TW201138831A - Modified granulocyte colony stimulating factor (G-CSF) - Google Patents

Abstract

This invention relates to novel protein conjugates, in particular, to novel 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 than current G-CSF formulations.

Description

201138831 VI. INSTRUCTIONS OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to a novel protein conjugate, in particular a PEGylated protein, and to methods of making and using same. One aspect of the present invention relates to a PEGylated _G_CSF which has an effect on the effectiveness and stability of the material. ~ [Prior Art] One of the most serious potential side effects of many types of chemotherapeutic drugs reduces the number of white blood cells (neutr〇penia). The hoarding of I. hypogonia may expose the patient to a serious infection and may interrupt chemotherapy. In fact, the complications associated with low white blood cell counts are the most common cause of dose reduction or delay in performing chemotherapy (see Link, et al. (2〇〇l) Cancer·; 92:1354 1367; “claw (10), Et al. (2003) J Clin Oncol. 21:4524 4531; and ―(10), et al. (2002) Am J Med. 112:406-411) 〇granular cell community stimulating factor (G_CSF) for development of antibacterial properties a major regulator of neutrophilic granulocytes (neutrophils). In 1983, 'G-CSF in mouse form was purified from explant tissues; and in 1985, unintentionally expressed high concentrations of G - Human equivalents of CSF grown in cancer cell lines grown in culture medium (see, eg, et al. (1985) PNAS USA 82: 1526-30). Human G-CSF was found to be a glycoprotein of about 19 kD' and Depending on the carbohydrate component, it is different in acidity. It was later found that the biological activity required the presence of a carbohydrate component from 1984 to 1986 to select human heavy & g_csf and identified 150941.doc 201138831. And expressed in E. c〇n cells, A human clinical trial to test the effects of this compound was finally achieved in patients suffering from chemotherapy-induced neutropenia. In 1991, the US FDA approved the use of recombinant human G_CSF produced in E. coli (called Hui Filgrastim 'trade name Neupogen®', and in 1993, Europe approved the relevant form of expression from Chinese hamster ovary cells (trade name lenograstim). It has been found that although there are many known Variant, but its core ~ protein contains 174 amino acids (see for example Ngata, et al. (1986)

Nature 319: 415-18; Souza, et al. (1986) Science 232: 61-5; U.S. Patent No. 4,999,291). U.S. Patent Application Serial No. 07/768,959, filed on Aug. 23, 1985, to the U.S. Patent Nos. 4,81,643, 4,999,291, 5,5,82,823 and 5,5,80,755. Some human multi-purpose G-CSF molecules and their manufacturing methods. These molecules form the basis of approved Neupogen and Neulastin products. In these cases, the possibility of PEGylation of the molecule was not discussed. In recent years, antigen-free water/gluten polymers such as polyethylene glycol ("PEG") have been used to covalently modify polypeptides of therapeutic and diagnostic importance. PEG is a polymer which is non-toxic, non-immunogenic, highly water-soluble, and easily cleared by itself. PEG has many applications and is commonly used in foods, cosmetics, beverages, and prescription drugs. The FDA has approved the use of pharmaceutical grade PEG' in the United States and is widely used as a biopharmaceutical carrier due to their high biocompatibility. PEGylation can modify certain properties of biopharmaceuticals without altering their function, thereby enhancing therapeutic efficacy. 150941.doc 201138831 Typically polyethylene glycol molecules are bound to proteins by reactive groups in proteins. An amine group such as that which is equivalent to an amine acid residue or at the N-terminus is suitable for such attachment. The peg molecule is attached to the amine group in the polypeptide by a methoxylated PEG ("mPEG") having a different reaction moiety. Such polymers include mPEG-succinimide succinate, mPEG-succinic acid carbonate, mPEG-imidate, and mPEG-cyanuridine chloride. A variety of chemical methods can be employed to couple the PEG to the active biopharmaceutical through the hydroxyl group at the end of the polymer chain. For example, it has been reported that covalent attachment of PEG to a therapeutic polypeptide can extend its in vivo half-life and/or reduce its immunogenicity and antigenicity: such as interleukin (Knauf, M. J. et al., J. Biol.

Chem. 1988, 263, 15, 064; Tsutsumi, Υ· et al., J. Controlled Release 1995, 33, 447), Interferon (Kita, Y. et al., Drug Des. Delivery 1990, 6, 157), Hydrogen Peroxide Enzymes (Abuchowski, A. et al., J. Biol. Chem. 1977, 252, 3, 582), superoxide dismutase (Beauchamp, C. 0 et al., Anal. Biochem. 1983, 131, 25), And adenosine deaminase (Chen, R. et al, Biochim. Biophy. Acta 1981, 660, 293). U.S. Pat. The acid group is randomly attached via a deuteration reaction, resulting in a guanamine linkage between the -PEG and the enzyme. Similarly, in the European Patent Publication EP 0 154 3 16, reductive alkylated lympholysin is described at neutral pH, which indicates about 13 〇/〇 of the eleven amino acid residues of the molecule. Repair

Decoration. European Publication EP 0 335 423 describes the thiolation of g-CSF using peg derivatives. I50941.doc 201138831 In order to improve the stability and bioavailability of the form and to ensure activity, site-specific rather than random PEGylation is required. Site-specific PEGylation by solid phase synthesis at the N-terminus, side chain and c-terminus of potent analogs of growth hormone releasing factor (Felix, A. M et al' Int. J. Peptide Protein Res· 1995, 46, 253). PCT WO 2006/094530, issued to Siegried, Inc., describes certain PEGylated protein conjugates and methods for their preparation. An example of a PEGylated G-CSF is provided, wherein the products are dipegylated at the terminus and B>^17 or at >1 and 1^535. Since Whirlpool blood is easily degraded in vivo, daily injections must be repeated during chemotherapy. Therefore, there is a need to enhance the pharmacokinetic properties of Whirlpool blood. The aldehyde-activated PEG has been utilized to specifically PEGylate the 〇-(:卯 位 位 , , , ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( Nos. 5,824,784 and 7'〇90,835. In 2002, the FDA approved the compound under the trade name Neulasta®. Although PEG- has been developed through site-specific attachment through amine and N-terminal linkages. Whirlpool blood, but the reaction time required for such reactions is long and relies heavily on acidic pH. WO 96/11953 states that the reaction conditions are designed to allow selective attachment of the polymer to the ends of the protein. Under the conditions described (wherein the pH is adjusted to 4. 〇), the composition consists essentially of a mono-pegylated product wherein the PEG is attached to the N-terminus, Lys35 or Lys4i. The PEGylated g_csf composition has been confirmed, for example, in Cindric, et al. (2〇〇7) J pham Bi〇med Analys 150941.doc 201138831 44 : 3 88-3 95. Some proposals have been made to provide stability G An alternative strategy for CSF molecules. Linking PEG to cysteine residues Some improvement in targeting is provided. thiol-reactive PEG (including PEG-maleimide) has been linked to G-CSF at the free cysteine residue of G-CSF. Veronese, et al. Human (2007) Bioconjugate Chem. 18: 1824-1830 states that G-CSF is PEGylated at Cysl7, which has been shown to increase agglutination, although agglutination is not covalently coagulated. Similarly, Hao, et al. (2006) Biodrugs 20: 357-363 illustrates the binding of PEG-maleimine to Cysl7, which has been shown to increase the half-life of the molecule. Site-specific mutations have been used to prepare site-specific polymer attachments. A method of polypeptides, for example, in U.S. Patent No. 6,646,11, the disclosure of which is incorporated herein incorporated by reference in its entirety in its entirety in its entirety in the the the the the the the the the the the the the the the the the Residues. The amino acid residues may include a per-amine acid, a face acid, a cysteine or an aspartic acid. As such, there is still a need for a PEGylated composition, and / Or a method for its preparation that produces a predictable and consistent product without complex N-terminal targets The problem 'and retains the activity of the original molecule. The present invention is based on the preparation of an unexpectedly active novel form of an early PEGylated protein, such as a particulate cell community stimulating factor (G-CSF). In the method of preparing a protein linked to pEG by a reductive alkylation method, the use of dimethyl occlusion ("DMS") limits the PEG junction to the N-terminus or adjacent sites. This method generally allows 150941.doc 201138831 PEG to be linked to the amino acid residue closest to the N-terminus. In particular, the present invention provides a G-CSF molecule comprising a PEG molecule linked to Lysl6 (or 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. In certain embodiments, the present invention relates to a composition comprising at least one class of G-CSF proteins, wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule, and at least 30% of the The composition is not PEGylated at the N-terminus. In a specific embodiment, each G-CSF molecule is covalently linked to a single polyethylene glycol molecule ("mono-PEGylated G-CSF"), and at least 30% of the composition is not The N-terminus is PEGylated. In another embodiment, the composition comprises at least 80% mono-glycolated G-CSF, wherein at least 30% of the composition is not PEGylated at the N-terminus. In another embodiment, the composition comprises at least 85%, at least 90%, or at least 95% mono-PEGylated G-CSF molecules, wherein at least 30% of the composition is not polymerized at the N-terminus Ethylene glycolation. In a particular embodiment of the composition, each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule via a specific lysine residue. In certain embodiments, the invention includes a composition having at least one G-CSF molecule covalently linked to at least one polyethylene glycol molecule via an amine linkage via an amine end of the G-CSF protein. In other embodiments, the invention includes a composition having at least one G-CSF molecule that transmits Lysl6 (Lysl 7 if the N-terminal methionine residue is calculated) and at least one polyethylene glycol molecule Covalent chain. In some embodiments of the composition, the ratio of the ratio of the PEGylated molecule at the N-terminus 150941.doc 201138831 to the second group (especially the second group PEGylated at Lys 16) Can be 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 comprises an amount that is not measurable using standard methods known in the related art. In certain embodiments, a substantially homogeneous composition is provided comprising a mono-pegylated protein wherein at least 30% of the protein molecules are not PEGylated at the N-terminus. In certain specific embodiments, the composition comprises at least 30% mono-pegylated protein PEGylated at a distance from an amine acid residue within 100 amino acids from the N-terminus. In a specific embodiment, the lysine is 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 19 or less, and 18 or less. Within 17, within 16, within 15, within 14, within 13, within 12, within 1丨, within 1〇, within 9, within 8, within 7, within 6, within Amino acid residues within 5, within 4, within 3 or within 2 amino acids. In certain embodiments, the present invention relates to a pharmaceutical formulation comprising at least one class of G-CSF proteins, wherein each G_CSF molecule is covalently linked to at least one polyethylene glycol molecule, and is optionally included A pharmaceutically acceptable carrier. In certain embodiments, the carrier is substantially free of protein. In certain embodiments, the pharmaceutical formulation comprises at least one class of g_csf peptones, wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule, and at least 30% of the composition is not N-terminally It is PEGylated. In a specific embodiment, each G_CSF molecule is covalently linked to a single polyethylene glycol 150941.doc 201138831 ("mono-PEGylated G-CSF"), and at least 30 Å/〇 The composition is not PEGylated at the N-terminus. In another embodiment, the formulation comprises at least 80% monoPEGylated G-CSF, wherein at least 30% of the composition is not PEGylated at the n-terminus. In another embodiment, the formulation comprises at least 85%, at least 9%, or at least 95 Å per gram of mono-pegylated G-CSF molecules, wherein at least 30% of the composition is not at the N-terminus It is pegylated. In a particular embodiment of the pharmaceutical formulation, each G_CSF molecule is covalently linked to at least one polyethylene glycol molecule via a particular lysine residue'. In some embodiments, the formulation comprises at least one G-CSF molecule that is covalently linked to at least one polyethylene glycol molecule via an amine chain via the amine end of the G-CSF protein. In other embodiments, the formulation comprises at least one G_csf molecule that is covalently linked to at least one polyethylene glycol molecule through Lys 16 (or Lysl7 if the N-terminal methionine residue is calculated). In some embodiments of the formulation, the ratio of the PEGylated molecule at the end of the second group to the second group (especially the second group PEGylated at Lys丨6) may be less than about 1 to about 100, about 10 to about 9 〇, about 2 to about 8 〇, about 30 to about 70, about 40 to about 60, about 50 to about 50, about 60 to about 40, about 7 to 、, , spoon 30, wherein less than about [includes quantities that are not measurable using standard methods known in the related art. It is contemplated that the G-CSF molecules and compositions of the present invention exhibit extended stability under standard storage conditions, i.e., can be stored for at least three months at a standard temperature (e.g., about 25. (:). In some embodiments) The protein-free carrier is serum-free, albumin-free or free of human serum albumin ("blood free 150941.doc -10- 201138831 clear, albumin-free or human serum albumin free" ("hsa_free")) In certain embodiments, the pharmaceutical formulation can be stored for a longer period of time, and as determined by methods described herein and/or methods known in the related art, G-CSF is not substantially / or measurable degradation. In some embodiments, after 4 months of storage at or below -20 ° C, the formulation of the 4th s of the month is stable (ie, does not show Degradable and/or does not exhibit substantial degradation. In other embodiments, the pharmaceutical formulation of the present invention has stability after storage for at least 1 G month at about 25 Torr or about 37 C. (ie does not show measurable degradation and/or does not show substantial decline The stability of the present veterinary drug formulation can be determined by any method known in the related art. In some embodiments, the method for determining the medicinal properties of the pharmaceutical formulation of the present invention is: using quinolinic acid ( "BCa") peptone assay to monitor changes in protein concentration over time. In other practical examples, the stability of the pharmaceutical formulations of the present invention is determined by: SDS PAGE analysis, indicating protein degradation (ie, G) - CSF conjugate degradation) as a function of time. In certain other embodiments, the stability is determined by HPLC analysis of the degradation of the product. In other embodiments, the method of determining the stability of the pharmaceutical formulation of the invention To: monitor the change in the formulation for $1 ± over time 'where the activity is determined by an in vitro or in vivo method known in the art for determining the activity of the formulation (eg, G-CSF activity). In a specific example of this embodiment, the method for assessing the activity of a pharmaceutical formulation of the invention comprising a plurality of G_5 substances is: a CSF-dependent murine 32D cell line 'Assessing the pharmaceutical formulation to live In other embodiments, the activity of the formulation is 150941.doc 201138831 is determined by measuring its ability to alter the number of white blood cells in an experimental animal, such as a hamster, in vivo. Therein is provided a pharmaceutical formulation comprising a mono-pegylated protein, wherein at least 30% of the protein molecules are not PEGylated at the N-terminus. In certain particular embodiments, the formulation comprises at least 30 % PEGylated protein which is PEGylated at a distance from the amino acid at the end of the amino acid. In a particular embodiment, the lysine is a distance N 80 or less, 7〇 or less, 6〇 or less, 5〇 or less, 40 or less, 30 or less, 2〇 or less, 19 or less, Within, Less than 17, Within 16, 15 Within, (4) within, (10), within 12, within η, within 1〇, 9 hearts, within, within 7, within 6, within 5, within 4, within 3 or 2 amino acid residues. In another aspect of the invention, a method of increasing the number of white blood cells of a recipient is provided which comprises administering to a recipient in need thereof a pharmaceutical formulation of the invention. In certain embodiments, the recipient is a human. In certain embodiments, the recipient suffers from or is at risk of developing neutropenia. In some other embodiments, the recipient is receiving treatment for an agent that reduces the number of white blood cells. In some embodiments, the endogenous degree of the recipient has decreased. In certain other embodiments, the recipient is receiving radiation therapy. The recipient may be suffering from lung cancer, lymphoma, breast cancer, bone marrow transplantation, testicular cancer, AIDS-related malignancies, bone marrow hematopoietic disorder, acute leukemia, congenital and periodic neutropenia or aplastic deprivation Enterprise (see M〇rtsyn, et al. (1998) Filgrastim (r_metHuG_csF, & 150941.doc 201138831

Clinical Practice, Second Edition, Marcel Dekker, New York, NY). In certain embodiments, the formulation is administered to a patient at risk of infection. In certain embodiments, a single dose of formulation is provided during chemotherapy. In some embodiments, a multi-dose formulation is provided during the chemotherapy process. In certain embodiments, the formulation is administered once daily, once a week, once every two weeks, or once a month. The formulation can be administered within 24 hours of a single dose of chemotherapy. In certain embodiments, the formulation is administered at least 14 days prior to receiving a dose of chemotherapy. In certain embodiments, the formulation is administered as an injection. In some embodiments, the formulation is suitable for subcutaneous administration. In other embodiments, Formulation 2 is suitable for intravenous administration. Formulations may also be provided orally. In other implementations, the patient receives a dose of at least once every two weeks, or at least about once a month, once a week, at least once every three weeks, at least once every three weeks. w κ 力 稞 关于 关于 关于 关于 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备 制备

The method of making the protein and the activation may also include removing substantially all of the unlinked protein conjugate. In some implementations, the protein is covalently linked to an activated polyethylene glycol-formaldehyde-activated water-soluble polymer. All unlinked water-soluble polymers on the f. In certain embodiments, polyethylene glycol-hydroformylation

. In certain embodiments, 150941.doc • 13-201138831 PEG is at least 10 kD. In certain other embodiments, the PEG is at least 20 kD. In a particular embodiment, the PEG is 3 〇 kD or 4 〇 kD. Any linker can be used to include an alkyl chain having 1 to 3 Å, 1 to 20, 1 to 15, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 carbon atom. The knot links the PEG to the protein. The process of the invention may be employed at reactions above the pH of methods known in the related art, such as at substantially neutral pH (about pH 7.0) (e.g., PEGylation of proteins using PEG aldehydes, see for example U.S. Patent No. 5,824,784). Such modifications to the methods known in the related art can provide manufacturing advantages in terms of cost, manufacturing efficiency, and/or ease of process. In certain embodiments, the reaction is carried out at a pH of from 3 to 8. In certain other embodiments, the reaction is carried out at a pH of from about 4 to about 7, or from about 4.5 to about 6, or about 5. In certain other embodiments, the reaction is carried out at a pH of 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). In another embodiment, the reaction buffer comprises a molar ratio of protein to activated water soluble polymer of from about 1 to about 3 to about 1 to about 6 Torr. In other embodiments, the reaction buffer comprises a molar ratio of protein to active water soluble polymer of from about 1 to about 4, from about 1 to about 5, from about 1 to about 6, from about 1 to about 7, and about 1 to about 8. about 1 to about 9, about 1 to about 1 〇, about 1 to about 15, about 1 to about 2, 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, and about 1 to about 60. In certain embodiments, the reaction buffer comprises a protein to activated water soluble polymer of from 150941.doc •14·201138831 in an ear ratio of from about 1 to about 7. In another implementation, Cobbe may remove substantially all of the unreacted water soluble polymer by methods known in the art, such as, for example, dialysis or chromatography. [Embodiment] Other advantages of the present invention will become apparent from the following detailed description of the invention. It will be apparent that the invention may be embodied in other and different embodiments, and many of the details may be modified. The invention may be practiced without some or all of the specific details. Therefore, the elaboration should be considered as illustrative rather than limiting. The present invention is based on the preparation of novel forms of mono-pegylated proteins, such as particulate cell community stimulating factor ("G_CSF"), which have unexpected activity. In the process of the invention for preparing a pEG-linked protein by reductive alkylation, the use of dimethyl sulfoxide ("dms") drives PEG binding to the end or its adjacent sites. In particular, the present invention provides a G-CSF molecule comprising a PEG molecule linked to Lysl6 (LySl7 if a terminal thioacetate is included). The invention also provides a method of linking an aldehyde-reactive pEG to a protein in the presence of DMSO. Different modifications can retain the activity of the original molecule and improve the medical properties of the molecule. Definitions As used herein, when the term "N-terminal", "amine-based" or the like is used to describe the content of a protein that is covalently linked to another molecule, it refers to α_ via the amino terminus of the protein. Amine covalent linkage. 150941.doc 15 201138831

As used herein, the term "wild type" or "natural type" refers to a protein or a peptide that is in its operational form or functional form and usually finds a natural functional form in the body. #楚^, 4术 s I also refers to a protein form that has not been artificially modified or altered. The 箸;} 寻 S I can also be about recombinant protein. Thus, the terms may refer to a "/~y frfr protein produced in an animal of origin relative to the nucleic acid and/or amino acid sequence of the protein, having altered glycosylation forms (including lack of glycosylation) ) protein. As used herein, unless otherwise indicated, the term "G-CSF" or a particulate neoplastic cell stimulating factor refers to a protein having an amino acid sequence as set forth in SEQ ID NO: 1 (Figure 1), or has substantial A homogenous amino acid sequence and having a biological property associated with stimulation of white blood cell production. As used herein, the terms include such intentionally modified proteins (eg, '''''''''''''''''''' -CSF has the addition, deletion, or substitution of an amino acid residue. These terms include natural and recombinantly produced human G_CSF. G-CSF refers to naturally occurring proteins or recombinant proteins, usually humans, obtained from any known source, such as tissue cultures, protein synthesis, natural or recombinant cell cultures. As used herein, the amino acid sequence is "substantially identical" as defined by the FASTA search method according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988), a sequence and Another amino acid sequence has at least 70% 'typically at least about 80%, and more typically at least about 90% identical. 0 150941.doc • 16 - 201138831 As used herein, the term "conjugate" refers to a protein or polypeptide line and A protein or polypeptide or a cluster thereof in which one or more other chemical groups attached to the conjugate bond interact to function. The G-CSF molecule can be conjugated to the polymer unit according to the present invention, including unmutated and mutated 昼"白暂,士私 J# y barren beak, such as but not limited to growth factors, antibodies, hormones specific 5 A therapeutically active protein such as, but not limited to, erythropoietin, interferon alpha, interferon P, interferon 丫, consensus interferon, G CSF GM-CSF, heme, interleukin (such as interleukin-2) And interleukin-6), tumor necrosis factor, various cytokines, growth factors (such as human growth factors and epidermal growth factor) 'immunoglobulin (such as thieves,

IgE IgM IgA, IgD), and/or structural and/or functional variants and/or fragments thereof, and protein-like or synthetic analogous protein forms thereof. Usually, 'a protein suitable for carrying out the present invention, such as G_CSF, may be in the form of early leave from a feeding animal; form an exogenous DNA sequence by genomic or CD-selection or by DNA σ* a product of a prokaryotic or eukaryotic host; 4 a product obtained by a chemical synthesis process or by activation of an endogenous gene. Therefore, 'protein can be & suede mussels derived from tissues, mammals, microbial cell cultures, plant cell cultures, transgenic animals, yeasts, fungi and/or transgenic plants, or Restructuring sources. Suitable prokaryotic hosts include a variety of fine g 'such as the large intestine g (E. eGli); suitable eukaryotic hosts include yeast, such as wine. ·, 乂 cerevisiae or pichia pastoris '·. Jungle animal cells, such as Chinese hamster ovary cells or monkey cells; such as Xiaoxuan, &, rabbit, Shanping, sheep, transgenic genes, 150941.doc 17 201138831; plant cell cultures and transgenic plants, such as Physcomitrellapatens (-species). Depending on the host' protein performance product used, it may be glycosylated by carbohydrates from mammals, plants or other eukaryotes, or may be unglycosylated. If the protein is G-CSF ' then the G-CSF performance product may also be included in the starting thiol amino acid residue at the station. While recombinant G_CSF is commonly used, especially from E. coli, the invention encompasses the use of any and all such forms of G-CSF. It has been reported that certain g_csf analogous organisms have biological functions' and they can also be joined in accordance with the present invention. Such G_CSF analogs may include those having an amino acid addition, deletion and/or substitution as compared to the G-CSF amino acid sequence according to SEq ID N〇. In certain embodiments, the sequence is aligned with SEQ ID No. 1, which includes an inserted amino acid, such as, for example, at positions 36, 37, and 38 of SEQ ID No. 1, inserted into VSE. In certain embodiments, the sequence is such as SEq id No. 2 (Fig. 2). Typically, the protein is a G-CSF-active, including a G-CSF mutant, a glycosylated G-CSF, an unglycosylated G-CSF, and/or a modified structure and/or function of GC SF. a variant. In another embodiment, the protein has the G-CSF amino acid sequence of SEQ ID NO. 1, which corresponds to recombinant G-CSF produced in bacteria, has 174 amino acids and additional N-terminal guanamine醯 residue. Also included are amino acid sequences other than SEQ ID NO. 1 (containing the methionine residue at position 1) but having G-CSF biological activity. G-CSF Conjugates In certain embodiments, the present invention is directed to a composition comprising less than one type of G-CSF protein to 150941.doc • 18 - 201138831, wherein & G_CSF molecule is associated with at least one polyethylene glycol The molecule is covalently linked and at least 30% of the composition is not PEGylated at the N-terminus. In a specific embodiment, each G_CSF molecule is covalently linked to a single polyethylene glycol molecule ("mono-glycolized G_CSF"), and at least 30% of the composition is not at the N-terminus It is pegylated. In another embodiment, the composition comprises at least 8% by weight of mono-glycolylated G-CSF, wherein at least 30% of the composition is not PEGylated at the N-terminus. In other embodiments, the composition comprises at least 85%, at least 900/. Or at least 95°/. A mono-PEGylated G_CSF molecule wherein at least 30% of the composition is not pegylated at the n-terminus. In a particular embodiment of the composition, each G_CSF molecule is covalently linked to at least one polyethylene glycol molecule through a particular lysine residue. In some real

In one embodiment, the invention comprises a composition having at least one G_CSF molecule covalently linked to at least one polyethylene glycol molecule via an amine linkage at the amine end of the G-CSF protein. In other embodiments, the invention comprises a group & matter having at least one G-CSF molecule, permeating through LySi 6 (or Lys 17 if the ruthenium V amine acid residue is calculated) and at least one polyethylene Covalent linkage of diol molecules. In some embodiments of the composition, the ratio of the PEGylated molecule at the N-terminus to the second group (especially the PEGylated molecular group at Lys 16) is less than about 1 to about 1 〇. 〇, about 〇 about 9 〇, about 2 〇 to about 8 〇, about 0 to about 70, about 40 to about 60, 'about 50 to about 50, about 60 to about 40, about 70 to about 30, of which Less than about! This includes quantities that are not measurable using standard methods known in the art. '50941.doc -19- 201138831 In a second embodiment, a substantially homogeneous composition comprising a mono-pegylated I white is provided. At least 30% of the protein molecules are not PEGylated at the N-terminus. In certain specific embodiments, the composition comprises at least 30 〇/〇 early PEGylated protein which is PEGylated by an amine acid residue within the amino acid. In a specific embodiment, the amine acid is 80 or less in the N-terminus, within 7 〇, within 6 、, within 5 、, within 40, within 30, within 2 、, within, within Within, within 17, within 16, within 15, within 14, within (3), within 12, within _, within 1 within 9, within 8, within 8, within 6 or within 6 Amino acids within 5, within 4, within 3 or within. Most polypeptides have multiple potential PEG-linked sites. Thus, although a homogenous group of 1PEG-protein conjugates has a pEG molecule Each protein knife is linked, but the chain is not necessarily located at the same position on each protein in the group. Similarly, although a homogenous group of diPEGylated protein conjugates has two peg molecules and each protein molecule Chains, but these chains are not necessarily located at the same position on each protein in the population. Some studies have indicated that 'PEGylation of Lysl7 may impair the / G-CSF of the recombinant G-CSF. For example, Reidhaar 〇ls〇 n et al ((ο%) Biochemistry 35 ·· 9034-9041) The scanning mutagenesis revealed that lysine 35 and lysine 41 are the key residues of biological activity, and structural simulation studies revealed that G-CSF may be PEGylated in cis-amino acid and lysine 24. Resulting in a decrease in the biological activity of the protein 'because it is located in the region of protein receptor interaction. Polymeric parts (such as polyethylene glycol) are attached to these positions due to 150941.doc -20- 201138831 In addition, Aritomi et al. This sterically hinders receptor binding (1999) Nature 401: 713-717^1 ^ W negative effects of Lys 1 7 modification on protein biological activity. PEG molecules are the object of the invention 'usually by reducing alkane The polymerase is prepared as a protein conjugate. Accordingly, the present invention provides a polymer-protein conjugate in which at least one nitrogen atom of a protein amine group is each bonded to a polymer unit via an amine chain. In the present invention, the term amine The group includes the first and second amine groups, particularly in the side chain of the amino acid, or the cis- 2 group, such as the NH 2 group in the side chain of the amine acid, NH 2 in the arginine group a tNH2_ group, or a histidine imidazole side chain The NH-group. The polymer unit usually comprises at least one polymer moiety and a linker moiety between the at least one polymer moiety and the amine chain. The linker moiety may be a straight chain or a branched chain. If the linker moiety is a branched chain, the 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, leucoates and malonate derivatives. The linker moiety is usually non-planar type so the polymer chain is not immobile. Typically, the linker moiety comprises a polyfunctional group containing up to 8 and more than 1 to 10 slave atoms. Heteroatoms such as nitrogen, oxygen or sulfur may also be included in the alkyl chain. The linker moiety can be cleavable at, for example, a carbon atom or a nitrogen atom. The branched bond moiety and the resulting branched bond polymer unit and the preparation method thereof are described in WO 95/11924 and WO 03/1049699. In one embodiment of the invention, the bond linker moiety comprises at least one sub-150941.doc 21 201138831 thiol attached to the nitrogen atom of the amine chain, for example: i to 12, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5 or less, such as: 4, 3, 2 or 1, and most often 2 methylene directly attached to the nitrogen of the amine chain On the atom. Polymers encompassed by the present invention include, but are not limited to, polyethylene glycol and its derivatives 'including PEG, methoxylated PEG ("mPEG"), PEG homopolymer, polyethylene glycol homopolymer And a copolymer of ethylene glycol and propylene glycol, wherein the homopolymer and the copolymer are alkyl-substituted or unsubstituted at the terminal. In certain embodiments, the polymer is mPEG and most commonly mono-methoxylated PEG. The water soluble polymer can be a linear, branched, or star polymer having a wide range of molecular weights. The size of the PEG can range from about 10 〇 kD. In a particular embodiment, the size of the PEG is from about 10 to about % kD. In some embodiments, the molecular weight of the polyethylene glycol moiety attached to the amine group is from 2 to 1 〇〇 kDa ', more typically from 5 to 60 kDa, and most typically from 1 〇 to 3 〇 kDa. In another embodiment of the invention, the amount of pendant ethylenoxide residues in the polyethylene glycol moiety is from about 40 to about 2270, more typically from about 11 to about 137, and most typically About 225 to about 680. The polymer portion is typically a polymer chain that is substantially non-antigenic or non-immunogenic. In addition, the polymer portion employed is typically selected from the group of water soluble polymers. Such selection has the advantage that the protein to which the water-soluble polymer portion is attached or bonded does not settle in an aqueous environment such as a physiological environment. In order to carry out the reductive densification reaction, the selected polymer should additionally have a single reactive brew to provide a controlled degree of polymerization as in the method of the invention. The polymer unit and the polymer portion may be branched or unbranched. 150941.doc -22· 201138831 r. In order for the final product to be used for therapeutic purposes, the polymer should be in the form of: Those skilled in the art will be able to select the polymer portion of the product based on considerations such as whether the polymer-protein conjugate will be used for /alpha therapy' and if so, consider the required dose, cycle time, protein resistance Hydrolyzed properties, and other considerations. The Litong Hang's polymer part can be selected from the group consisting of: 聚聚diol ί5 injured 'xi part, such as dextran and its derivatives, poly- and its derived ketone parts, such as poly Acetylpyrrolidone; cellulose fraction such as hydroxymethylcellulose, polyvinyl alcohol, poly-1,3-dioxolane, water-1,3,6-dioxane, ethylene_ma An anhydride copolymer, a polyamino acid moiety; and/or a polypropylene guanamine moiety; and/or other similar non-immunogenic polymer moiety (homopolymer or random copolymer) and/or Its derivatives. These polymers can also function or be active when included in the present invention. In particular, the polymer portion is a polyalkylene glycol moiety. The term polyalkylene glycol means a polyalkylene glycol group or a polyalkylene glycol moiety in which the alkylene group is a straight chain or a branched chain group. The term polyalkylene glycol also includes polyalkylene glycols formed from a mixture of alkanediols, such as polymers containing a mixture of polyethylene and poly(propyl) propyl groups, and poly(extended isopropyl), polyethylidene and polycondensation. A polymer of an isobutyl mixture. The polyalkylene glycol portion of the polymer protein conjugate according to the present invention is usually a polyethylene glycol moiety or a polyethylene glycol residue formed by removing two terminal hydroxyl groups. The group belonging to the group is an a-substituted polyalkylene oxide derivative such as methoxypolyethylene glycol (mPEG); or other polyalkylene oxide derivative substituted by a suitable alkyl group, such as Etc. containing a mono- or bi-terminal crC4 group I50941.doc -23· 201138831. Typically, it is a linear, non-antigenic polymer such as a monodecyl polyethylene glycol homopolymer. Alternative polyalkylene oxides, such as other polyethylene glycol homopolymers, polyethylene glycol heteropolymers, other alkyl polyalkylene oxide block copolymers, and polyalkylene oxides a copolymer of block copolymers. In order to covalently attach polyethylene glycol (PEG) and similar poly(alkylene oxide) to a molecule (specifically, a protein), the hydroxyl end of the polymer must first be converted to a reactive functional group. This process is referred to as "activation" in the text and the product is called "activated PEG". For example, the methoxylated PEG ("mPEG")' can be activated to be covalently attached to the amine group by methods well known in the art, i.e., the mPEG can be modified to contain different reactive moieties. Part, suitable for subsequent attachment to a protein via an amino acid residue comprising an available amine residue (eg, an amine sulfhydryl residue). The peg reagent used in the method according to the invention is generally a reagent having the formula: 丨_CH0, wherein R is hydrazine, a lower alkyl group, an aryl group or any suitable protecting group; n is an integer, It represents the number of ethyl epoxide residues in the polyethylene glycol moiety; m is an integer 'which indicates the number of methylene groups; Llg〇, ν, s and/or the branched or unbranched linker moiety L2 is a branch or a non-branched linker part, which may or may not exist; and 丫 is an integer, with the constraint that y is i when L2 is absent, and when L2 is present , y is at least 1. In a particular embodiment, the PEG is a PEG acetaldehyde, most typically a methoxy-PEG acetaldehyde. The structure of polyethylene glycol_formaldehyde is as follows: 150941.doc •24· 201138831

H, C

The ligation method provides a method of preparing a protein f conjugate, and a conjugate prepared by the method, the method comprising: reacting a protein with an activated polyethylene glycol in a reaction buffer containing DMSO The protein-quality plate is covalently linked to the activated water-soluble polymer. The method can also include removing substantially all of the unchained water soluble polymer to obtain the protein dimer. In certain embodiments, the polyethylene glycol-formaldehyde is

. In some embodiments, peg is at least 10 kD. In certain other embodiments, the pEG is at least 2 turns ratio. In a particular embodiment, the PEG is 30 kD or 40 kD. The reducing agent for reactive alkylation is usually selected from, but not limited to,

NaCNBH4 or NaBH4. Tongwei, the reaction is carried out at a protein concentration of 〇 5 to 1 mg/ml, more usually 1 to 10 mg/m b and most usually 3 to 7 mg/ml. The reaction = can be carried out at a molar ratio of the protein-forming polymer of from 1:1 to 1:400, and usually 1: 1: 30 and most usually: i5y: 3 Torr. In another embodiment, the reaction buffer comprises a molar ratio of protein to activated water-soluble polymer of from about 1 to about 3 to about 1 to about 60. In other embodiments, the 'reaction buffer comprises a protein to the activated water soluble polymer at a ratio of about 4 to about 4, about 1 to about 5, m to about 6, W to about 7, about I50941.doc •25·201138831 1 than about 8, about! More than about 9, about! Approximately 1 〇, about "匕 (4), about i is about 20, about U is about 25, about! About 30, about] about 35, about about deduction, about 1 to about 45, about! , about U^55, ^ about (9). In some embodiments, the hearing buffer; the night contains protein to the activated water-soluble polymer molar ratio of about 1 to about 7. In another embodiment Substantially all of the unreacted water-soluble polymer can be removed by methods known in the art, such as, for example, dialysis or chromatography. Although the #S self-quality can be obtained by the method described herein. Glycols 'However, the invention specifically encompasses pegylation of therapeutic polypeptides.

For some implementations, the therapeutic protein used in the method of the present invention may be, for example, a protease, a pituitary hormone protease inhibitor, a production (P1, a community stimulating factor, a hormone, a coagulation factor +, an anticoagulation@子, Neurotrophic factor, rheumatoid factor, CD protein f, osteoinductive, interleukin, growth factor, interferon' cytokine, insulin-like long-term factor, chemokine, immunoglobulin, gonadotropin, interleukin , Echemotactin, interferon, a lipid-binding protein allergen, or a combination of other substances. Specific non-limiting examples of such therapeutic proteins include interferon-α2Α, interferon_α2Β, interferon β, interferon gamma, insulin-like growth factor _I (iGF_1}, insulin-like growth factor-2 (IGF-2), insulin, human growth hormone (hGH), transforming growth factor (TGF), erythropoietin ( Ep〇), ciliary neurotransformation factor (CNTF), thrombopoietin (τρ〇), brain-derived neurite outgrowth factor (BDNF) IL-1, insulin releasing hormone (insuiintr〇pin), I 2 nerve victory Primitive god Factor (GDNF), IL_丨RA, tissue fibrin I5094I.doc •26- 201138831 lysogen activator gu ,, (} superoxide dismutase (S〇D), urokinase, catalase, chain Kinase, fibroblast growth factor (FGF), heme neuron canine growth factor, adenine deaminase (NGF), granulosa cell giant cell community stimulating factor (GM_CSF), bovine growth hormone (bgh), granular Cell community stimulating factor (G_CSF), vasopressin, platelet-derived growth factor (PDGF), protein that enhances bactericidal/permeability (Βρι), L-aspartate prolylase, arginase, uric acid Enzyme, 丫_interferon, phenylalanine aminolysis enzyme, ie, vesicle proliferating hormone, proinsulin, epidermal growth factor, fibroblast growth factor, nerve growth factor (NGF), tumor necrosis factor, blood serotonin, thyroid Glandular hormone (pTH, including human p-butyl H), bone morphogenetic protein, hematopoietic growth factor, luteinizing hormone, membrane high

Glucagon, glucagon-like peptide-HGLP", Y^ypYY), VIIIC factor, ιχ factor, tissue factor, and Wen Weber's factor

Willebrand factor), protein c, atrial natriuretic factor, pulmonary surfactant protein, bombesin, prothrombin, enkephalinase, mullerian-inhibiting agent, pubic relaxin A chain, pubic relaxation Hormone B chain, pubic flaccid relaxin, deoxyribonuclease, inhibin, activin, vascular epidermal growth factor, integrin, protein VIII or D, bone-derived neurotrophic factor (BDNF), neurotrophin 3, _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, G - CSF, a biologically active fragment of any of the foregoing, or a combination of the foregoing. In order to maintain the pH in a preferred range, the reaction can be carried out in the presence of a buffer. For example, the buffer may be selected from the group consisting of phosphate buffer, acetate buffer, 150941.doc • 27·201138831 HEPES, MES, or other similar buffer. The reaction buffer may typically be a standard buffer containing no amine component, such as phosphate buffered saline (PBS). The reaction buffer typically comprises a salt (e.g., a Na salt) at a concentration of from about 0.1 mM to about 100 mM, most typically from about 1 mM to about 50 mM, and more typically from about 10 mM to about 20 mM. In certain embodiments, the polypeptide is mixed with the dried activated water soluble polymer with agitation. The pH of the reaction buffer is typically from about 6.5 to about 8.5 or from about 6.6 to about 7.5. In certain embodiments, the reaction buffer has a neutral pH of about 7.0. The reaction buffer additionally contains an organic solvent, particularly dimethyl sulfoxide ("DMSO"). DMSO can be present in the reaction mixture at a concentration of 5-80%, usually 10-40% (v/v). DMSO is widely used as a general-purpose solvent, but it is generally not used to affect the pegylation reaction, and is not particularly useful for preferentially affecting the reaction away from the N-terminal PEGylation site of the protein. β ρ〇τ disclosure case w〇08/0192 14 A method for selectively preparing a modified erythropoietin in a dMS containing buffer. The PEG chain provides a selective attachment of the amine-based ester to the PEG-EPO at the N-terminus of the protein. Conversely, the reaction conditions described herein, including the use of aldehyde-bonded PE^, appear to specifically drive the covalent attachment of activated pEG to a particular lysine site. Furthermore, it has been found that the addition of DMSO to the reaction buffer can be altered. Position of PEGylation 1. In particular, the addition of _ to the reaction buffer will drive the reaction toward the preferential concentration of polyethylene glycol 2 in the protein. Thus, the method of the present invention can selectively modify the amino acid residues in the related protein, particularly the lyophilic acid residues near the terminal end of the modified white amine. Such selective modification of such proteins is beneficial because it is generally known that two water-soluble polymers (e.g., PEG) are linked to proteins via amine groups, resulting in loss of activity. The loss of activity normally associated with PEGylation is due to the previously targeted random targeted amide acid reaction. Random modification of the lysine residue may adversely alter protein function due to substantially altering the tertiary structure or morphology of the protein. While not wishing to be bound by any theory in any way, the reaction conditions disclosed herein appear to selectively modify the protein at the selected residue or at the amino terminus (generally believed to not contribute to protein activity). Specific pegylation of proteins with aldehyde activated PEG has previously been reported. However, this kind of reaction is only low? 11 specific, but lost specificity at pH 7 or above. The <amp;> method is particularly useful for PEGylation and/or ligation of pH sensitive proteins. The temperature at which the reaction is carried out is usually up to 5 Torr, and more usually the temperature is 2. 〇 to 8 ° C, and most usually about 4 t: ^ The choice of a specific temperature can affect the reaction time, the selected temperature is usually prepared by the polymer · protein connection: should use the amine chain and polymer units The two amine-based gas atoms of the joined protein f. In the reactions described herein, the activated water soluble polymer is present in excess of the molar amount ' and thus will require removal of unreacted excess activated water soluble polymer from the newly formed protein conjugate. As used herein, "removing substantially all of the unlinked water soluble polymer" refers to a generally known method for carrying out such separation methods, such as removal of about 80% unlinked water solubility by dialysis. The polymer, typically removed by about 90%, more typically by about 95%, and most typically by about 99%. Illustrative, 'Pharmaceutical Formulations 150941.doc -29- 201138831 In certain embodiments, the present invention relates to a pharmaceutical formulation comprising at least one class of G-CSF proteins, wherein each G-CSF molecule is associated with at least one The covalent linkage of the ethylene glycol molecule is optionally included in a pharmaceutically acceptable carrier. In certain embodiments, the carrier is substantially free of protein. In certain embodiments, the pharmaceutical formulation comprises at least one class of G-CSF proteins, wherein each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule, and at least 30% of the composition is not at the n-terminus It is pegylated. In a particular embodiment, 'each G-CSF molecule is covalently linked to a single polyethylene glycol molecule ("mono-PEGylated G-CSF"), and at least 30% of the composition is not N The end is PEGylated. In another embodiment, the formulation comprises at least 80% monoPEGylated g-CSF, wherein at least 30% of the composition is not PEGylated at the N-terminus. In other embodiments, the formulation comprises at least 85°/. , at least 90% or at least 95°/. The mono-PEGylated G-CSF molecule' wherein at least 3% of the composition is not polyethylene glycolated at the n-terminus. In a particular embodiment of the pharmaceutical formulation, each G-CSF molecule is covalently linked to at least one polyethylene glycol molecule via a particular lysine residue. In certain embodiments, the formulation comprises at least one G-CSF molecule covalently bonded to at least one polyethylene glycol molecule via an amine linkage at the amine end of the G-CSF protein. In other embodiments, the formulation comprises at least one G_CSF molecule 'covalently linked to at least one polyethylene glycol molecule through Lysl6 (or Lys 17 if the n-terminal methionine residue is calculated). In some embodiments of the formulation, the ratio of the PEGylated molecule at the N-terminus to the second group (especially the second group of PEGylated at Lysl6) 150941.doc 201138831 is less than about 1 More than about 100, about 10 to about 90, about 20 to about 80, about 3 to about 7, about 4 to about 60, about 50, about 60 to about 40, about 7 to about 3, Less than about 1 in the sputum includes quantities that are not measurable using standard methods known in the related art. It is intended that the present & G GSF molecules and compositions exhibit long-term safety lines under standard storage conditions, i.e., at standard temperatures (e.g., about 25. 〇 for example, at least three months of storage. In some embodiments, The protein-containing carrier is serum-free, albumin-free or free of human serum albumin ("hsa-free"). In some embodiments, the pharmaceutical formulation can be stored for a longer period of time, and as herein The method described and/or the method known in the related art determines that there is no substantial and/or measurable degradation of G_CSF. In some embodiments, it is determined after storage for at least 15 months at about -2 Torr or 4 Torr. The pharmaceutical formulation of the present invention = stability (i.e., does not exhibit measurable degradation and/or does not show, and degradation in the periplasm). In other embodiments +, such as about Μ. The pharmaceutical formulation of the present invention has stability (i.e., does not exhibit measurable degradation and/or does not exhibit substantial degradation) after storage at about 37 ° C for at least 10 months. Any method known in the art for determining the stability of the formulation of the j drug of the present invention. In one embodiment, the method for determining the stability of a pharmaceutical formulation of the present invention is to monitor the change in protein concentration over time, such as by bisquinolinecarboxylic acid ("bca") protein analysis. In other embodiments, 'inventive f The method for determining the stability of a pharmaceutical formulation is as follows: The change in protein degradation (i.e., g_csf conjugate degradation) over time is determined by SDS PAGE analysis. In some other embodiments, the stability can be determined by: The degradation of the product is analyzed by HpLC. In other examples 150941.doc 31 201138831, the stability of the pharmaceutical formulation of the present invention is determined by monitoring the activity of the formulation over time, wherein the activity is determined by the related art. Any in vitro or in vivo method for determining the activity of the formulation (e.g., G-CSF activity) is known. In a particular example according to this embodiment, the invention comprises a plurality of G-CSF-conjugates. The method for assessing the activity of a formulation is to assess the ability of the pharmaceutical formulation in an in vitro biological assay using a G-CSF-dependent murine 32D cell line. In an embodiment, the assay activity is determined by measuring its ability to alter the number of blood cells in an experimental animal, such as a hamster, in vivo. In certain embodiments, a pharmaceutical formulation comprising a monomeric ethylene is provided. Alcoholized protein, wherein at least 3% of the protein molecules are not PEGylated at the N-terminus. In certain particular embodiments, the formulation comprises at least a PEGylated protein that is ligated to The lyophilic acid residue is PEGylated from within the amino acid. In some embodiments, the lysine is within 80, less than 7, within, within 50, within the N-terminus, 40 or less, 3〇 or less, 2〇 or less, within , within, within, within, within, within, within, within, within, within, within, within, within, within, within, within, within, within, within, within, within, within, within Within 1 、, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 internal amino acid residues. The formulations of the present invention can be mixed or combined with other pharmaceutically acceptable carriers or vehicles by methods known in the art to render them suitable for injection. Some of the pharmaceutically acceptable carriers for formulating the products of the present invention are physiological saline, human gold albumin, human blood-damaged proteins and the like. The present invention also relates to a pharmaceutical composition comprising 150941.doc-32·201138831 comprising an excipient and/or a carrier as described above. Happy to be # -3⁄4 ik li W ‘People The acceptable carrier can be water, suspension, and lotion. Examples of non-aqueous solutions are C (?? didiol, vegetable oil (such as olive oil), and organic ester for injection: water: carrier includes water, alcohol solution / aqueous solution, emulsion, physiological saline and buffered medium. Enteral vehicle steel solution, Ringer's dextrose Wnger.s deXtrose), grape t and gasified sodium, lactated Ringer's solution (10) (10) Ringer, s) or no U oil. The intravenous vehicle includes a fluid and a nutrient replenisher, an electrolyte replenisher (such as a supplemental solution in which the forest format is used as a substrate), and the like. Preservatives or other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like. The pharmaceutical compositions comprise an effective amount of a polymer-egg self-contained conjugate of the invention, and a pharmaceutically acceptable diluent, preservative, co-solvent, emulsifying agent and/or carrier. Such compositions include dilutions of various buffer contents (such as Tris_Hci, acetate buffer, discate buffer, pH buffer, and ionic strength buffer); additives such as detergents and co-solvents, such as Tween 8〇, polysorbate 8〇), antioxidants (such as ascorbic acid and sodium metabisulfite), preservatives (such as benzyl alcohol) and fillers (such as lactose or mannitol); and the incorporation of materials into the polymerizability A granular preparation of the compound (such as polylactic acid, polyglycolic acid, etc.), or incorporated into a liposome. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance of the polymer protein conjugates according to the present invention. The 150941.doc •33-201138831 protein f-conjugate and the pharmaceutically acceptable carrier or vehicle prepared according to the present invention may be formulated by a method known in the related art to form a drug suitable for injection, and Things. See, for example, W097/09996, W097/40850, 8660, and w〇99/〇74〇1. The compound can be formulated in a 1 mM sodium phosphate/potassium phosphate buffer containing 7, for example, mM sodium hydride penetrant. The pharmaceutical composition may contain a preservative as needed. The pharmaceutical composition substantially comprises a conjugate, a multi-charge contained in a pharmaceutically acceptable buffer suitable for maintaining a solution pH in the range of 5.5 to about 7 Å, a machine anion, and optionally Or a plurality of pharmaceutically acceptable carriers and/or excipients. Method of Use In another aspect of the invention, a method of increasing the number of white fines in a recipient is provided 'including acceptance of need The pharmaceutical formulation of the invention is administered. In certain embodiments, the recipient is a human. In certain embodiments, the recipient suffers from or is at risk of developing neutropenia. In certain other embodiments The recipient is receiving a drug that reduces the number of white blood cells (7). In some embodiments, the endogenous concentration of the recipient is reduced. In some other embodiments, the recipient is receiving radiation therapy. The recipient is suffering from lung cancer, lymphoma, breast cancer, bone marrow transplantation, testicular cancer, malignant tumor associated with AIDS, bone marrow hematopoietic disorder, acute leukemia, congenital and periodic neutropenia or aplastic anemia See Mortsyn, et al. (1998) Filgrastim (r_metHuG_CSF). In "clinical practice (Clinical Practice)," second edition, Marcel Dekker company,

New York, NY). In certain embodiments, a formulation of the invention is administered to a patient at risk of infection. 150941.doc • 34· 201138831 In certain embodiments, _, chemotherapeutic therapy provides a single dose of the formulation. In the case of Yu Yi, the number of gents is too high. In the case of chemotherapy, it is difficult to invest in multiple doses. 2 In some implementations, the frequency of the formulation is once a week, every two weeks, once or every month. - times. The formulation can be used in a chemical itch method - 4* m + ', , day, and inside. In certain embodiments, the formulation is administered at least 14 days prior to the on-agent chemotherapy. In certain embodiments, the formulation is administered as an injectable solution. In some embodiments, the formulation is suitable for subcutaneous administration. In other embodiments, the formulation is administered intravenously. Formulations in oral form may also be provided, and at least about weekly to other times, the patient receives at least once every two weeks, at least about once every three weeks, or at least about once a month. A therapeutically effective amount is one that produces the amount of conjugate necessary to cause an increase in the production of white blood cells by the bone marrow cells. The exact number of conjugates needs to be referenced to factors such as the exact species of condition to be treated, the condition of the patient to be treated, and other ingredients in the composition. The formulated conjugated "J</RTI> s weekly ligand strength should be effective in administering a human patient who is experiencing a condition characterized by low white blood cell production or defects. The average therapeutically effective amount of the conjugate may vary and is specifically based on the advice and prescription of a qualified physician. For example, it may be administered, for example, once per kilogram of body weight per gram of body weight to 10 pg, typically 1 to 3 pg per kilogram of body weight. Alternatively, the pharmaceutical compositions of the present invention may contain a fixed dose of the compound, for example, from 1 to 10 mg' or from 2 to 9 or about 6 mg in a fixed dose formulation suitable for recipients above 45 kg. However, it is well known that the formulation of the pharmaceutical composition comprising the conjugate of the present invention should be effective in administering a variety of ways to treat a condition characterized by low white blood cell production or defects. Human patient. The average therapeutically effective amount of the conjugate may vary and should be based on the advice and prescription of a qualified physician. EXAMPLES Example 1: A process for producing different PEG-GCSF (d-PEG-GCSF) in the presence of DMSO using aldehyde-PEG (30K). The monoethoxylated PEG (molecular weight 30,000 Daltons) was activated to the aldehyde compound (Ald-PEG-30K) and used as a acetylation reagent. The GCSF produced in E. coli is used as a polypeptide. A concentration of 0.37 mg/ml was prepared from GCSF-protein (7 mg) in a reaction buffer containing 15% DMSO, 10 mM sodium acetate-MES buffer, pH 5. The polypeptide was mixed with PEG reagent (30 mg/mL solution in 0.5 mM HCL) with stirring to achieve a final PEG/GCSF molar ratio of 16:1. NaCNBH3 was added and maintained at a concentration of 22 mM for 24 hours at 4 °C. The reaction product was purified, and unreacted PEG molecules were removed by SP-column chromatography at pH 4 in 10 mM sodium acetate buffer. The bound PEGylated molecule is eluted by a stepwise gradient of: (1) 50 mM NaCl, pH 4; (2) 25 mM NaCl, pH 5; (3) 40 mM NaCl, pH 5.5; 70 mM NaCl, pH 5.5; and (5) 50 mM NaCl, pH 6.5. The PEGylated GCSF product is dissolved during the first and second steps. Figure 3 shows a PAGE (polyacrylamide gel electrophoresis) image of the purified sample, which is a single band of material. The use of DMSO in the process of the invention produces a single mono-pegylated product of 150941.doc • 36·201138831 having a total molecular weight of about 80 KD. The linear 30K PEG in the cross-linked acrylamide has a hydrodynamic size of about 60 kD' and the spherical GCSF protein has a molecular weight of 19 kD. Similar products produced without DMSO were also shown as a single species with the same molecular weight (see Figure 4). The abbreviation 'd-PEG-GCSF' in the examples refers to a product obtained by a method comprising DMSO and acid-PEG (30K). And NeulastaTM refers to a product obtained by a method which does not contain DMSO and aldehyde-PEG (2〇k). Neulasta is the trade name for Amgen. Example 2: Two samples (DM_82〇5 and 82〇1 ICi6 groups) were used to demonstrate that d_PEG-GCSF is equivalent to the in vitro study of Neu丨asta. Two samples (DM8205 group and 8210K16 group) were prepared by the method shown in Example 1. Using cell strains that require GCSF for growth, d-PEG-GCSF was compared to the control group peg-GCSF in a cell proliferation assay (obtained in a conventional method, the name is Neulasta®, group number: p〇8〇〇) 98) Biological activity. Tissue culture cells NFS-60 were monolayer grown in a medium consisting of 1% FBS (fetal calf serum) and 1% penicillin-streptomycin contained in RPMI medium in a microwell assay plate. A fixed number of cells (one per well, one per well) were contacted with various concentrations of test compound and compared to the growth of cells in the wells containing only the medium in the case of cell growth in contact with the drug. After incubation for 72 hours at 3 rc in a humidified incubator at 5% C 2 , the cells were contacted with a medium containing 5 mg/ml of Μττ (*) and in the same soil. The culture was further incubated for 4.5 hours. The metabolism of MTT was stopped by the addition of acidified 25% SDS aqueous solution. The assay plate was left at room temperature 150941.doc -37·201138831 to allow the cells to dissolve. The dissolved material was gently shaken for 5 minutes to homogenize, and the absorbance of the contents in each well was read in a spectrophotometer set at a wavelength of 570 nm. The absorbance OD is plotted against the concentration of the corresponding test compound to which the cells are exposed. (Figure 5). Determine the ED5 〇 dose. The results are shown in Table 1. One unit of in vitro activity was based on ED5 〇 = 0.01 ng/ml of Neupogen activity. 0 Statistical analysis showed that the difference observed was greater than 95% confidence (P < 0.001) significance. The data indicates that the biological activity of the 8201K16 group is comparable to that of Neulasta, while the biological activity of DM8205 is superior to that of Neulasta. Table 1: ED50 concentration of d-PEG-GCSF and Neulasta stimulated by mM-well cells by MTT assay. (*) MTT is an abbreviation for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole rust bromide; it is obtained from Sigma (MO, catalog number M2128). Sample ED5〇 (ng/mL) Control (Neulasta) 0.0177 DM8205 0.0163 8201K16 0.0175 Neupogen 0.01 Example 3: Description d-PEG-GCSF (group 1PN7019) is equivalent to an in vitro study of Neulasta. The in vitro activity of the second group of d-PEG-GCSF (lPN7019) was tested in a cell proliferation assay in the same manner as described in Example 2. The results are shown in Figure 6. The ED5〇 concentration of PEG-GCSF (Group 7019) was 99% of the ED50 concentration of Neulasta 150941.doc -38 - 201138831 » Therefore, the activity was comparable; the difference was within the statistical error range. Example 4: d_PI: G-GCSF (group 1PN7606) and Neulasta were compared in vitro and in vivo. The group d_pEG_GCSF (1PN7606) was prepared by a method comprising DMSO (Prol〇ng method). The samples were compared in an in vitro NFS-60 cell proliferation assay using the same method as the method presented in the above examples. The data is shown in Figure 7. The ED50 concentration was compared with the ED5 of the 7606 group (97% of the Neuaasta. Therefore, the in vitro activity of the d-PEG-GCSF (group 1PN7606) was slightly better than that of Neulasta. These samples were prepared more than 3 months before the test. The in vivo assay was used to compare the biological activity of two compounds to stimulate the absolute number of neutrophils (ANC). In this analysis, on day ,, C57BL/6N mice received a single dose of 1 〇〇mg/kg ring. Phospholipid (CPA)' to induce neutropenia. Subsequently, mice were treated with d-PEG-GCSF (test) or Neulasta (control) 24 hours after administration of cpA. For the next 4 consecutive days, The test substance or control injection was administered every 24 hours every day. Blood samples were taken at 〇, 3〇, 54, 78, 1, 2, and 126 hours. Samples at zero time point were used as baseline readings. One treatment group has 1 animal. The blood sample is analyzed using a veterinary cytometer, • to estimate the total number of WBCs (white blood cells). Peripheral blood smears are examined to estimate the number of cells sorted. The percentage of neutrophils in the blood is calculated. Absolute neutrophils (ANC). Trapezoidal rule 'calculating each ANC animals relative to the area under the curve of the time curve (AUC). AUC to dose plotted with respect to the data, as shown in FIG. I50941.doc -39- 201138831

The data shows that during treatment, the AUC of d_PEG_GCSF and Neulasta are both above the baseline value. The average of d-peg-GCSF (lPN7606) appears to be better 'however' in this example, the test compound has similar in vivo efficacy as Neulasta. Example 5: Comparison of PEGylation positions The PEGylation positions of the samples studied in the previous examples were examined. The sample is digested with protease V8 and the PEG-containing fragment is separated by HPLC using standard methods as is well known in the art. The sequence of the PEGylated fragment is then determined using the Edman Degradation method as is well known in the art. The sample was added to the Prosorb membrane and utilized from 494 from Applied Biosystem.

The Procise Protein Sequencer/140C Analyzer determines its sequence by comparing the resulting sequence to the theoretical sequence. A fragment from the N-terminus to the 19th residue was found to contain PEG. Thus, PEG can only be conjugated to the N-terminus (alphaamine) or to the amino acid 16 "further digesting the fragment by chymotrypsin. If a sequence such as XXLE or LXXLE is determined, the PEGylation site is determined to be the terminus. If no lysine is detected, or if the concentration is much lower than that of leucine, the PEGylation site is determined to be Lys-16. Please note that the first sequence sequence we calculated is Thr. If the nickname site calculated by us is Met, the lysine 16 should be referred to as lysine 17. The results are summarized in Table 2 below. 150941.doc -40- 201138831 Table 2: Percentage of groups in d-peg_GCVSF relative to NeiHasta's sequence distribution product compared to Neulasta sample group PEG-Ald DMSO N-terminal lysine-16 activity DM8205 30K 52% 48 % Better 8201K16 3 OK 33% 67% Equivalent 1PN7019 30K Mixed 18% 75% Equivalent 1PN7606 3 OK 42% 58% Equivalent or better Neulasta 20K No 100% 0% Equivalent Example 6: Determination of in vitro activity. The in vitro biological assay of G-CSF is a mitotic assay using G-CSF-dependent murine 32D cell strain. The cells were stored in Iscoves medium containing 5% FBS and 20 ng/ml G-CSF. Cells were prepared by rinsing twice with growth medium lacking G-CSF prior to sample addition. An extended twelve point G-CSF standard curve was prepared from 48 to 0.5 ng/ml (equivalent to 4800-5 0 IU/ml). Four dilutions (1000 to 3000 IU/ml) of each sample were prepared and evaluated three times in accordance with the estimation of the straight line portion of the standard curve. The PEGylated G-CSF sample can be diluted to about one-quarter to one-tenth of the unpegylated G-CSF. Each dilution or standard of a sample having a volume of 40 μM was added to a suitable well in a 96-well microtiter assay plate containing 10,000 cells/well. After incubation for 48 hours at 37 ° C and 5.5% C02, 0.5 pmCi thiol-3H-thymidine was added to each well. After 18 hours, the assay plates were collected and counted. A dose-response curve (G-CSF concentration 150941.doc 41 201138831 log value versus background CPM) was generated and a linear regression analysis was performed on the points belonging to the straight line portion of the standard curve. The concentration of the unknown test sample is determined using the linear equation obtained and using dilution factor correction. Example 7: Determination of in vivo activity. In vivo testing was performed by a single subcutaneous injection of a mg"mg/kg sample of male golden hamsters. Blood was collected from the tails of each animal, and the blood samples were subjected to complete blood count on the same day as the samples were collected. Calculate the average number of white blood cells. According to the CRC standard math table, the second edition (7) is written by the CRC Publishing Company, Boca Rat〇n, Fla 1981, page 125, to calculate the average area of the net WBC below the curve after a single subcutaneous injection. Example 8: Stability study. The stability can be assessed by product degradation as observed by SEC-HPLC. The pegylated G-CSF was studied for 16 days at 4 t under two pH values (pH 4.0 and pH 6.0). Increasing the pH to 6 〇 provides an environment for accelerated stability analysis. For the sample of ρΗ 6.0, the mono-pegylated G-CSF prepared as described above was placed in a solution containing 2 mM sodium phosphate, 5 mM sodium acetate, 2.5% mannitol, 〇.〇〇 5% 1 [ ^ Delete _8 〇 buffer _6 〇) The final protein concentration was 0.25 mg / ml ^ 1 ml aliquots were stored in 3 mi sterile injection glass vials. Each glass vial was stored at Dou and 29 C for up to 16 days. Stability was assessed by the SEC_HpLC tracking method. The subsequent measured value of the right remains the same as the initial (time = 〇) measurement (as determined by visual observation), and the sample is considered to be stable during the time period. [Simplified illustration] Figure 1 Mainly fully processed human The amino acid sequence of the granulocyte community stimulating factor 15094I.doc -42· 201138831 ("G-CSF") (SEQ m N〇: 〇. Figure 2 Sequence data of the G_CSF protein used in the κ case. Figure 3 The purified d_peg_GcsF prepared according to the present invention is subjected to eneamine gel electrophoresis. Line 1: molecular weight size labeling (indicated by kD, 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, Line 2: purified d_peg_GCSF; line 3: without GCSF. 14.4); modified Figure 4 for purified 1^ without using DMS〇, but using the existing method of sample pEG and reaction conditions 8_(3(:817) for polyacrylamide gel electrophoresis. Line 1: molecular weight size label (in kD, 200, 116.3, 97.4, 66.3 '55.4, 36.5, 31, 21.5, 14.4), line 2 : Purified peg-GCSF. Figure 5 (^ ugly - 〇 € 8? and control (仏 111 & 31 &);) The proliferation of 860 cells. Figure 6d-PEG-GCSF and control (Neulasta) on NFS60 cells proliferation. Figure 7d-PEG-GCSF and Neulasta proliferation effect on NFS60 cells. Figure 8 in the injection In vivo analysis of ANC within 5 days after test compound (d_P]EG-GCSF and control) 150941.doc •43· 201138831 Sequence Listing <11〇>American Pune Pharmaceutical Company <120> Modified Granular cell community stimulating factor (G-CSF) <130> 13939-105002 <140> 099133189 <141> 2010-09-29 <150> 61/247,389 <151> 2009-09-30 <160> 4 <170> Patentln version 3.5 <210> 1 <211> 175 <212> PRT <2]3>Human<400>

Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu 15 10 15

Lys Cys Leu Glu Gin Va) Arg Lys !le Gin Gly Asp Gly Ala Ala Leu 20 25 30

Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45

Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser 50 55 60

Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 65 70 75 80

Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie 85 90 95

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu G)n Leu Asp Val Ala 100 105 110

Asp Phe Ala Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly Met Ala 115 120 125

Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140

Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser 145 150 155 160

Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 165 170 175 <210> 2 <211> 531 <212> DNA <213> Human 150941.doc <220> 48 201138831 <221&gt CDS <222> (1)..(525) <400> 2 atg act cca tta ggt cct get tet tet ctg ccg caa age ttt ctg ctg

Met Thr Pro Leu Gly Pro Aia"er Ser Leu Pro Gin Ser Phe Leu 1 5 H) 15 aaa tgt ctg gaa cag gtt cgt aaa ate cag ggt gac ggt get gca ctg

Lys Cys Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu 20 25 30 caa gaa aaa ctg tgc get act tac aaa ctg tgc cat ccg gaa gag ctg

Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Uu 35 40 45 gta ctg ctg ggt cat tet ett ggg ate ccg tgg get ccg ctg tet tet

Val Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Uu Ser Ser 50 55 60 igt cca tet caa get ett cag ctg get ggt tgt ctg tet caa ctg cat

Cys Pro Ser Gin Ala Leu Gin Uu Ala Gly Cys Leu Ser Gin Leu His 65 70 75 S〇 tet ggt ctg itc ctg tat cag ggt ett ctg caa get ctg gaa ggt ate

Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He 85 90 . 95 tet ccg gaa ctg ggt ccg act ctg gac act ctg cag eta gat gta get

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 100 105 Π0 gac ttt get act act att tgg caa cag atg gaa gag etc ggt atg gca

Asp Phe Ala Thr Thr lie Trp Gin Gin Met Glu Clu Leu Gly Met Ala 115 120 125 cca get ctg caa ccg act caa ggt get atg ccg gca ttc get tet gca

Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 ttc cag cgt cgt gca gga ggt gta ctg gtt get tet cat ctg caa tet

Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser 145 150 155 160 lie ctg gaa gta tet tac cgt gtt ctg cgt cat ctg gee cag ccg

Phe Leu Clu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 165 170 175 taatag <210> 3 <211> 207 <212> PRT <213> Human <400> 3

Met Ala Gly Pro Ala Thr Gin Ser Pro Met Lys Leu Mel Ala Leu Gin ! 5 10 15

Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gin Clu Ala Thr Pro 20 25 30

Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Cys Leu 35 40 45

Glu Gin Val Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 50 55 60

Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 65 70 75 80 150941.doc 96 144 192 240 288 336 384 432 480 525 531 201138831

Val Leu Leu Gly His Ser Leu Gly He Pro Trp Ala Pro Leu Ser Ser 85 90 95

Cys Pro Ser G)n Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 100 105 110

Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly Me 115 120 125

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 130 135 140

Asp Phe Ala Thr Thr Me Trp Gin Gin Met Glu Glu Leu Gly Met Ala 145 150 155 160

Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala 165 170 175

Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser 180 185 190

Phe Leu Glu Val Ser Tyr Arg Va) Leu Arg His Leu Ala Gin Pro 195 200 205 <210> 4 <211> 204 <212> PRT <213> Human <400>

Met Ala Gly Pro Ala Thr Gin Ser Pro Met Lys Leu Met Ala Leu Gin 15 10 15

Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gin Glu Ala Thr Pro 20 25 30

Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Cys Leu 35 40 45

Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys 50 55 60

Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu 65 70 75 80

Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser 85 90 95

Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu 100 105 110

Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie Ser Pro Glu 115 120 125

Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Va) Ala Asp Phe Ala 130 135 140 150941.doc 201138831

Thr Thr lie Trp Gin Gin Met Giu Glu Leu Gly Met Ala Pro Ala Leu 145 150 155 160

Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg 165 170 175

Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu 180 185 190

Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 195 200 J 5094].doc

Claims (1)

201138831 VII. Scope of Application: 1. A composition comprising granulosa cell community stimulating factor ("G-CSF") molecules, including the covalent linkage of polyethylene glycol ("PEG") molecules in the closest An amino acid residue at the N-terminus, wherein at least 30% of the G-CSF molecules are not PEGylated at the N-terminus. 2. The composition of claim 1, wherein each G-CSF molecule is covalently linked to a single PEG molecule. 3. The composition of claim 1 wherein at least 80% of the G-CSF molecules are mono-PEGylated. 4. The composition of claim 1 wherein at least 85% of the G-CSF molecules are mono-PEGylated. 5. The composition of claim 1 wherein at least 90% of the G-CSF molecules are mono-PEGylated. 6. If the composition is claimed, at least 95% of the gcsf molecules are mono-PEGylated. 7. A composition according to the formula, wherein the lytic acid residue is 6 residues. Such as. The composition of claim 1, wherein if the N-terminal methionine residue is counted, the lysine residue is a Lys 17 residue. A 9' composition comprising a population of mono-pegylated proteins, wherein the protein molecules to v30/〇 are not PEGylated at the N-terminus. The composition of claim 9, wherein at least 3% by weight of the monopegylated protein is PEGylated with an amino acid residue within one of the amino acids of the quinone. The composition of π § 9, wherein at least 3% by weight of the mono-pegylated 150941.doc 201138831 protein is PEGylated at the N-terminal 50 amino acids. 12. The composition of claim 9, wherein at least 30% of the monopegylated proteins are PEGylated with an amino acid residue within the N-terminal 20 amino acids. 13. The composition of claim i, wherein the ratio of the p-PEGylated G-CSF molecule at the n-terminus to the G-CSF molecule PEGylated to the most proximal N-terminal is less than about 1 ratio About 100. 14. As requested! The composition wherein the ratio of the p-PEGylated G-CSF molecule at the n-terminus to the G-CSF molecule which is pegylated by the amino acid closest to the N-terminus is about 1 Torr to about 90. 15. As requested! The composition wherein the proportion of the G-CSF molecule at the n-end of the pegylated G-CSF molecule relative to the G-CSF molecule which is pegylated at the N-terminus is less than about 20 to about 80. 16_, as in the claim composition, wherein the ratio of the p-PEGylated G-CSF molecule at the n-terminus to the G-CSF molecule that is pegylated with respect to the amino acid closest to the N-terminus is less than about 3 Å. 70. 17. A pharmaceutical formulation comprising the composition of claim 1 and a protein-free carrier. 18. The pharmaceutical formulation of claim π, wherein the formulation is stable at about _2 (TC remains stable for at least 15 months. 19. The pharmaceutical formulation of claim 17, wherein the formulation is at about 4 ° C The drug is still stable after at least 15 months of storage. 20. The pharmaceutical formulation of claim 7, wherein the formulation is stable at about 251 storage 150941.doc 201138831 at least ίο months. 21·If the request is 丨7 Formulation wherein the formulation is stable at about 37. The sputum remains stable after at least 10 months of storage. 22. A method of increasing the number of white blood cells of the recipient comprises administering to the recipient of the need, such as a request 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Neutropenia) The risk of or suffering from neutropenia. 25. The method of claim 22, wherein the recipient is receiving a treatment for reducing the number of white blood cells. 26. The method of claim 22, wherein the recipient The concentration of source G_CSF is reduced. 27_If requested The method of item 22 wherein the recipient is receiving radiation therapy. 28. The method of claim 22 wherein the recipient is suffering from lung cancer, lymphoma, breast cancer, bone marrow transplantation, testicular cancer, AIDS-related malignancy, bone marrow Myelodysplastic disorders, acute leukemia, congenital and periodic neutropenia or aplastic anemia. 29. The method of claim 22, wherein the formulation is administered by injection. 30. The method of claim 22, wherein the formulation is administered subcutaneously. 3 1. The method of claim 22, wherein the formulation is administered orally. 32. The method of claim 22, wherein The formulation is administered intravenously. 3 3 _ - A method of preparing a protein conjugate comprising reacting a protein with a PEG-aldehyde activated by 150941.doc 201138831 in a reaction buffer comprising DMSO, thereby allowing the protein to be Covalently linked to the PEG polymer. 34. A conjugate formed by the method of claim 33. 3. 5. The method of claim 33, wherein substantially all unlinked PEG poly is removed 6. The method of claim 33, wherein the PEG is at least 10 kD. The method of claim 33, wherein the PEG is at least 20 kD. The method of item 33, wherein the PEG is 30 kD. 3. The method of claim 33, wherein the PEG is at least 40 kD. 40. A method of preparing a G-CSF protein conjugate comprising reacting a G-CSF protein with an activated PEG-aldehyde in a reaction buffer comprising DMSO, thereby rendering the G-CSF protein and the PEG polymer Covalent chain. 41. A conjugate formed by the method of claim 40. 42. The method of claim 40, wherein substantially all of the unlinked PEG polymer is removed to obtain the G-CSF protein conjugate. 43. The method of claim 40, wherein the PEG is at least 10 kD. 44. The method of claim 40, wherein the PEG is at least 20 kD. 45. The method of claim 40, wherein the PEG is 30 kD. 46. The method of claim 40, wherein the PEG is at least 40 kD. 150941.doc