer further comprises an organic solvent, and in particular, dimethyl sulfoxide ("DMSO"). The DMSO may be present in the reaction mixture in concentration of 5-80% and, typically 10-40% (v/v). DMSO is widely used as a general solvent, but not typically for affecting pegylation reactions, in particular, for preferentially affecting the resulting sites of pegylation of said reaction away from a protein N-terminal. PCT Publication WO 08/019214 describes a process of selective preparation of N-terminal modified erythropoietin in a DMSO-containing buffer. The PEG linkage provided a carbamate linked PEG-EPO selectively at the N-terminal of the protein.

[0074] In contrast, the reaction conditions described herein, including use with an aldehyde-conjugated PEG, appear to specifically drive the covalent conjugation of the activated PEG toward particular lysine sites. Moreover it has now been found that addition of DMSO to the reaction buffer alters the sites of pegylation. In particular, the addition of DMSO to the reaction buffer as described herein drives the reaction toward the preferential pegylation of the protein at Lysl7. Accordingly, the methods of the invention allow for selective modification of specific amino groups of the protein of interest, in particular, modification of lysine residues near the amino terminus of the protein. This selective modification of such proteins is beneficial in that it has been generally understood that associating water-soluble polymers, e.g., PEG, with proteins via amino-groups resulted in a loss of activity. It is believed that loss of activity commonly associated with pegylation was due to the random, lysine-targeted reactions of the prior art. Random modification of lysine residues may inadvertently alter protein function by substantially altering the tertiary structure or morphology of said protein. While not wishing to be limited to any theory in any way, the reaction conditions disclosed herein appear to enable the selective modification of a protein at select residues or at its amino terminus, which is not generally believed to contribute to the activity of a protein.

[0075] Specific mono-pegylation of proteins has previously been reported using aldehyde activated PEG. However, such reaction were only specific at low pH, losing specificity at a pH of 7 or higher, a. Accordingly, the methods of the instant invention may be of particular use in the pegylation and/or conjugation of pH sensitive proteins.

[0076] The reaction is typically performed at a temperature from 2°C to 50°C, more usually at a temperature from 2°C to 8°C and most typically about 4°C. The selection of a specific temperature may affect the reaction time, which is to be chosen such that polymer- protein conjugates with two nitrogen atoms of amino groups of the protein being conjugated with a polymer unit via an amine linkage are generally prepared.

[0077] In the reactions described herein, the activated water-soluble polymer will be present in molar excess and as such, the unreacted excess activated water-soluble polymer will need to be removed from newly formed protein conjugates. As used herein, "removing substantially all unlinked water-soluble polymer" refers to generally known methods for carrying out such a separation, e.g., through dialysis. Generally, about 80% of unlinked water-soluble polymer is removed, typically about 90%> is removed, more typically about 95%) is removed and most typically about 99% is removed.

PHARMACEUTICAL FORMULATIONS

[0078] In certain embodiments, the invention relates to a pharmaceutical formulation comprising at least one population of G-CSF proteins wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule, optionally in a pharmaceutically acceptable carrier. In certain embodiments, the carrier is substantially protein free. In certain embodiments, the pharmaceutical formulation comprises at least one population of G-CSF proteins wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule and at least a 30% of the composition is not N-terminal pegylated. In a specific embodiment, each G-CSF molecule is covalently linked to a single polyethylene glycol molecule ("monopegylated G-CSF") and at least 30%> of the composition is not N-terminally pegylated. In another embodiment, the formulation comprises at least 80% monopegylated G-CSF wherein at least 30% of the composition is not N-terminally pegylated. In further embodiments, the formulation comprises at least 85%, at least 90% or at least 95%) monopegylated G-CSF molecules wherein at least 30%> of the composition is not N-terminally pegylated.

[0079] In specific embodiments of the pharmaceutical formulation, each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule through a particular lysine residue. In certain embodiments, the formulation includes at least one G-CSF molecule covalently linked to at least one polyethylene glycol molecule via the amino terminus of the G-CSF protein through an amine linkage. In other embodiments, the formulation comprises at least one G-CSF molecule covalently linked to at least one polyethylene glycol molecule through Lysl6 (Lysl7 if an N-terminal methionine residue is counted).

[0080] In some embodiments of the formulation, the ratio of the N-terminally pegylated molecules to a second population, especially a second population at Lysl6 can range from less than about 1 to about 100, about 10 to about 90, about 20 to about 80, about 30 to about 70, about 40 to about 60, about 50 to about 50, about 60 to about 40, about 70 to about 30, wherein less than about 1 includes an amount undetectable using standard methods known in the art.

[0081] The G-CSF molecules and compositions of the invention are expected to exhibit prolonged stability under standard storage conditions, i.e., storage at standard temperature, e.g., about 25 °C for example for at least three months. In certain embodiments, the protein-free carrier is serum-free, albumin-free or human serum albumin-free ("hsa- free")). In certain embodiments, the pharmaceutical formulations may be stored for extended period of time without substantial and/or detectable degradation of G-CSF as determined by methods described herein and/or known in the art. In certain embodiments, the pharmaceutical formulations of the invention are stable (i.e., do not exhibit detectable and/or do not exhibit substantial degradation) as determined at least 15 months after storage at about - 20°C or 4°C. In other embodiments, the pharmaceutical formulations of the invention are stable (i.e., do not exhibit detectable and/or do not exhibit substantial degradation) as determined at least 10 months after storage at about 25°C or about 37°C. The stability of pharmaceutical formulations of the invention may be assessed by any method known in the art. In certain embodiments, the stability of the pharmaceutical formulations of the invention is assessed by monitoring alteration in the protein concentration over time as determined by a bicinchoninic acid ("BCA") protein assay. In other embodiments, the stability of the pharmaceutical formulations of the invention is assessed by indication of protein degradation (i.e., G-CSF conjugate degradation) over time as determined by SDS PAGE analysis. In certain other embodiments, the stability can be measured by analyzing breakdown of the product by an HPLC. In still other embodiments, the stability of the pharmaceutical formulations of the invention is assessed by monitoring the activity of said formulation over time, wherein said activity is determined by any in vitro or in vivo method known in the art for determination of activity of said formulation (e.g., G-CSF activity). In a specific example in accordance with this embodiment, the activity of a pharmaceutical formulation of the invention comprising a plurality of G-CSF-conjugates is evaluated by the ability of said pharmaceutical formulation in an invitro bioassay utilizing a G-CSF dependent clone of murine 32D cells. In other embodiments, the acitivyt of the formulation is measured in vivo in its capacity to alter white blood cell count in experimental animals, such as hamsters.

[0082] In certain embodiments, a pharmaceutical formulation is provided comprising a monopegylated protein wherein at least 30% of the protein molecules are not pegylated at the N-terminal. In certain specific embodiments, the formulation comprises at least 30% monopegylated proteins which are pegylated at a lysine residue within 100 amino acids of the N-terminal. In specific embodiments, the lysine is within 80, within 70, within 60, within 50, within 40, within 30, within 20, within 19, within 18, within 17, within 16, within 15, within 14, within 13, within 12, within 11, within 10, within 9, within 8, within 7, within 6, within 5, within 4, within 3 or within 2 amino acid residues from the N-terminal.

[0083] The formulations of the invention may be further rendered suitable for injection by mixture or combination with an additional pharmaceutically acceptable carrier or vehicle by methods known in the art. Among the pharmaceutically acceptable carriers for formulating the products of the invention are saline, human serum album, human plasma proteins, etc. The invention also relates to pharmaceutical compositions comprising a conjugate as described above and a pharmaceutically acceptable excipient and/or carrier. Such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

[0084] Pharmaceutical compositions comprising effective amounts of polymer- protein conjugates of the present invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions includes diluents of various buffer content, such as Tris-HC 1 , acetate, phosphate, pH and ionic strength; additives such as detergents and solubilizing agents such as Tween 80, Polysorbate 80, antioxidants such as ascorbic acid and sodium metabisulfite, preservatives such as benzyl alcohol and bulking substances such as lactose or mannitol; incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the polymerprotein conjugates according to the present invention. [0085] The protein conjugates prepared in accordance with this invention may be formulated in pharmaceutical compositions suitable for injection with a pharmaceutically acceptable carrier or vehicle by methods known in the art. See, e.g., W097/09996,

W097/40850, W098/58660, and W099/07401. The compounds of the present invention may be formulated, for example, in 10 mM sodium/potassium phosphate buffer at pH 7 containing a tonicity agent, e.g. 132 mM sodium chloride. Optionally, the pharmaceutical composition may contain a preservative.

[0086] The pharmaceutical compositions generally comprise a conjugate, a multiply charged inorganic anion in a pharmaceutically acceptable buffer suitable to keep the solution pH in the range of from about 5.5 to about 7.0, and optionally one or more pharmaceutically acceptable carriers and/or excipients.

METHODS OF USE

[0087] In another aspect of the invention, a method of increasing white cell count in a host is provided comprising administering a pharmaceutical formulation of the invention to a host in need thereof. In certain embodiments, the host is human. In certain embodiments, the hosts are at risk of or suffering from neutropenia. In certain other embodiments, the hosts are being treated with an agent that decreases their white blood cell count. In certain embodiments, the hosts have decreased endogenous levels of G-CSF. In certain other embodiments, the hosts are undergoing radiation treatment. The hosts can be suffering from lung cancer, lymphoma, breast cancer, bone marrow transplantation, testicular cancer, AIDS- related malignancies, myleodysplasitc disorders, acute leukemia, congenital and cyclic neutropenias or aplastic anemia (see Mortsyn, et al.(1998) Filgrastim (r-metHuG-CSF). In Clinical Practice, 2 ^nd Ed., Marcel Dekker, Inc., New York, NY). In certain embodiments, the formulation is administered to a patient at risk of infection.

[0088] In certain embodiments, the formulation is provided in a single dose during a course of chemotherapy. In some embodiments, the formulation is provided as multiple doses over the course of chemotherapy. In certain embodiments, the formulation is administered once daily, once weekly, once every two weeks or once a month. The formulation can be administered within twenty four hours of a dose of chemotherapy. In certain embodiments, the formulation is administered at least 14 days before a dose of chemotherapy.

[0089] In certain embodiments, the formulation is administered as an injection. In some embodiments, the formulation is suitable for subcutaneous administration. In other embodiments, the formulation is suitable for intravenous administration. The formulation can also be provided as an orally available form, receives a dose at least about once a week. In other embodiments, the patient receives a dose at least about once every two weeks, at least about once every three weeks, or at least about once every month.

[0090] The therapeutically effective amount is that amount of conjugate necessary for the in vivo biological activity of causing bone marrow cells to increase production of white blood cells. The exact amount of conjugate is a matter of preference subject to such factors as the exact type of condition being treated, the condition of the patient being treated, as well as other ingredients in the composition. The pharmaceutical formulations containing the conjugate may be formulated at a strength effective for administration by various means to a human patient experiencing disorders characterized by low or defective white blood cell production. Average therapeutically effective amounts of the conjugate may vary and in particular should be based upon the recommendations and prescription of a qualified physician. For example, 0.01 to 10 μg per kg body weight, typically 0.1 to 3 μg per kg body weight, may be administered e.g. once a chemotherapy cycle. Alternatively, the

pharmaceutical compositions of the invention may contain a fixed dose of the conjugate, e.g. from 1 to 10 mg, or from 2-9 or about 6mg in a fixed dose formulation useful for a host over 45kg. However, the skilled artisan will recognize that the pharmaceutical compositions containing the conjugates of the invention may be formulated at a strength effective for administration by various means to a human patient experiencing disorders characterized by low or defective white blood cell production. Average therapeutically effective amounts of the conjugate may vary and should be based upon the recommendations and prescription of a qualified physician.

[0091]

EXAMPLES

Example 1: Procedure for the generation of differentiated PEG-GCSF (d-PEG-GCSF) using aldehvde-PEGPOK) in the presence of DMSO.

[0092] Mono-methoxylated PEG (molecular weight 30,000 Dalton) activated as an aldehyde (Ald-PEG-30K) was used as the pegylating reagent. GCSF produced in E.coli was used as the polypeptide. The GCSF-protein (7 mg) was prepared in a reaction buffer containing 15% DMSO, 10 mM sodium acetate-MES buffer, pH 5, at a concentration of 0.37 mg/ml. This polypeptide was mixed with the PEG reagent (30 mg/mL in 0.5 mM HCL) under stirring to reach the final PEG/GCSF molar ratio of 16: 1. NaCNBH ₃ was added and maintained at 22 mM, for 24 hours at 4°C. The products of the reaction were purified, and un-reacted PEG molecule removed by SP-column chromatography at pH4, in 10 mM sodium acetate buffer. The bound pegylated molecules was eluted by step gradient of: (1) 50 mM NaCl, pH4, (2) 25 mM NaCl, pH5, (3) 40 mM NaCl, pH5.5, (4) 70 mM NaCl, pH5.5 and (5) 50 mM NaCl, pH6.5. The pegylated GCSF product was eluted between first and second step. Figure 3 shows the PAGE (polyacrylamide gel electrophoresis) image of purified sample, as a single band species.

[0093] Using DMSO in the process generated a product that is a single

monopegylated species at total molecular weight of about 80KD. The hydrodynamic size of linear 30K PEG in the crosslinked acrylamide is about 60kD, and the globular GCSF protein has 19kD molecular weight. A similar product produced without using DMSO also shows single species at the same molecular weight (see Figure 4). For abbreviation in the examples, d-PEG-GCSF refers to the product made with aldehyde-PEG(30K) produced with the DMSO included in the process And Neulasta™ refers to the product made with aldehyde-PEG (20K) produced without DMSO in the process. Neulasta is a Trade Mark of Amgen.

Example 2: In vitro study to show d-PEG-GCSF is equivalent to Neulasta with two samples, (lot DM-8205 and 8201K16)

[0094] Two samples were prepared using the process as outlined in Example 1 (lots

DM8205 and 8210K16). The bioactivity of the d-PEG-GCSF vs. reference peg-GCSF which was produced in conventional method (as represented by Neulasta®, lot number: P080098) was compared in the cell proliferation assay using a cell line that requires GCSF for growth. [0095] Tissue culture cells, NFS-60, were growing as monolayers on microwells in the medium consisting of 10% FBS (fetal bovine serum) in medium-RPMI and 1% penicillin- streptomycin. A fixed number of cells (10,000 per well) was exposed to various

concentrations of test compounds and the growth of drug-exposed cells was compared to the growth in wells with medium only. After 72 hours of incubation at 37°C in a 5% C0 ₂ humidified incubator, the cells were exposed to medium containing 5 mg/ml of MTT(*) and further incubated for 4.5 hours in the same incubator. The metabolism of MTT by the cells were quenched by addition of acidified 25% SDS in water. The plates were left at room temperature to dissolve the cells. The dissolve materials were gently shaken to homogeneity for 5 minutes, and the absorbance of the content in each well were read on a

spectrophotometer set at wavelength 570 nm. The absorbed OD was plotted vs. the

corresponding test compound level to which the cells were exposed. (Figure 5). The ED ₅₀ doses were determined. The result is presented in Table- 1. One unit of the in vitro activity is based on Neupogen activity of ED ₅₀ =0.01 ng/ml.

[0096] Statistical analysis shows a confidence level of greater than 95% ( P<0.001) that the observed differences are significant. The data suggests that the bioactivity of Lot 8201K16 is comparable to that of Neulasta, while bioactivity of DM8205 is better than Neulasta.

Table 1 : ED50 levels of d-PEG-GCSF vs Neulasta in the stimulation of growth of NFS-60 cells as assayed by MTT. (*) MTT is abbreviation for 3-(4,5-Dimethyl-2-thiazolyl)-2,5- diphenyl-2HterazoliumBromide); obtained from Sigma (MO, catalog M2128).

Example 3: In vitro study to show d-PEG-GCSF is equivalent to Neulasta Got

1PN7019).

[0097] A second lot of d-PEG-GCSF (1PN7019) was tested for its in vitro activity in the cell proliferation assay in the same way as described in Example 2. The result is shown in Figure 6. The ED ₅₀ value of PEG-GCSF (Lot 7019) was 99% of the ED ₅₀ value of Neulasta. Therefore, the activity of Peg-GCSF is comparable with that of Neulasta; their differences are within the statistical error.

Example 4: In vitro and in vivo comparison d-PEG-GCSF and Neulasta (lot 1PN7606).

[0098] One lot of d-PEG-GCSF (1PN7606) was produced by a process that included

DMSO (Prolong process). The samples were compared in the in vitro proliferation assay of NFS-60 cells, using the same method as outlined in the above examples. The data is shown in Figure 7. Comparing the ED ₅₀ levels, lot 7606 has ED ₅₀ that is 97% that of Neulasta.

Therefore, d-PEG-GCSF (lot 1PN7606) had slightly better in vitro activity than Neulasta. These samples were prepared more than 3 months before the testing.

[0099] The two compounds were then compared in an in vivo assay for bioactivity for the stimulation of absolute neutrophil count (ANC). In this assay, on day 0, C57BL/6N mice were injected with cyclophosphamide (CPA) at 100 mg/kg as a single dose to induce neutropenia. These mice were then treated with d-PEG-GCSF (test) or Neulasta (reference) 24 hours after the administration of CPA. Subsequently, daily injection of the test or the reference was administered at an interval of 24 hours for 4 subsequent days. The blood samples were withdrawn at 0, 30, 54, 78, 102, and 126 hours. The samples at time zero served as the baseline reading. There were 10 animals per treatment group. The blood samples were analyzed for estimation of total WBC (white blood cells) using a veterinary cell counter. Peripheral blood smears were examined for estimation of the differential cell counts. Absolute neutrophil counts (ANC) were calculated using the percentage of neutrophils in the blood. The area under the curve (AUC) of ANC vs. time was calculated using trapezoidal rule for each animal. The AUC data is plotted against the dosage as shown in Figure 8.

[00100] The data shows that the AUC rises above the baseline level over the treatment period for both d-PEG-GCSF and Neulasta. The mean values seemed in favor of d-peg- GCSF(1PN7606), however, the test compound and Neulasta had similar in vivo potency in this experiment.

Example 5: Peorlation site comparisons.

[00101] The samples studied in previous samples were examined for their pegylation sites. Samples were digested with protease V8, the PEG containing fragments were isolated by HPLC using standard methods as is well known in the field. The pegylated fragments were then sequenced using Edman Degradation method as is well known in the art. The samples were loaded onto Prosorb membrane and sequenced using 494 Procise Protein Sequencer/140C analyzer from Applied Biosystem, Inc. The resulting sequences were compared with the theoretical sequences. The N-terminus fragment up to residue 19 was found to contain PEG. Therefore, PEG can only be conjugated to either N terminus (alpha amine) or lysine 16. This fragment was further digested by chymotrypsin. If a sequence of XXLE or LXXLE were detected then pegylation site was assigned to N terminus. If lysine was not detected or detected at much lower levels than leucine , then pegylation site of Lys- 16 was assigned. Note that we count our sequence site as 1 at Thr. If we were to count met at site 1 than lysine 16 would be assigned as lysine 17. The result is summarized Table 2 below.

Table 2: Sequence assignment of d-peg -GCVSF vs Neulasta

Example 6: Measurements of In vitro activity.

[00102] The G-CSF in vitro bioassay is a mitogenic assay utilizing a G-CSF dependent clone of murine 32D cells. Cells are maintained in Iscoves medium containing 5% FBS and 20 ng/ml G-CSF. Prior to sample addition, cells are prepared by rinsing twice with growth medium lacking G-CSF. An extended twelve point G-CSF standard curve is prepared, ranging from 48 to 0.5ng/ml (equivalent to 4800 to 50 IU/ml). Four dilutions, estimated to fall within the linear portion of the standard curve, (1000 to 3000 IU/ml), are prepared for each sample and run in triplicate. The pegylated G-CSF samples can be diluted

approximately 4-10 times less than non-pegylated G-CSF. A volume of 40 ul of each dilution of sample or standard is added to appropriate wells of a 96 well microtiter plate containing 10,000 cells/well. After forty-eight hours at 37°C and 5.5% C0 ₂ 0.5umCi of methyl- ³ H- thymidine is added to each well. Eighteen hours later, the plates are harvested and counted. A dose response curve (log G-CSF concentration vs. CPM-background) is generated and linear regression analysis of points which fall in the linear portion of the standard curve performed. Concentrations of unknown test samples can be determined using the resulting linear equation and correction for the dilution factor.

Example 7. Measurements of In vivo activity.

[00103] The in vivo testing can be carried out by dosing male golden hamsters with a

0.1 mg/kg of sample, using a single subcutaneous injection. Animals are subjected to terminal bleeds and serum samples subject to a complete blood count on the same day that the samples are collected. The average white blood cell counts are calculated. The net average WBC area under the curve after the single subcutaneous injection is calculated according to CRC Standard Mathematical Tables, 26th Ed. (Beyer, W. H., Ed.) CRC Press Inc., Boca Raton, Fla. 1981. p. 125.

Example 8: Stability Studies

[00104] Stability can be assessed in terms of breakdown of product, as visualized using

SEC-HPLC. Pegylated G-CSF are studied in two pH levels, pH 4.0 and pH 6.0 at 4°C, each for up to 16 days. Elevating the pH to 6.0 provides an environment for accelerated stability assays. For the pH 6.0 samples, monopegylated G-CSF as prepared above are placed in a buffer containing 20 mM sodium phosphate, 5 mM sodium acetate, 2.5 % mannitol, 0.005 % TWEEN80, pH 6.0 at a final protein concentration of 0.25 mg/ml. One ml aliquots are stored in 3 ml sterile injection vials. Vials of each are stored at 4°C. and 29°C. for up to 16 days. Stability is assessed by SEC-HPLC tracings. If the later measurements stay the same (as ascertained by visual inspection) as the initial (Time=0) measurements, the sample is considered to be stable for that length of time.