EA047322B1 - METHOD FOR OBTAINING MODIFIED THERAPEUTIC PROTEIN (VERSIONS) - Google Patents

Abstract

The invention relates to methods for producing a modified therapeutic protein comprising an activated water-soluble polymer conjugated with one or more oxidized carbohydrate constituents of the therapeutic protein, wherein said water-soluble polymer is polysialic acid (PSA).

Description

Field of invention

The present invention relates to substances and methods for conjugating a water-soluble polymer to a protein.

State of the art

The preparation of conjugates by forming a covalent bond between a water-soluble polymer and a therapeutic protein can be accomplished by a variety of chemical methods. PEGylation of polypeptide drugs protects them during circulation and improves their pharmacodynamic and pharmacokinetic profiles (Harris and Chess, Nat Rev Drug Discov. 2003;2:214-21). In the process of PEGylation, repeating ethylene glycol monomers (polyethylene glycol (PEG)) are attached to a polypeptide drug substance. PEG molecules have a large hydrodynamic volume (5-10 times the size of globular proteins), have high solubility in water and high hydration ability, are non-toxic, non-immunogenic and are quickly eliminated from the body. PEGylation of molecules can result in increased drug resistance to enzymatic degradation, increased in vivo half-life, decreased dosing frequency, decreased immunogenicity, increased physical and thermal stability, increased solubility, increased stability in liquids, and decreased aggregation. The first PEGylated drug substances were approved by the FDA in the early 1990s. Since then, the FDA has approved several PEGylated drug substances for oral, injectable, and topical use.

Polysialic acid (PSA), also referred to as colominic acid (CA), is a naturally occurring polysaccharide. This substance is a homopolymer of N-acetylneuraminic acid with an α(2^8) keto acid bond and contains vicinal diol groups at the non-reducing end. This substance is negatively charged and is a natural component of the human body. This substance can be easily obtained, in large quantities and with predetermined physical properties, from bacteria (US Patent No. 5846951). Due to the fact that PSC obtained with the help of bacteria is chemically and immunologically identical to PSC synthesized in the human body, bacterial PSC is non-immunogenic even when bound to proteins. Unlike other polymers, PSA acid is capable of biodegradation. Covalent binding of colominic acid to catalase and asparaginase has been shown to increase enzymatic stability in the presence of proteolytic enzymes or in blood plasma. Comparative in vivo studies with polysialylated and unmodified asparaginase revealed that polysialylation increases the half-life of this enzyme (Fernandes and Gregoriadis, Int J Pharm. 2001;217:215-24).

An overview of the binding of PEG derivatives to peptides and proteins is presented in the article by Roberts et al. (Adv Drug Deliv Rev 2002;54:459-76). One approach for linking water-soluble polymers to therapeutic proteins is to conjugate the polymers via carbohydrate moieties of the protein. The vicinal hydroxyl (OH) groups of carbohydrates in proteins can be readily oxidized by sodium periodate (NaIO4) to form reactive aldehyde groups (Rothfus et Smith, J Biol Chem 1963;238:140210; van Lenten et Ashwell, J Biol Chem 1971;246:1889- 94). The polymer can then combine with the aldehyde groups of the carbohydrate using reagents containing, for example, an active hydrazide group (Wilchek M and Bayer EA, Methods Enzymol 1987;138:429-42). More recent technology involves the use of reagents containing aminooxy groups that react with aldehydes to form oxime bonds (WO 96/40662, WO2008/025856).

Additional examples describing the conjugation of a water-soluble polymer to a therapeutic protein are described in WO 06/071801, which reports the oxidation of carbohydrate components of von Willebrand factor and subsequent coupling to PEG using hydrazide chemistry techniques; US Patent Publication No. 2009/007 6237, which reports oxidation of rFVIII, subsequent coupling to PEG and other water-soluble polymers (eg, PSA, HES, dextran) using hydrazide chemistry techniques; in the application WO 2008/025856, which reports the oxidation of various coagulation factors, for example, rFIX, FVIII and FVIIa, subsequent coupling with, for example, PEG using aminooxy chemistry methods by forming an oxime bond, and in US patent No. 5621039, in which reported oxidation of FIX and subsequent coupling to PEG using hydrazide chemistry techniques.

An improved method has recently been described involving mild periodate oxidation of sialic acids to form aldehydes, followed by reaction with an aminooxy group-containing reagent in the presence of catalytic amounts of aniline (Dirksen A., and Dawson PE, Bioconjugate Chem. 2008;19,2543-8 ; and Zeng Y et al., Nature Methods 2009;6:207-9). Aniline catalysis dramatically accelerates oxime coupling, allowing the use of very low reagent concentrations. The use of nucleophilic catalysts is also described in Dirksen, A., et al., J Am Chem Soc., 128:15602-3 (2006); Dirksen, A., et al., Angew chem. Int Ed., 45:7581-4 (2006); Kohler, J.J., ChemBioChem., 10:2147-50 (2009); Giuseppone, N., et al., J Am Chem Soc., 127:5528-39 (2005); and Thygesen, M.B., et al., J Org Chem., 75:1752-5 (2010).

- 1 047322

Although aniline catalysis can accelerate oxime coupling, allowing for short reaction times and the use of low concentrations of aminooxy reagent, aniline has toxic properties that must be taken into account, for example, when preparing a conjugated therapeutic protein that forms the basis of a pharmaceutical drug. For example, aniline has been shown to induce methemoglobinemia (Harrison, J.H., and Jollow, D.J., Molecular Pharmacology, 32(3) 423-431, 1987). Long-term dietary supplementation of aniline in rats has been shown to induce tumor formation in the spleen (Goodman, DG., et al., J Natl Cancer Inst., 73(1):265-73, 1984). In vitro studies have also shown that aniline promotes the induction of chromosomal mutations and has potential genotoxic activity (Bombhard E.M. et Herbold B, Critical Reviews in Toxicology 35,783-835, 2005).

These facts continue to require the development of substances and methods for conjugating water-soluble polymers to proteins that improve the pharmacodynamic and pharmacokinetic properties of proteins while minimizing the costs associated with the use of various reagents and minimizing the health risks of the consumer patient.

The essence of the invention

The present invention relates to a method for producing a modified therapeutic protein comprising an activated water-soluble polymer conjugated to one or more oxidized carbohydrate moieties of the therapeutic protein, the method comprising:

a) the first stage, including adjusting the pH of the solution containing the therapeutic protein to a pH value of 6.0, wherein the concentration of the therapeutic protein is 1.0 mg/ml;

b) a second step comprising adding an excess concentration of activated water-soluble polymer to the solution obtained in the first step over a maximum period of 15 minutes, the excess concentration being 50 molar excess, under conditions including a temperature of 22°C, no light and mixing;

c) a third step comprising adding m-toluidine to the solution from the second step, wherein m-toluidine is added to provide a final concentration of 10 mM under conditions including a temperature of 22° C., no light and stirring;

d) a fourth step comprising oxidizing one or more carbohydrate moieties of the therapeutic protein by adding the oxidizing agent sodium periodate (NaIO ₄ ) to the solution from the third step to provide a final concentration of 400 μM;

e) a fifth step, where the therapeutic protein is incubated with the activated water-soluble polymer, m-toluidine and NaIO4 under conditions that allow the activated water-soluble polymer to conjugate with one or more oxidized carbohydrate moieties of the therapeutic protein, said conditions including a time period of 2 hours, a temperature of 22° C; in the absence of light and under agitation, where one or more carbohydrate moieties of the therapeutic protein are oxidized by an oxidizing agent; and wherein an oxime bond is formed between the oxidized carbohydrate moiety and an active aminooxy group on the water-soluble polymer and said formation of the oxime bond is catalyzed by m-toluidine; And

f) a sixth step, wherein the conjugation of the water-soluble polymer to one or more oxidized carbohydrate moieties of the therapeutic protein is stopped in the fifth step by the addition of L-cysteine; wherein L-cysteine is added in an amount to provide a final concentration of 10 mM, under conditions including a time period of 60 minutes; temperature from 22°C; lack of light and mixing;

wherein said water-soluble polymer is polysialic acid (PSA), wherein said activated water-soluble polymer contains an active aminooxy group and is prepared by a process comprising:

1) incubating a solution containing an oxidized water-soluble polymer with an activated aminooxy linker containing an active aminooxy group, under conditions that ensure the formation of a stable oxime bond between the oxidized water-soluble polymer and the activated aminooxy linker, and these conditions include a period of time from 1 minute to 24 h; temperature from 2 to 8°C; in the presence or absence of light, presence or absence of stirring; due to which a water-soluble polymer containing an active aminooxy group is formed; And

2) purification of the activated water-soluble polymer containing an active aminooxy group by a method selected from the group consisting of chromatography, filtration, dialysis and precipitation, at a temperature of 2 to 8°C.

In a preferred embodiment of the proposed method, a solution containing an oxidized water-soluble polymer and an activated aminooxy linker is incubated at 4°C for 1 hour in the absence of light with stirring.

In a preferred embodiment of the proposed method, the activated water-soluble polymer containing the active aminooxy group is purified using anion exchange chromatography at a temperature of 4°C.

- 2 047322

In a preferred embodiment of the proposed method, the water-soluble polymer is oxidized by incubation with NaIO4 to form an oxidized water-soluble polymer.

In a preferred embodiment of the proposed method, the activated aminooxy linker is selected from the group consisting of:

a) 3-oxa-pentane-1,5-dioxyamine linker of the formula:

b) 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula:

and c) a 3,6,9,12,15-pentaoxa-heptadecane-1,17-dioxyamine linker of the formula:

.0^ F /°x.

h ₂ n o o o nh ₂ where the water-soluble polymer is PSA and PSA is oxidized by incubation with an oxidizing agent to form a terminal aldehyde group at the ^reducing end of PSA.

In this case, the aminooxy linker is 3-oxa-pentane-1,5-dioxyamine.

In a preferred embodiment of the proposed method, an activated water-soluble polymer containing an active aminooxy group is obtained by a method including:

a) incubating a solution containing the oxidized water-soluble polymer with an activated aminooxy linker containing an active aminooxy group under conditions that ensure the formation of a stable oxime bond between the oxidized water-soluble polymer and the activated aminooxy linker, said conditions including a period of 1 hour; temperature 4°C; lack of light and mixing; thereby forming a water-soluble polymer containing an active aminooxy group; And

b) purification of an activated water-soluble polymer containing an active aminooxy group using anion exchange chromatography at a temperature of 4°C; wherein the activated aminooxy linker is a 3-oxa-pentane-1,5-dioxyamine linker of the formula:

thereby forming an oxime bond between the water-soluble polymer and the aminooxy linker.

In another aspect, the present invention relates to a method for producing a modified therapeutic protein comprising an activated water-soluble polymer conjugated to one or more oxidized carbohydrate moieties of the therapeutic protein, the method comprising:

a) the first stage, including adjusting the pH of the solution containing the therapeutic protein to a pH value of 6.0, wherein the concentration of the therapeutic protein is 1.0 mg/ml;

b) a second step comprising adding an excess concentration of activated water-soluble polymer to the solution from the first step for a maximum period of time of 15 minutes, the excess concentration being 50 molar excess, under conditions including a temperature of 22°C; lack of light and mixing;

c) a third step comprising adding m-toluidine to the solution from the second step, wherein m-toluidine is added to provide a final concentration of 10 mM under conditions including a temperature of 22°C; lack of light and mixing.

d) a fourth step comprising oxidizing one or more carbohydrate moieties of the therapeutic protein by adding the oxidizing agent sodium periodate (NaIO ₄ ) to the solution from the third step to provide a final concentration of 400 μM;

e) a fifth step, wherein the therapeutic protein is incubated with the activated water-soluble polymer, m-toluidine and NaIO ₄ under conditions that allow the activated water-soluble polymer to be conjugated with one or more oxidized carbohydrate moieties of the therapeutic protein, said conditions including a time period of 2 hours, a temperature of 22 °C; absence of light and agitation, where one or more carbohydrate moieties of the therapeutic protein are oxidized by an oxidizing agent; and wherein an oxime bond is formed between the oxidized carbohydrate moiety and an active aminooxy group on the water-soluble polymer, and said formation of the oxime bond is catalyzed by m-toluidine; And

f) a sixth step, wherein the conjugation of the water-soluble polymer to one or more oxidized carbohydrate moieties of the therapeutic protein is stopped in the fifth step by the addition of L-cysteine; wherein L-cysteine is added in an amount to provide a final stopping agent concentration of 10 mM, under conditions including a time period of 60 minutes; temperature from 22°C; lack of light and mixing;

wherein said activated water-soluble polymer is polysialic acid (PSA);

wherein said activated water-soluble polymer contains an active aminooxy group and is prepared by a process comprising:

- 3 047322

1) incubating a solution containing an unoxidized water-soluble polymer with an activated aminooxy linker containing an active aminooxy group under conditions that ensure the formation of a stable oxime bond between the unoxidized water-soluble polymer and the activated aminooxy linker, said conditions including incubation at a temperature of 22°C in for 2 hours; lack of light and mixing; due to which a water-soluble polymer containing an active aminooxy group is formed; And

2) purification of the activated water-soluble polymer containing an active aminooxy group by a method selected from the group consisting of chromatography, filtration, dialysis and precipitation.

In a preferred embodiment of the proposed method, the activated water-soluble polymer is prepared by a process including the additional step of adding additional activated aminooxy linker containing the active aminooxy group immediately before increasing the temperature.

In a preferred embodiment of the proposed method, the activated water-soluble polymer containing the active aminooxy group is purified by a method selected from the group consisting of dialysis, ultrafiltration/diafiltration (UV/DF) and chromatography at a temperature of 22°C.

In a preferred embodiment of the proposed method, the activated water-soluble polymer containing the active aminooxy group is further purified by a method selected from the group consisting of dialysis, ultrafiltration/diafiltration (UF/DF) or chromatography at 4°C.

In a preferred embodiment of the proposed method, an activated water-soluble polymer containing an active aminooxy group is obtained by a method including:

a) incubating a solution containing an unoxidized water-soluble polymer with an activated aminooxy linker containing an active aminooxy group under conditions that ensure the formation of a stable oxime bond between the unoxidized water-soluble polymer and the activated aminooxy linker, said conditions including incubation at 22°C for 2 hours in the absence of light, with stirring; where the method further includes a stage in which the temperature of the solution is increased to a temperature of 32 to 37°C and incubated for an additional 12-24 hours; thereby forming a water-soluble polymer containing an active aminooxy group; And

b) purification of a water-soluble polymer containing an active aminooxy group by dialysis at a temperature of 22°C; wherein the activated aminooxy linker is a 3-oxa-pentane-1,5dioxyamine linker of the formula:

thereby forming an oxime bond between the unoxidized water-soluble polymer and the aminooxy linker.

In both of the proposed methods, the therapeutic protein is selected from the group consisting of: Factor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), von Willebrand Factor (VWF), Factor FV (FV), Factor X (Fx), Factor XI (FXI), Factor XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1, tissue factor (TF), ADAMTS 13 protease, IL-1 alpha, IL-1 beta, IL -2, IL-3, IL-4, IL-5, IL-6, IL-11, colony-stimulating factor-1 (CSF-1), M-CSF, FSC, GM-CSF, granulocyte colony-stimulating factor (G-CSF ), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8, IL-9, IL-10, IL-12, IL- 13, IL-14, IL-15, IL16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL- 32 alpha, IL-33, thrombopoietin (TPO), Ang-1, Ang-2, Ang-4, Ang-Y, angiopoietin-like polypeptide 1 (ANGPTL1), angiopoietin-like polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide 3 (ANGPTL3), angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin-like polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide 6 (ANGPTL6), angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin, vascular endothelial growth factor (VEGF), angiogenin, activin A, activin B, activin C, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein- 9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, The receptors of the II of the bone morphogenetic protein, the neurotrophic factor of the brain, cardiotrophin-1, the ciliary neurotrophic factor, the ciliary neurotrophic factor receptor, the crypto protein protein, the cytokine of the chemotxic factor of 1 neutrophils, which is induced by the chemotxic factor of 2 temples of 2 temples, is an induced kins of the chemotactic factor of 2β neutrophils , endothelial cell growth factor β, endothelin 1, epidermal growth factor, epigen, epiregulin, neutrophil attractant of epithelial origin, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, growth factor

- 4 047322 fibroblasts 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblast growth factor 13, fibroblast growth factor 16 , fibroblast growth factor 17, fibroblast growth factor 19, fibroblast growth factor 20, fibroblast growth factor 21, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell lineage neurotrophic factor receptor α1, glial cell lineage neurotrophic factor receptor α2 growth-related growth-associated protein α, growth-associated protein β, growth-associated protein γ, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, hepatoma-derived growth factor, insulin-like growth factor I, insulin-like factor receptor growth, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor a, nerve growth factor, nerve growth factor receptor, neuropoietin, neurotrophin-3, neurotrophin-4, oncostatin M ( OSM), placental growth factor, placental growth factor 2, platelet-derived endothelial cell growth factor, platelet-derived growth factor, platelet-derived growth factor chain A, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor chain B, platelet-derived growth factor BB, receptor a platelet-derived growth factor, platelet-derived growth factor receptor β, stimulating growth factor of B-cell precursors, stem cell factor (SCF), stem cell factor receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, thymic stromal lymphopoietin (TSLP), tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasmogen activator receptor, phospholipase activating protein (PUP), insulin, ricin lectin, prolactin, human chorionic gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue plasminogen activator, IgG, IgE, IgM, IgA and IgD, α-galactosidase, β-galactosidase, DNase, fetuin, luteinizing hormone, estrogen, insulin, albumin, lipoproteins, fetoprotein, transferrin, thrombopoietin, urokinase, integrin, thrombin, leptin, Humira (adalimumab), Prolia (denosumab), Enbrel (etanercept) and a protein from the table or a biologically active fragment thereof.

In preferred embodiments of the proposed methods, the water-soluble polymer consists of 10-300 units of sialic acid.

In preferred embodiments of the proposed methods, the therapeutic protein is FIX or a biologically active fragment thereof.

In preferred embodiments of the proposed methods, the therapeutic protein is FVIIa or a biologically active fragment thereof.

In preferred embodiments of the proposed methods, the therapeutic protein is FVIII or a biologically active fragment thereof.

In preferred embodiments, the proposed methods further include the step of purifying the modified therapeutic protein.

In preferred embodiments of the proposed methods, the modified therapeutic protein is purified by a method selected from the group consisting of chromatography, filtration and precipitation.

In preferred embodiments of the proposed methods, the modified therapeutic protein is purified using chromatography; where an antichaotropic salt is used at the stage of loading the chromatographic medium and at the stage of washing the chromatographic medium; the method contains one or more washing stages, in which the flow direction is selected in an upward direction and the flow rate is from 0.2 cm/min to 6.7 cm/min; and one or more elution stages in which the flow direction is selected in a downward direction and the flow rate is from 0.2 cm/min to 6.7 cm/min; further comprising concentrating the modified therapeutic protein by ultrafiltration/diafiltration (UF/DF).

Figures

In fig. 1 shows the primary structure of coagulation factor IX (SEQ ID NO: 1).

In fig. Figure 2 shows the binding of oxidized rFIX to aminooxy-PSA.

In fig. Figure 3 shows the synthesis of water-soluble di-aminoxyl linkers 3-oxa-pentane-1,5-dioxyamine and 3,6,9-trioxa-undecane-1,11-dioxyamine.

In fig. Figure 4 shows the preparation of aminooxy-PSA.

In fig. Figure 5 shows a visual representation of PSA-FIX conjugates obtained in the presence of different catalysts by SDS-PAGE-ELECTROPHORESIS. a) Comparison of aniline with m-toduidine at

- 5 047322 using different concentrations. b) Comparison of aniline with o-aminobenzoic acid, maminobenzoic acid, p-aminobenzoic acid, p-aminobenzamide and sulfanilic acid,

c) Comparison of aniline and m-toluidine with o-anisidine and m-anisidine.

In fig. Figure 6 shows the percentage of polysialylation with different nucleophilic catalysts.

Detailed description of the invention

The pharmacological and immunological properties of therapeutic proteins can be improved by chemical modification and conjugation with polymeric compounds such as: polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), hydroxyalkyl starch (HAS), hydroxyl ethyl starch (HES), carbohydrate, polysaccharides, pullulan, chitosan , hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyldextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene anhydride maleic hydride acids, polystyrene-maleic anhydride copolymer, poly(1hydroxymethylethylene-hydroxymethylformal) (PHF), 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate (MPA). The properties of the resulting conjugates are usually highly dependent on the structure and size of the polymer. Therefore, polymers with a defined and narrow size distribution are generally preferred in this field. Synthetic polymers such as PEG are easily produced with a narrow size distribution, whereas PSC can be purified in a manner that results in a final PSC preparation with a narrow size distribution. In addition, PEGylation reagents with defined polymer chains and narrow size distributions are commercially available at reasonable prices.

The addition of a soluble polymer, such as polysialylation, is one approach to improve the properties of therapeutic proteins such as the blood coagulation protein FIX, as well as other coagulation proteins (eg, VWF, FVIIa (see, for example, US publication 2008/0221032A1, included herein by reference) and FVIII).

Therapeutic proteins.

In certain embodiments, the therapeutic protein is selected from the group consisting of: Factor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), von Willebrand Factor (VWF), Factor FV (FV), Factor X (FX) , Factor XI (FXI), Factor XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1, tissue factor (TF), ADAMTS 13 protease, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony-stimulating factor-1 (CSF-1), M-CSF, FSC, GM-CSF, granulocyte colony-stimulating factor (G- CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8, IL-9, IL-10, IL-12, IL -13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31 , IL-32 alpha, IL-33, thrombopoietin (TPO), Ang-1, Ang-2, Ang-4, Ang-Y, angiopoietin-like polypeptide 1 (ANGPTL1), angiopoietin-like polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide 3 (ANGPTL3 ), angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin-like polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide 6 (ANGPTL6), angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin, vascular endothelial growth factor (VEGF), angiogenin, activin A, activin B, activin C , bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB morphogenetic protein, bone morphogenetic protein receptor II, brain-derived neurotrophic factor, cardiotrophin1, ciliary neurotrophic factor, ciliary neurotrophic factor receptor, crypto protein, cryptic protein, cytokine-inducible neutrophil chemotactic factor 1, cytokine-inducible neutrophil chemotactic factor 2α, cytokine-inducible chemotactic factor 2β neutrophils, endothelial cell growth factor β, endothelin 1, epidermal growth factor, epigen, epiregulin, neutrophil attractant of epithelial origin, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, factor fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblast growth factor 13, fibroblast growth factor 16, fibroblast growth factor 17, fibroblast growth factor 19, factor fibroblast growth factor 20, fibroblast growth factor 21, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell line neurotrophic factor receptor al, glial cell line neurotrophic factor receptor α2 growth-associated protein, growth-associated protein a, growth-associated protein β, growth-associated protein γ, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, hepatoma-derived growth factor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, protein , insulin-like growth factor binding, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor a, nerve growth factor, nerve growth factor receptor, neuropoietin, neurotrophin-3, neurotrophin-4, oncostatin M (OSM), placental growth factor, placental growth factor 2, platelet-derived endothelial cell growth factor, platelet-derived growth factor, platelet-derived growth factor chain A, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor chain B, platelet-derived growth factor BB, platelet-derived growth factor receptor α, platelet-derived growth factor receptor β growth factor, B-cell progenitor growth factor stimulating factor, stem cell factor (SCF), stem cell factor receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1 .2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III , thymic stromal lymphopoietin (TSLP), tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasmogen activator receptor, phospholipase activating protein (PUP), insulin, ricin lectin, prolactin, human chorionic gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone , tissue plasminogen activator, IgG, IgE, IgM, IgA and IgD, α-galactosidase, β-galactosidase, DNase, fetuin, luteinizing hormone, estrogen, insulin, albumin, lipoproteins, fetoprotein, transferrin, thrombopoietin, urokinase, integrin, thrombin, leptin, Humira (adalimumab), Prolia (denosumab), Enbrel (etanercept) and a protein from the table or a biologically active fragment thereof.

Production of Therapeutic Proteins Production of therapeutic proteins includes any method known in the art: (i) production of recombinant DNA by genetic engineering, (ii) introduction of recombinant DNA into prokaryotic or eukaryotic cells by, for example and without limitation, transfection, electroporation or microinjection , (iii) culturing said transformed cells, (iv) expressing the therapeutic protein, for example constitutively or by induction, and (v) isolating said blood coagulation protein, for example from a culture medium or by harvesting the transformed cells in order to obtain purified therapeutic protein.

In other aspects of the invention, the therapeutic protein is produced by expression in a suitable prokaryotic or eukaryotic host system capable of producing a pharmacologically acceptable blood coagulation protein molecule. Examples of eukaryotic cells are mammalian cells such as: CHO, CoS, HEK 293, BHK, SK-Hep and HepG2.

A wide variety of vectors are used to produce therapeutic proteins and are selected from eukaryotic and prokaryotic expression vectors. Examples of prokaryotic expression vectors include plasmids such as, but not limited to, pRSET, pET, and pBAD, in which the promoters used in the prokaryotic expression vectors include one or more of, but not limited to, lac, trc, trp, recA, or araBAD. Examples of eukaryotic expression vectors include: (i) for expression in yeast, vectors such as and without limitation pAO, pPIC, pYES or pMET using promoters such as and without limitation AOX1, GAP, GAL1 or AUG1; (ii) for expression in insect cells, vectors such as but not limited to pMT, pAc5, pIB, pMIB or pBAC, using promoters such as but not limited to PH, p10, MT, Ac5, OpIE2, gp64 or polh, and (iii) for expression in mammalian cells, vectors such as, but not limited to, pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derived from, in one aspect of the invention, viral systems such as, but not limited to, vaccinia virus, adeno-associated viruses, herpes viruses or retroviruses using promoters such as and without limitation CMV, SV40, EF-1, UbC, RSV, ADV, BPV and β-actin.

Introduction.

In one embodiment, the conjugated therapeutic protein of the present invention may be administered by injection, such as intravenous, intramuscular, or intraperitoneal injection.

For administration of a composition containing a conjugated therapeutic protein of the present invention to a human or experimental animal, in one aspect, the composition contains one or more pharmaceutically acceptable carriers. The terms pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that are stable, inhibit protein degradation such as aggregation and degradation of drugs, and, in addition, do not cause allergic or other adverse reactions when administered by routes generally known in the art, as described below. Pharmaceutically acceptable carriers include any and all clinically used solvents, dispersing media, coatings, antimicrobial and antifungal agents, isotonic and absorption retarding agents and the like, including agents

- 7 047322 disclosed above.

As used herein, the term effective amount is intended to mean a dose suitable for treating a disease or disorder, or alleviating a symptom of a disease or disorder. In one embodiment, the term effective amount refers to a dose suitable for treating a mammal suffering from a bleeding disorder described herein.

Such compositions may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intracisternal injection or infusion procedure. It also involves administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a specific site. Typically, the compositions are substantially free of pyrogens as well as other impurities that may be harmful to the recipient.

Single or multiple administrations of the compositions can be carried out at the dose level and according to the schedule selected by the attending physician. For the prevention or treatment of a disease, the appropriate dose will depend on the type of disease requiring treatment as described above, the severity and course of the disease, whether the drug is being used for prophylactic or therapeutic purposes, previous treatment, the patient's medical history, and response to the drug. and, the discretion of the staff physician.

The present invention also provides a pharmaceutical composition containing an effective amount of a conjugated therapeutic protein as defined herein. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier, diluent, salt, buffer or excipient. The pharmaceutical composition can be used to treat the disorders defined above, accompanied by increased bleeding. The pharmaceutical composition of the present invention may be a solution or a lyophilized preparation. Solutions of this pharmaceutical composition may be subjected to any suitable lyophilization process.

As a further aspect, the invention includes kits that contain the composition of the present invention packaged in a manner that facilitates its use for administration to subjects. In one embodiment of the invention, such a kit includes a substance or composition described herein (for example, a composition containing a conjugated therapeutic protein) packaged in a container, such as a closed vial or container with a label affixed to the container or placed in a package that describes the use of a substance or composition in the practical implementation of this method of the invention. In one embodiment of the invention, this kit contains a first container with a composition containing a conjugated therapeutic protein and a second container with a physiologically acceptable reconstitution solution for the composition in the first container. In one aspect of the invention, the substance or composition is packaged in unit dose form. The kit may further include a device suitable for administering the composition according to a specific route of administration. Preferably, the kit contains a label that describes the use of the therapeutic protein or peptide composition.

Water-soluble polymers.

Sialic acid and PSA.

PSCs are composed of polymers (usually homopolymers) of N-acetylneuraminic acid. The secondary amino group usually carries an acetyl group, but may carry a glycolyl group instead. Possible hydroxyl group substituents include acetyl, lactyl, ethyl, sulfate and phosphate groups.

But ^he

N-Acetylneuraminic acid

Neu5Ac

Structure of sialic acid (N-acetylneuraminic acid).

PSCs and mPSCs typically comprise linear polymers consisting primarily of N-acetylneuraminic acid units linked by 2,8- or 2,9-glycosidic linkages or combinations thereof (eg, alternating 2,8- and 2,9-linkages). In particular, α-2,8 glycosidic linkages are preferred for PSCs and mPSCs. Such PSCs and mPSCs are conveniently prepared from colominic acids, which are referred to herein as CC and mAC. Conventional PSCs and mPSCs contain at least 2, preferably at least 5, preferably at least 10, and most preferably at least 20 N-acetylneuraminic acid moieties. So they can contain

- 8 047322 from 2 to 300 N-acetylneuraminic acid fragments, preferably from 5 to 200 N-acetylneuraminic acid fragments or most preferably from 10 to 100 N-acetylneuraminic acid fragments. PSCs and mPSCs are predominantly free of sugar components other than N-acetylneuraminic acid. Therefore, PSA and CC preferably contain at least 90%, more preferably at least 95%, and most preferably at least 98% N-acetylneuraminic acid moieties.

When PSC and CK contain fragments other than N-acetylneuraminic acid (as, for example, in mPSC and mKK), they are predominantly located at one or both ends of the polymer chain. Such other fragments may, for example, be fragments derived from the terminal fragments of N-acetylneuraminic acid by oxidation or reduction.

For example, WO-A-0187922 describes such mPSCs and mCCs in which the non-reducing terminal N-acetylneuraminic acid residue is converted to an aldehyde group by reaction with sodium periodate. In addition, WO 2005/016974 describes such mPSCs and mCCs in which the reducing residue of the terminal N-acetylneuraminic acid is reduced to a reduced open ring on the reducing residue of the terminal N-acetylneuraminic acid, thereby forming a vicinal diol group, followed by oxidation to convert the vicinal diol group into an aldehyde group.

Sialic acid-rich glycoproteins bind selectin in humans and other organisms. They play an important role in the human body during influenza infections. For example, sialic acid can hide mannose antigens from mannose-binding lectin on the surface of host cells or bacteria. This prevents complement activation. Sialic acids also hide the penultimate residue of galactose, thereby preventing the rapid clearance of this glycoprotein by the galactose receptor of liver parenchymal cells.

Structure of colominic acid (N-acetylneuraminic acid homopolymer).

Colomic acids (a subclass of PSA) are homopolymers of N-acetylneuraminic acid (NANA) with an α (2^8) ketoside bond and are synthesized, in particular, by certain Escherichia coli strains carrying the K1 antigen. Colomic acids have many physiological functions. They are important as raw materials for the production of medicines and cosmetics.

Comparative in vivo studies with polysialylated and unmodified asparaginase revealed that polysialylation increases the half-life of this enzyme (Fernandes and Gregoriadis, Biochimica Biophysica Acta 1341: 26-34, 1997).

As used herein, the term sialic acid fragments is considered to include sialic acid monomers and polymers (polysaccharides) that are soluble in aqueous solution or suspension and have little or no harmful effects, such as side effects, in mammals when administered with a PSA-protein conjugate blood coagulation in a pharmaceutically effective amount. These polymers are characterized, in one aspect, as containing 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 sialic acid residues . In certain aspects, various sialic acid residues are linked into a chain.

In one embodiment, the sialic acid portion of the polysaccharide compound is highly hydrophilic, and in another embodiment, the entire compound is highly hydrophilic. Hydrophilicity is imparted mainly by the protruding carboxyl groups of sialic acid residues, as well as by hydroxyl groups. The saccharide moiety may contain other functional groups such as amine, hydroxyl or sulfate groups or combinations thereof. These groups may be present on naturally occurring saccharide compounds or introduced into derived polysaccharide compounds.

The naturally occurring polymer PSA is available as a polydisperse formulation exhibiting a wide molecular size distribution (e.g., Sigma C-5762) and high polydispersity (PD). Because polysaccharides are typically synthesized in bacteria, which carry an inherent risk of co-secreting endotoxins, purification of long polymer chains of sialic acid may increase the likelihood of increased endotoxin content. Short PSA molecules with 1-4 sialic acid residues can also be produced synthetically (Kang SH et al., Chem Commun. 2000;227-8; Ress DK and Linhardt RJ, Current Organic Synthesis. 2004;1:31-46) , while minimizing

- 9 047322 there is a risk of obtaining high concentrations of endotoxins. Despite this, PSC preparations with a narrow molecular size distribution and low polydispersity, which are also free of endotoxins, can now be produced. Polysaccharide compounds of particular use for this invention are, in one aspect, synthesized by bacteria. Some of these naturally occurring polysaccharides are known as glycolipids. In one embodiment, the polysaccharide compounds are substantially free of terminal galactose residues.

Methods of joining.

The therapeutic protein can be covalently linked to polysaccharide compounds using any of a variety of techniques known to those skilled in the art. In various aspects of the present invention, sialic acid moieties are coupled to a therapeutic protein, for example, FIX, FVIII, FVIIa or VWF, for example, in the manner described in US Pat. No. 4,356,170, which is incorporated herein by reference.

Other techniques for linking PSC to polypeptides are also known and are contemplated by this invention. For example, US Patent Publication No. 2007/0282096 describes the conjugation of an amine or hydrazide derivative, such as PSA, to proteins. In addition, US Patent Publication 2007/0191597 describes PSA derivatives containing an aldehyde group at the reducing end to react with substrates (eg, proteins). The contents of these references are incorporated herein by reference in their entirety.

Various methods are disclosed from column 7, line 15 to column 8, line 5 of US Pat. No. 5,846,951 (the contents of which are incorporated by reference herein in their entirety). Typical techniques include coupling via a peptide bond between a carboxyl group on one or the other molecule of a blood coagulation protein or polysaccharide and an amino group of a blood coagulation protein or polysaccharide, or an ester bond between a carboxyl group of a blood coagulation protein or polysaccharide and a hydroxyl group of a therapeutic protein or polysaccharide. Another link by which the therapeutic protein is covalently bound to the polysaccharide compound is through a Schiff base between the free amino group of the blood coagulation protein reacting with the aldehyde group formed at the non-reducing end of the polysaccharide upon oxidation with periodate (Jennings HJ and Lugowski C, J Immunol. 1981; 127:1011-8; Fernandes AI and Gregoriadis G, Biochim Biophys Acta 1997;1341;26-34). The resulting Schiff base is, in one aspect of the invention, stabilized by specific reduction of NaCNBH ₃ to form a secondary amine. An alternative approach is the formation of terminal free amino groups in the PSA molecule by reductive amination with NH ₄ Cl after preliminary oxidation. Bifunctional reagents can be used to link two amine or two hydroxyl groups. For example, a PSA molecule containing an amino group is combined with the amino groups of a protein with reagents such as BS3 (bis(sulfosuccinimidyl) suberate (Pierce, Rockford, IL). For example, to link the amine and thiol groups, heterobifunctional cross-linking reagents such as sulfo-EMCS (^^-maleimidocaproyloxy)sulfosuccinimide ester/Pierce) are additionally used.

In another approach, PSA hydrazide is prepared and linked to a carbohydrate moiety of the protein after preliminary oxidation and formation of aldehyde bonds.

As described above, the free amino group of the therapeutic protein reacts with the 1-carboxyl group of the sialic acid residue to form a peptidyl bond, or an ester bond is formed between the 1-carboxyl group of the acid and the hydroxyl or other suitable active group of the blood coagulation protein. Alternatively, the carboxyl group forms a peptide bond with the deacetylated 5-amino group, or the aldehyde group of the therapeutic protein molecule forms a Schiff base with the N-deacetylated 5-amino group of the sialic acid residue.

Alternatively, the polysaccharide compound is non-covalently linked to the therapeutic protein. For example, the polysaccharide compound and the pharmaceutically active compound in one aspect of the invention are linked via hydrophobic interactions. Other non-covalent bonds involve electrostatic interactions with oppositely charged ions attracting each other.

In various embodiments, the therapeutic protein is bound or associated with the polysaccharide compound in stoichiometric amounts (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:7 , 1:8, 1:9 or 1:10, etc.). In various embodiments, 1-6, 7-12, or 13-20 polysaccharides are associated with the blood coagulation protein. In still other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more polysaccharides are linked to blood coagulation protein.

In various embodiments, the therapeutic protein is modified to introduce glycosylation sites (ie, sites other than the native glycosylation sites). Such modifications can be made using standard molecular biology techniques known in the art. Moreover, the therapeutic protein may be glycosylated in vivo or in vitro before conjugation to a water-soluble polymer using one or more carbohydrate moieties. These glycosylated sites may serve as targets for conjugation

- 10 047322 proteins with water-soluble polymers (US Patent Publication No. 20090028822, US Patent Publication No. 2009/0093399, US Patent Publication No. 200 9/0081188, Patent Publication

US No. 2007/0254836, US Patent Publication No. 2006/0111279 and DeFrees S. et al., Glycobiology, 2006, 16, 9, 833-43). For example, a protein that is not naturally glycosylated in vivo (eg, a protein that is not a glycoprotein) can be glycosylated as described above.

E. Aminooxy binding.

In one embodiment, the reaction of hydroxylamine or hydroxylamine derivatives with aldehydes (eg, a carbohydrate moiety after oxidation with sodium periodate) to form an oxime group is used to produce blood coagulation protein conjugates. For example, a glycoprotein (eg, a therapeutic protein of the present invention) is first oxidized by an oxidizing agent such as sodium periodate (NaIO ₄ ) (Rothfus JA et Smith EL., J Biol Chem 1963, 238, 1402-10 and Van Lenten L and Ashwell G ., J Biol Chem 1971, 246, 1889-94). Periodate oxidation of glycoproteins is based on the classical Malaprade reaction, described in 1928, the oxidation of vicinal diols with periodate to form an active aldehyde group (Malaprade L., Analytical application, Bull Soc Chim France, 1928, 43, 683-96). Additional examples of such an oxidizing agent are: lead tetraacetate (Pb(OAc) ₄ ), manganese acetate (MnO(Ac) ₃ ), cobalt acetate (Co(OAc) ₂ ), thallium acetate (TlOAc), cerium sulfate (Ce(SO ₄ ) ₂ ) (US Patent 4,367,309) or potassium perruthenate (KRuO ₄ ) (Marko et al., J Am Chem Soc 1997,119, 12661-2). The term oxidizing agent refers to a mild oxidizing compound that is capable of oxidizing vicinal diols in carbohydrates, resulting in the formation of active aldehyde groups upon reaction under physiological conditions.

The second stage is the binding of a polymer containing an aminooxy group to an oxidized carbohydrate moiety to form an oxime bond. In one embodiment of the invention, this step can be performed in the presence of catalytic amounts of the nucleophilic catalyst aniline or aniline derivatives (Dirksen A et Dawson PE, Bioconjugate Chem. 2008; Zeng Y et al., Nature Methods 2009;6:207-9). Aniline catalysis greatly accelerates oxime ligation, allowing the use of very low reagent concentrations. In another embodiment of the invention, the oxime bond is stabilized by reduction of NaCNBH ₃ to form an alkoxyamine bond (Fig. 2). Additional catalysts are described below.

Additional information on aminooxy technology can be found at the following links, the contents of each of which are included in this document in full: EP 1681303A1 (HAA-coupled erythropoietin); WO 2005/014024 (polymer-protein conjugates linked by an oxime linking group); WO96/40662 (compounds containing aminooxy linker and their use in conjugates); WO 2008/025856 (modified proteins); Peri F et al., Tetrahedron 1998, 54, 12269-78; KublerKielb J et. Pozsgay V., J Org Chem 2005, 70, 6887-90; Lees A et al., Vaccine 2006, 24(6), 716-29; and Heredia KL et al., Macromoecules 2007, 40(14), 4772-9.

The present disclosure provides numerous methods for linking a water-soluble polymer to an aminooxy linker. For example, this document describes the coupling of a linker to the reducing or non-reducing end of a water-soluble example, such as PSA. The binding site (eg, reducing or non-reducing end) is determined by one or more conditions (eg, time and temperature) of the binding process, as well as the state (eg, native or oxidized) of the water-soluble polymer. In one embodiment, the oxidized water-soluble polymer, such as PSA, is coupled at its non-reducing end to the aminooxy linker by conducting the coupling reaction at a reduced temperature (eg, between 2-8°C). In another embodiment, a native (eg, non-oxidized) water-soluble polymer, such as PSA, is coupled at its non-reducing end to an aminooxy linker by conducting the coupling reaction at an elevated temperature (eg, between 22-37°C). The above embodiments are described in more detail below and in the examples.

This document describes that the reaction of oxidized PSA with a diaminooxy linker exhibits two reactions: a fast reaction of the aldehyde group at the non-reducing end and a slow reaction at the reducing end. If native PSA (which is not oxidized and does not contain an active aldehyde group) reacts with the reducing end at room temperature, PSA derivatives can be formed. Thus, in various embodiments of the invention, to minimize the unwanted side reaction at the reducing end of a water-soluble polymer such as PSA, the preparation of the PSA-aminooxy linker reagent is performed at a temperature between 2-8°C.

Another embodiment of the present disclosure provides for derivatization of native PSA at the reducing end. Herein, it is assumed that native PSA (which is not oxidized by NaIO4 and therefore does not contain a free aldehyde group at its non-reducing end) reacts with the diaminooxy linker at room temperature and derivatization of PSA at its reducing end can be achieved. This coupling occurs by ring opening at the reducing end and subsequent formation of the oxime (this side reaction is described above and is the cause of the byproduct in the aminooxy-PSA reagent). This reaction can occur with native PSC, resulting in a degree of modification of up to approximately 70%.

- 11 047322

The following structure was determined as the main product by 13C-NMR spectroscopy:

This reaction can be transferred to other carbohydrates like dextran and starch or other polysaccharides containing reducing end groups. The use of a nucleophilic catalyst like m-toluidine or aniline is also contemplated. Thus, this paper proposes the preparation of aminooxy-PSA reagents using native PSA (i.e., without prior oxidation), which can then be used for the chemical modification of therapeutic proteins.

Therefore, various embodiments of the present disclosure provide methods that describe binding conditions (e.g., incubation temperature of 2-8°C) of a diaminooxy linker to a water-soluble polymer, such as oxidized PSA, that are favorable for binding to any non-reducing end or, in one alternative an embodiment in which the binding conditions (eg, room temperature incubation) of the diaminooxy linker to a water-soluble polymer, such as native unoxidized PSA, are favorable for binding to any reducing end.

In various embodiments, the water-soluble polymer that is bound to the oxidized carbohydrate moiety of a therapeutic protein (e.g., FVIII, FVIIa, or FIX) according to the aminooxy technology described herein includes, but is not limited to: polyethylene glycol (PEG), branched PEG, polysialic with acid (PSK), carbohydrate, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextrane, carboxymethylenexide (PAO), polyalliclycol (PAG), polypropylene glycol, polyoxazoline, polyaczasolin, polyacroloron Lin, polyvinyl alcohol ( PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-maleic anhydride copolymer, polystyrene-maleic anhydride copolymer, poly(1-hydroxymethylethylenehydroxymethylformal) (PHF), 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate (MPA).

Nucleophilic catalysts.

As described herein, the conjugation of water-soluble polymers to therapeutic proteins can be catalyzed by aniline. Aniline strongly catalyzes the aqueous reactions of aldehydes and ketones with amines to form stable imines such as hydrazones and oximes. The following diagram compares the non-catalyzed and aniline-catalyzed oxime ligation reaction (Kohler JJ, ChemBioChem 2009;10:2147-50):

"pfovami

However, given the numerous health risks associated with aniline, the use of alternative catalysts is desirable. The present invention provides aniline derivatives as alternative oxime ligation catalysts. Such aniline derivatives include m-toluidine.

- 12 047322

In one embodiment, m-toluidine (known as meta-toluidine, mmethylaniline, 3-methylaniline, or 3-amino-1-methylbenzene) is used to catalyze the conjugation reactions described herein. M-toluidine and aniline have similar physical properties and especially the same pKa value (m-toluidine: pKa 4.73, aniline: pKa 4.63).

The nullophilic catalysts of this invention are suitable for oxime ligation (eg, using aminooxy coupling) or hydrazone formation (eg, using hydrazide chemistry methods). In various embodiments of the invention, a nucleophilic catalyst is proposed for the conjugation reaction at a concentration of 0.1; 0.2; 0.3; 0.5; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0; eleven; 12, 13; 14; 15; 16; 17; 18; 19; 20; 25; thirty; 35; 40; 45 or 50 mM. In one embodiment of the invention, the nucleophilic catalyst is provided at a concentration between 1 and 10 mM. In various embodiments of the invention, the pH range of the conjugation reaction is within the range of 4.5; 5.0; 5.5; 6.0; 6.5; 7.0 and 7.5. In one embodiment of the invention, the pH value is between 5.5 and 6.5.

Purification of conjugated proteins.

Various embodiments of the invention require purification of a protein that has been incubated with an oxidizing agent and/or a therapeutic protein that has been conjugated to a water-soluble polymer in accordance with the present disclosure. Numerous purification techniques are known in the art, which include, but are not limited to, chromatographic techniques such as ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography and affinity chromatography or combinations thereof, filtration techniques (eg, UV/DF) and precipitation techniques, as well as dialysis techniques and any combination of the above methods (Guide to Protein Purification, Meth. Enzymology volume 463 (ed. Burgess RR and Deutscher MP), ^2nd ed., Academic Press 2009).

The following examples are not intended to be limiting, but are merely examples of specific embodiments of this invention.

Examples

Example 12.

Polysialylation of rFVIII using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

50 mg of rFVIII was transferred into the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO4 was added to this solution to obtain a final concentration of 200 µM. Oxidation was carried out at RT for 30 min in the dark with gentle shaking. The reaction was then stopped with cysteine (final concentration: 10 mM) for 60 min at RT. The resulting solution was transferred to a 20 mL IOC column (Merck EMD TMAE (M)), which was equilibrated with Buffer A (20 mM Hepas, 5 mM CaCl ₂ , pH 7.0). The column was equilibrated with 5 OC of Buffer Solution A. Oxidized rFVIII was eluted with Buffer Solution B (20 mM Hepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing rFVIII were collected. Protein content was determined (Coomassie, Bradford method), adjusted to 1 mg/ml using reaction buffer, and pH adjusted to 6.0 by dropwise addition of 0.5 M HCl. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was then added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The coupling reaction was carried out for 2 h in the dark with gentle shaking at room temperature. Excess aminooxy-PSA reagent was removed using CHB. The conductivity of the reaction mixture was increased to 130 mS/cm by adding a buffer solution containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture was applied to a column filled with 80 ml phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate was eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, PSA-rFVIII-containing fractions were collected and subjected to UV/DP using a 30 KDa membrane made from regenerated cellulose (88 cm ² , Millipore). The resulting preparation was studied by analytical methods by measuring total protein (Coomassie, Bradford method) and chromogenic activity of FVIII. It was determined that the PSC-FVIII conjugate showed a specific activity of >70% compared to the activity of native rFVIII.

Method 2:

mg of recombinant factor VIII (rFVIII), obtained during the production of ADVATE in GEPES buffer solution (50 mM GEPES, ~350 mM sodium chloride, 5 mM calcium chloride, 0.1% polysorbate 80, pH 7.4), dissolved in reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is carried out for 30±5 minutes at temperature (T) T= +22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 min at

T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized rFVIII was further purified by anion exchange chromatography on EMD TMAE (M) support (Merck). The resulting mixture is diluted with Buffer Solution A (20 mM Hepas, 5 mM CaCl ₂ , pH 6.5) to obtain a conductivity of 5 mS/cm. This solution is applied to an IOC column (layer thickness: 5.4 cm) with a column volume of 10 ml using a flow rate of 1.5 cm/min. The column is then washed (flow rate: 1.5 cm/min) with a 5 OK mixture of 92:8 (w/w) Buffer A and Buffer B (20 mM Gepes, 5 mM CaCl ₂ , 1.0 M NaCl, pH 7.0). The oxidized rFVIII is then eluted with a 50:50 (w/w) mixture of Buffer A and Buffer B, followed by a post-elution step of 5 OC Buffer B. The elution steps are carried out using a flow rate of 1.0 cm/min.

Next, the reagent aminohydroxypolysialic acid (ΠΟΚ-ΟΝΗ ₂ ) in a 50-fold molar excess is added to the eluate containing purified oxidized rFVIII with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking.

The resulting PSC-rFVIII conjugate was purified by hydrophobic interaction chromatography (HIC) using low-substitute phenylsepharose FF resin (GE Healthcare) packaged in a column manufactured by GE Healthcare with a bed thickness (h) of 15 cm and a final column volume (FC) of 81 ml .

Ammonium acetate is added to the reaction mixture by adding 50 mM Hepes buffer solution containing 350 mM sodium chloride, 8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reaction mixture are mixed with 1 volume of a buffer system containing ammonium acetate, and the pH of the solution is adjusted to pH 6.9 by adding dropwise 0.5 N. aqueous NaOH solution. This mixture is applied to the CHB column at a flow rate of 1 cm/min, followed by a wash step using >3 OK of equilibration buffer solution (50 mM Hepas, 350 mM sodium chloride, 2.5 M ammonium acetate, 5 mM calcium chloride, pH 6 ,9).

To remove reaction by-products and the antichaotropic salt, a second washing step of >5 OK of wash buffer solution 1 (50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9) is carried out in an upward flow mode at a flow rate of 2 cm/min . Then the purified PSA-rFVIII conjugate is eluted in a downward flow mode, using a step gradient from 40% wash buffer solution 2 (50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and 60% elution buffer solution (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow rate of 1 cm/min. The elution process of the PSA-rFVIII conjugate is monitored at 280 nm UV, and the eluate containing the conjugate is collected in a volume of <4 OK. The post-elution step is carried out with >3 OK of elution buffer solution under the same conditions to separate the by-product and/or unmodified rFVIII from the main product.

Finally, the purified conjugate is concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a molecular weight cut-off of 30 kDa (88 cm ² , Millipore).

The conjugate obtained using this technique was examined analytically by measuring total protein, FVIII chromogenic activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay). Specific activity >50% and PSC degree >5.0 were calculated for this conjugate.

Method 3:

50 mg of rFVIII was transferred into the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The coupling reaction was carried out for 2 h in the dark with gentle shaking at room temperature. The reaction was then stopped with cysteine (final concentration: 10 mM) for 60 min at RT. Then the conductivity of the reaction mixture was increased to 130 mS/cm by adding a buffer solution containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture was applied to a column filled with 80 ml phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate was eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, PSA-rFVIII-containing fractions were collected and subjected to UV/DP using a 30 kDa membrane made from regenerated cellulose (88 cm ² , Millipore). The resulting preparation was studied by analytical methods by measuring total protein (Bradford method) and chromogenic activity of FVIII. For the PSA-rFVIII conjugate, the specific activity was determined to be >70% compared to the activity

- 14 047322 native rFVIII.

Method 4:

mg of recombinant factor VIII (rFVIII) obtained from the ADVATE manufacturing process in 50 mM GEPES buffer solution (50 mM GEPES, ~350 mM sodium chloride, 5 mM calcium chloride, 0.1% polysorbate 80, pH 7.4), dissolved in reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution was adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, the reagent aminooxy-polysialic acid (ΠΟΚ-ΟΝΗ ₂ ) in 50-fold molar excess was added to this rFVIII solution with gentle stirring for a maximum period of time (t) of 15 minutes. Then, an aqueous solution of m-toluidine (50 mM) was added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution was added to obtain a concentration of 400 μM.

The reaction mixture was incubated for 120±10 min in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction was stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 min.

The resulting PSC-rFVIII conjugate was purified by hydrophobic interaction chromatography (HIC) using low-substitute phenylsepharose FF resin (GE Healthcare) packaged in a column manufactured by GE Healthcare with a bed thickness (h) of 15 cm and a final column volume (FC) of 81 ml .

Ammonium acetate was added to the reaction mixture by adding 50 mM Hepes buffer solution containing 350 mM sodium chloride, 8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two volumes of the reaction mixture were mixed with 1 volume of a buffer system containing ammonium acetate, and the pH of the solution was adjusted to pH 6.9 by adding dropwise 0.5 N. aqueous NaOH solution. This mixture was applied to the CHB column at a flow rate of 1 cm/min, followed by a wash step using >3 OK of equilibration buffer solution (50 mM Hepas, 350 mM sodium chloride, 2.5 M ammonium acetate, 5 mM calcium chloride, pH 6 ,9).

To remove reaction by-products and the antichaotropic salt, a second washing step of >5 OK of wash buffer solution 1 (50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9) was carried out in an upward flow mode at a flow rate of 2 cm/min . Then the purified rFVIII conjugate was eluted in a downward flow mode using a step gradient of 40% wash buffer solution 2 (50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and 60% elution buffer solution ( 20 mM Gapes, 5 mM calcium chloride, pH 7.5) at a flow rate of 1 cm/min. The elution process of the PSA-rFVIII conjugate was monitored at 280 nm UV, and the eluate containing the conjugate was collected in a volume of <4 OC. The post-elution step was carried out with >3 TC of elution buffer under the same conditions to separate by-product and/or unmodified rFVIII from the main product.

Finally, the purified conjugate was concentrated by ultra-/diafiltration (UF/DF) using a membrane made of regenerated cellulose with a molecular weight cut-off of 30 kDa (88 cm ² , Millipore).

The conjugates obtained using this technique were examined analytically by measuring total protein, FVIII chromogenic activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Analysis data (average of 6 consecutive series):

Technological yield (Bradford method): 58.9%.

Technological yield (chrome act. FVIII): 46.4%.

Specific activity: (chrom. act. FVIII/mg protein): 4148 IU/mg.

Specific activity (% of original substance): 79.9%.

Degree of PSA content (mol/mol): 8.1.

Example 18.

Polysialylation of EPO using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The initial concentration of erythropoietin (EPO) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO4 is added to this solution to obtain a final concentration of 200 µM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products, or alternatively transferred to a 20 mL IOC column (Merck EMD TMAE (M)) which equilibrate 15 047322 is sewn with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is balanced with 5 OK

Buffer solution A. Oxidized EPO is eluted with Buffer solution B (20 mM Gepes, 5 mM

CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing EPO are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy-PSA reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-EPO containing fractions were collected and subjected to UV/DF using a membrane made from regenerated cellulose (POMM 10 kDa, 50 cm ² , Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, Method 1 is performed as follows.

mg of EPO is dissolved in 5 ml of histidine buffer solution, pH 6.0 (20 mM L-histidine, 150 mM NaCl). Then 100 μl of an aqueous solution of sodium periodate (5 mM) is added, and the reaction mixture is incubated for 1 hour in the dark at 4°C with gentle stirring, and the reaction is stopped for 15 minutes at room temperature by adding 50 μl of a 1 M aqueous solution of cysteine. The mixture is then subjected to UV/DF using Vivaspin 15R 10 kDa centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

The concentrate (approx. 7 ml) containing oxidized EPO is mixed with 2 ml of an aqueous solution of mtoluidine (50 mM) and incubated for 30 min at room temperature. Then the aminooxy-PSA reagent with an MW of 20 kDa (described above) is added until a 5-fold molar excess of the reagent is obtained. This mixture is incubated for 2.5 hours at RT in the dark with gentle stirring.

Free EPO is removed using anion exchange chromatography (AOX). The reaction mixture is diluted with 20 ml of Buffer A (50 mM Gapes, pH 7.5) and applied to a 20 ml HiPrep QFF 16/10 column (GE Healthcare, Fairfield, CT), pre-equilibrated with Buffer A. The column is then eluted with Buffer. B (50 mM Gapes, 1 M NaCl, pH 7.5). Free EPO is eluted by washing the column with 25% Buffer Solution B, and the conjugate with 50% Buffer Solution B. The conductivity of the conjugate-containing fractions is subsequently increased to ~190 mS/cm with Buffer Solution C (50 mM Hepes, 5 M NaCl, pH 6.9) and applied to a 20 mL HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer D (50 mM Gapes, 3 M NaCl, pH 6.9). The free PSA reagent is washed out with Buffer Solution D in an amount of 5 OK. Next, the conjugate is eluted with 100% Buffer Solution E (50 mM Gapes, pH 7.4). Conjugate-containing fractions were concentrated by UV/DP using a 10 kDa membrane made from regenerated cellulose (88 cm ² , 10 kDa cutoff/Millipore). The final stage of diafiltration is carried out against a histidine buffer solution with pH 7.2 containing 150 mM NaCl. The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art. For the PSA-EPO conjugate, a specific activity of >50% was determined compared to the activity of native EPO. The conjugate was further analyzed analytically by size exclusion HPLC using an Agilent 1200 HPLC system equipped with a Shodex KW 803 column under previously described conditions (Kolarich et al, Transfusion 2006;46:1959-77). It was shown that the resulting preparation does not contain free EPO.

Method 2:

EPO is transferred or dissolved in a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is performed for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized EPO is further purified by ion exchange chromatography. The eluate fractions containing oxidized EPO are collected and used in the conjugation reaction.

To the eluate containing purified oxidized EPO, add the reagent aminooxypolysialic acid (PSA-OIN ₂ ) in a 50-fold molar excess with gentle stirring in

- 16 047322 for a maximum period of time (t) 15 min. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-EPO conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-EPO conjugate are collected and concentrated by ultra/diafilteration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Erythropoietin (EPO) is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) is added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO4 (final concentration: 400 µM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, the PSA-EPO containing fractions were collected and subjected to UV/DF using a membrane made from regenerated cellulose (POMM 10 kDa, 88 cm ² , Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 3 is performed as follows. 10 mg of EPO is dissolved in 8 ml of histidine buffer solution, pH 6.0 (20 mM L-histidine, 150 mM NaCl). Then add 200 μl of an aqueous solution of sodium periodate (5 mM) and 2 ml of an aqueous solution of mtoluidine (50 mM). Next, the aminooxy-PSA reagent with an MW of 20 kDa (described above) is added until a 5-fold molar excess of the reagent is obtained. The mixture is incubated for 2 hours in the dark at room temperature with gentle stirring, and the reaction is stopped for 15 minutes at room temperature by adding 100 μl of a 1 M aqueous solution of cysteine.

Free EPO is removed using anion exchange chromatography (AOX). The reaction mixture is diluted with 20 ml of Buffer A (50 mM Gapes, pH 7.5) and applied to a 20 ml HiPrep QFF 16/10 column (GE Healthcare, Fairfield, CT), pre-equilibrated with Buffer A. The column is then eluted with Buffer. B (50 mM Gapes, 1 M NaCl, pH 7.5). Free EPO is eluted by washing the column with 25% Buffer Solution B, and the conjugate with 50% Buffer Solution B. The conductivity of the conjugate-containing fractions is subsequently increased to ~190 mS/cm with Buffer Solution C (50 mM Hepes, 5 M NaCl, pH 6.9) and applied to a 20 mL HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer D (50 mM Hepas, 3 M NaCl, pH 6.9). The free PSA reagent is washed out with Buffer Solution D in an amount of 5 OK. Next, the conjugate is eluted with 100% Buffer Solution E (50 mM Gapes, pH 7.4). Conjugate-containing fractions were concentrated by UV/DP using a 10 kDa membrane made from regenerated cellulose (88 cm ² , cutoff 10 kDa, Millipore). The final stage of diafiltration is carried out against a histidine buffer solution with pH 7.2 containing 150 mM NaCl. The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field. For the PSA-EPO conjugate, a specific activity of >50% was determined compared to the activity of native EPO. The conjugate was further analyzed analytically by size exclusion HPLC using an Agilent 1200 HPLC system equipped with a Shodex KW 803 column under previously described conditions (Kolarich et al, Transfusion 2006;46:1959-77). It was shown that the resulting preparation does not contain free EPO.

Method 4:

EPO is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, the aminooxy-polysialic acid (PSA-OCN ₂ ) reagent is added to this EPO solution in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then, an aqueous solution of m-toluidine (50 mM) is added over 15 minutes until

- 17 047322 final concentration 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting PSA-EPO conjugate is purified by ion exchange chromatography. The PSA-EPO-containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose (POMM 10 kDa, 88 cm ² , Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 19.

Polysialylation of Ang-2 using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The original concentration (Ang-2) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration units to remove excess periodate, quencher and by-products or alternatively transferred to a 20 mL IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM Gapes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized Ang-2 is eluted with Buffer Solution B (20 mM Gepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing Ang-2 were collected. Determine the protein content (Coomassie, Bradford method) and adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-An^ containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. Angiopoietin-2 (Ang-2) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. The eluate fractions containing the PSA-A^-2 conjugate were collected and subjected to UV/DP using a regenerated cellulose membrane (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

- 18 047322

Method 2:

Ang-2 is transferred or dissolved in reaction buffer (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is carried out for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized Ang-2 is further purified by ion exchange chromatography. The eluate fractions containing oxidized Ang-2 are collected and used in the conjugation reaction.

To the eluate containing purified oxidized Ang-2, add the reagent aminohydroxypolysialic acid (ΠΟ'Κ-ΟΝΗ ₂ ) in 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting conjugate nGR-Ang-? additionally purified by ion exchange chromatography.

Fractions containing the nCK-Ang-2 conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Angiopoietin-2 (Ang-2) is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenyl Sepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, nCK-Ang-2-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 3 is performed as follows. Angiopoietin-2 (Ang-2) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. PCK-Ang-2-containing eluate fractions were collected and subjected to UV/DF using a membrane made of regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

Method 4:

Ang-2 is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid (APCK-ONH ₂ ) reagent in 50-fold molar excess is added to this Ang-2 solution with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, add 40 mM aqueous sodium periodate solution until half

- 19 047322 concentration values of 400 µM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting conjugate nGR-Ang-? purified by ion exchange chromatography. PSA-Ang^-containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 20.

Polysialylation of VEGF using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The initial concentration of vascular endothelial growth factor (VEGF) is transferred to a reaction buffer solution (for example, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized VEGF is eluted with Buffer Solution B (20 mM Gepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing VEGF are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M NaOH.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-VEGF-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. Vascular endothelial growth factor (VEGF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. The eluate fractions containing PSA-VEGF were collected and subjected to UV/DP using a membrane made of regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

Method 2:

VEGF is transferred or dissolved in a reaction buffer solution (for example, 50 mM Hepes, 350 mM

- 20 047322 sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is performed for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized VEGF is further purified by ion exchange chromatography. The eluate fractions containing oxidized VEGF are collected and used in the conjugation reaction.

To the eluate containing the purified oxidized VEGF, add the reagent aminohydroxypolysialic acid (ΠΟΚ-ΟΝΗ ₂ ) in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-FRES conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-VEGF conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Vascular endothelial growth factor (VEGF) is transferred into a reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, the PSA-VEGF-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

In an alternative embodiment of the invention, method 3 is performed as follows. Vascular endothelial growth factor (VEGF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. The eluate fractions containing PSA-VEGF were collected and subjected to UV/DP using a membrane made of regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

Method 4:

VEGF is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid (APS-ONH ₂ ) reagent is added to this VEGF solution in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting PSA-FRES conjugate is purified by ion exchange chromatography. Containing

PSC-FRES eluate fractions were collected and concentrated by UV/DAF using a membrane made from regenerated cellulose (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 21:

Polysialylation of EGF using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The initial concentration of epidermal growth factor (EGF) is transferred to a reaction buffer solution (for example, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized EGF is eluted with Buffer Solution B (20 mM Geps, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing EGF are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-EGF-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. Epidermal growth factor (EGF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. The PSA-EGF-containing eluate fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

Method 2:

EGF is transferred or dissolved in a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes until the concentration is obtained

- 22 047322

200 µM. The oxidation reaction is performed for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized EGF is further purified by ion exchange chromatography. The eluate fractions containing oxidized EGF are collected and used in the conjugation reaction.

To the eluate containing purified oxidized EGF, add the reagent aminohydroxypolysialic acid (ΠΟ'Κ-ΟΝΗ ₂ ) in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then, an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-EGF conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-EGF conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Epidermal growth factor (EGF) is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, the PSA-EGF-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

In an alternative embodiment of the invention, method 3 is performed as follows. Epidermal growth factor (EGF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. The conjugate-containing eluate fractions were collected and subjected to UV/DF using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

Method 4:

EGF is dissolved or transferred into a reaction buffer solution (for example, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid (APS-ONH ₂ ) reagent is added to this EGF solution in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

- 23 047322

The resulting EGF conjugate is purified by ion exchange chromatography. PSCEFR-containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 22.

Polysialylation of NGF using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The initial concentration of nerve growth factor (NGF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO.4 is added to this solution to obtain a final concentration of 200 µM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized NGF is eluted with Buffer Solution B (20 mM Gepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing NGF are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-NGF-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined analytically by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. Nerve growth factor (NGF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. The eluate fractions containing PSA-NGF were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

Method 2:

NGF is transferred or dissolved in a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is performed for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) in

- 24 047322 within 15 minutes at T=+22±2°C until a final concentration of 10 mM is obtained and incubation for

60±5 min.

Oxidized NGF is further purified by ion exchange chromatography. Eluate fractions containing oxidized NGF are collected and used in the conjugation reaction.

To the eluate containing purified oxidized NGF, add the reagent aminohydroxypolysialic acid (ΠΟΚ-ΟΝΗ ₂ ) in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-NGF conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-NGF conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Nerve growth factor (NGF) is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, the PSA-NGF-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 3 is performed as follows. Nerve growth factor (NGF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT, and the conjugate is purified by ion exchange chromatography. The PSA-NGF-containing eluate fractions were then collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

Method 4:

NGF is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid (APS-ONH ₂ ) reagent is added to this NGF solution in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting NGF conjugate is purified by ion exchange chromatography. PSCFRN-containing eluate fractions are collected and concentrated by ultra-/diafiltration (UV/DF) using

- 25 047322 membrane made from regenerated cellulose (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 23.

Polysialylation of HGH using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

This document describes that the amino acid sequence of human growth hormone (HGH) is first modified to introduce at least one glycosylation site. After purification, HGH is glycosylated in vitro according to methods known in the art.

The original concentration of human growth hormone (HGH) is transferred to a reaction buffer solution (for example, 50 mM GAPES, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized HGH is eluted with Buffer Solution B (20 mM Gepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing hGH are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-HGH containing fractions were collected and subjected to UV/DF using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. This document describes that the amino acid sequence of human growth hormone (HGH) is first modified to introduce at least one glycosylation site. After purification, HGH is glycosylated in vitro according to methods known in the art. The hGH is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO4 is added to this solution to obtain a final concentration of 200 µM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. The eluate fractions containing PSA-HGH were collected and subjected to UV/DF using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

Method 2:

This document describes that the amino acid sequence of human growth hormone (HGH) is first modified to introduce at least one glycosylation site. After purification, HGH is glycosylated in vitro according to methods known in the art.

- 26 047322

HGH is transferred or dissolved in a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is carried out for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized hGH is further purified by ion exchange chromatography. Eluate fractions containing oxidized hGH are collected and used in the conjugation reaction.

To the eluate containing purified oxidized hGH, add the reagent aminooxy-polysialic acid (ΠΟ'Κ-ΟΝΗ ₂ ) in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-HGH conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-HGH conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

This document describes that the amino acid sequence of human growth hormone (HGH) is first modified to introduce at least one glycosylation site. After purification, HGH is glycosylated in vitro according to methods known in the art.

Human growth hormone (HGH) is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, the PSA-HGH containing fractions were collected and subjected to UV/DF using a membrane made from regenerated cellulose (Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 3 is performed as follows. This document describes that the amino acid sequence of human growth hormone (HGH) is first modified to introduce at least one glycosylation site. After purification, HGH is glycosylated in vitro according to methods known in the art. The hGH is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of mtoluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. The PSA-HGH containing eluate fractions are then collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

Method 4:

This document describes that the amino acid sequence of human growth hormone (HGH) is first modified to introduce at least one glycosylation site. After

- 27 047322 purification HGH is glycosylated in vitro in accordance with methods known in this field.

HGH is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, the reagent aminooxy-polysialic acid (ΠΟΚ-ΟΝΗ ₂ ) is added to this solution of hGH in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting HGH conjugate is purified by ion exchange chromatography. The PSGHRP-containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 24.

Polysialylation of TNF-alpha using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

The original concentration of tumor necrosis factor alpha (TNF-alpha) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized TNF-alpha is eluted with Buffer Solution B (20 mM Hepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing TNF-alpha are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-TNF-alpha-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined analytically by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. Tumor necrosis factor alpha (TNF-alpha) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. Containing

The PSA-TNF-alpha fractions of the eluate were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

Method 2:

TNF-alpha is transferred or dissolved in reaction buffer (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is performed for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized TNF-alpha is further purified by ion exchange chromatography. The eluate fractions containing oxidized TNF-alpha are collected and used in the conjugation reaction.

To the eluate containing purified oxidized TNF-alpha, the aminohydroxypolysialic acid reagent (PSA-ONHj) is added in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine is added over 15 minutes. 50 mM) to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120 ± 10 min at pH 6.0 in the dark at a temperature (T) T = + 22 ± 2 ° C with gentle shaking (protein concentration: 1 mg/ml ).

The resulting PSA-TNF-alpha conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-TNF-alpha conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Tumor necrosis factor alpha (TNF-alpha) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, the PSA-TNF-alpha-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

In an alternative embodiment of the invention, method 3 is performed as follows. Tumor necrosis factor alpha (TNF-alpha) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. The eluate fractions containing PSA-TNF-alpha were collected and subjected to UV/DP using a membrane made of regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

Method 4:

TNF-alpha is dissolved or transferred into a reaction buffer (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve the final protein concentration

- 29 047322

1.0±0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous solution

HCl.

Next, aminooxy-polysialic acid (PSCONH ₂ ) reagent in 50-fold molar excess is added to this TNF-alpha solution with gentle stirring for a maximum period of time (t) of 15 minutes. Then, an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting TNF-alpha conjugate is purified by ion exchange chromatography. The PSA-TNF-alpha-containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 25.

Polysialylation of insulin using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

This document describes that the amino acid sequence of insulin is first modified to introduce at least one glycosylation site. After purification, insulin is glycosylated in vitro according to methods known in the art. The original concentration of insulin is transferred to a reaction buffer solution (for example, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized insulin is eluted with Buffer Solution B (20 mM Gepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing insulin are collected. Protein content is determined (Coomassie, Bradford method), adjusted to 1 mg/ml using reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl2. Finally, the PSA-insulin-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. This document describes that the amino acid sequence of insulin is first modified to introduce at least one glycosylation site. After purification, insulin is glycosylated in vitro according to methods known in the art. Insulin is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

After this, the solution is subjected to UV/DF using centrifugal filtration devices

- 30 047322

Vivaspin to remove excess periodate, reaction quenchers and their by-products.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. The eluate fractions containing PSA insulin were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

Method 2:

This document describes that the amino acid sequence of insulin is first modified to introduce at least one glycosylation site. After purification, insulin is glycosylated in vitro according to methods known in the art.

Insulin is transferred or dissolved in a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is carried out for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized insulin is further purified by ion exchange chromatography. The eluate fractions containing oxidized insulin are collected and used in the conjugation reaction.

To the eluate containing purified oxidized insulin, add aminohydroxypolysialic acid (APS-ON^) reagent in 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-insulin conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-insulin conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

This document describes that the amino acid sequence of insulin is first modified to introduce at least one glycosylation site. After purification, insulin is glycosylated in vitro according to methods known in the art.

Insulin is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) is added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, the PSA-insulin-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

In an alternative embodiment of the invention, method 3 is performed as follows. This document describes that the amino acid sequence of insulin is first modified to introduce at least one glycosylation site. After purification, insulin is glycosylated in vitro according to methods known in the art.

Insulin is transferred into a reaction buffer solution (for example, 50 mM Gapes, 350 mM chloride per

- 31 047322 sodium, 5 mM calcium chloride, pH 6.0) and dilute it to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. The eluate fractions containing PSA insulin were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

Method 4:

This document describes that the amino acid sequence of insulin is first modified to introduce at least one glycosylation site. After purification, insulin is glycosylated in vitro according to methods known in the art.

Insulin is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid (PSCONH2) reagent in 50-fold molar excess is added to this insulin solution with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting insulin conjugate is purified by ion exchange chromatography. The PSA-insulin-containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 26.

Polysialylation of interferon-alpha using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The initial concentration of interferon-alpha is transferred to a reaction buffer solution (for example, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO4 is added to this solution to obtain a final concentration of 200 µM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized interferon-alpha is eluted with Buffer Solution B (20 mM Gepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing interferon-alpha are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-interferon-alpha containing fractions are collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. Interferon-alpha is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. The eluate fractions containing PSA-interferon-alpha were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

Method 2:

Interferon-alpha is transferred or dissolved in a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is carried out for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of Lcysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized interferon-alpha is further purified by ion exchange chromatography. The eluate fractions containing oxidized interferon-alpha are collected and used in the conjugation reaction.

To the eluate containing purified oxidized interferon-gamma, add the aminohydroxypolysialic acid (APS-OMP ₂ ) reagent in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 0.6 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-interferon-alpha conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-interferon-alpha conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

Method 3:

Interferon-alpha is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, PSA-interferon-alpha-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

In an alternative embodiment of the invention, method 3 is performed as follows. Interferon-alpha is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml.

- 33 047322

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. The eluate fractions containing PSA-interferon-alpha were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

Method 4:

Interferon-alpha is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid (HCK-ONH ₂ ) reagent in 50-fold molar excess is added to this interferon-alpha solution with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting interferon-alpha conjugate is purified by ion exchange chromatography. The PSA-interferon-alpha containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 27.

Polysialylation of interferon-gamma using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

mg of interferon-gamma is dissolved in 5 ml of histidine buffer solution, pH 6.0 (20 mM Lhistidine, 150 mM NaCl). Then 100 μl of an aqueous solution of sodium periodate (5 mM) is added and the reaction mixture is incubated for 1 hour in the dark at 4°C with gentle stirring and the reaction is stopped for 15 minutes at room temperature by adding 50 μl of a 1 M aqueous solution of cysteine. The mixture is then subjected to UV/DF using Vivaspin 15R 10 kDa centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

The concentrate (approx. 7 ml) containing oxidized interferon-gamma is mixed with 2 ml of an aqueous solution of m-toluidine (50 mM) and incubated for 30 minutes at room temperature. Then the aminooxy-PSA reagent with an MW of 20 kDa (described above) is added until a 5-fold molar excess of the reagent is obtained. This mixture is incubated for 2.5 hours at RT in the dark with gentle stirring.

Free interferon-gamma is removed using cation exchange chromatography (CEC). The reaction mixture is diluted with 20 ml of Buffer A (50 mM Gapes, pH 6.5) and applied to a 20 ml HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer A. The column is then eluted with Buffer B (50 mM Gapes, 1 M NaCl, pH 6.5). Free interferon-gamma is eluted by washing the column with 25% Buffer Solution B, and the conjugate with 50% Buffer Solution B. The conductivity of the conjugate-containing fractions is subsequently increased to ~190 mS/cm with Buffer Solution C (50 mM Hepes, 5 M NaCl, pH 6.9 ) and applied to a 20 mL HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer D (50 mM Gapes, 3 M NaCl, pH 6.9). The free PSA reagent is washed out with Buffer Solution D in an amount of 5 OK. Next, the conjugate is eluted with 100% Buffer Solution E (50 mM Gapes, pH 6.9). Conjugate-containing fractions were concentrated by UV/DP using a 10 kDa membrane made from regenerated cellulose (88 cm ² , cutoff 10 kDa, Millipore). The final stage of diafiltration is carried out against a histidine buffer solution with pH 6.9 containing 150 mM NaCl. The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field. For conjugate

- 34 047322

PSC-interferon-gamma determined specific activity >50% compared to the activity of native interferon-gamma. The conjugate was further analyzed analytically by size exclusion HPLC using an Agilent 1200 HPLC system equipped with a Shodex KW 803 column under previously described conditions (Kolarich et al, Transfusion 2006;46:1959-77). It was shown that the resulting preparation does not contain free interferon-gamma.

Method 2:

mg of interferon-gamma is dissolved in 8 ml of histidine buffer solution, pH 6.0 (20 mM Lhistidine, 150 mM NaCl). Then add 200 μl of an aqueous solution of sodium periodate (5 mM) and 2 ml of an aqueous solution of m-toluidine (50 mM). Next, the aminooxy-PSA reagent with an MW of 20 kDa (described above) is added until a 5-fold molar excess of the reagent is obtained. The mixture is incubated for 2 hours in the dark at room temperature with gentle stirring, and the reaction is stopped for 15 minutes at room temperature by adding 100 μl of a 1 M aqueous solution of cysteine.

Free interferon-gamma is removed using cation exchange chromatography (CEX). The reaction mixture is diluted with 20 ml of Buffer A (50 mM Gapes, pH 6.5) and applied to a 20 ml HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer A. The column is then eluted with Buffer B (50 mM Gapes, 1 M NaCl, pH 6.5). Free interferon-gamma is eluted by washing the column with 25% Buffer Solution B, and the conjugate with 50% Buffer Solution B. The conductivity of the conjugate-containing fractions is subsequently increased to ~190 mS/cm with Buffer Solution C (50 mM Hepes, 5 M NaCl, pH 6.9 ) and applied to a 20 mL HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer D (50 mM Gapes, 3 M NaCl, pH 6.9). The free PSA reagent is washed out with Buffer Solution D in an amount of 5 OK. Next, the conjugate is eluted with 100% Buffer Solution E (50 mM Gapes, pH 6.9). Conjugate-containing fractions were concentrated by UV/DP using a 10 kDa membrane made from regenerated cellulose (88 cm ² , 10 kDa cutoff/Millipore). The final stage of diafiltration is carried out against a histidine buffer solution with pH 6.9 containing 150 mM NaCl. The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field. For the PSCinterferon-gamma conjugate, a specific activity of >50% was determined compared to the activity of native interferon-gamma. The conjugate was further analyzed analytically by size exclusion HPLC using an Agilent 1200 HPLC system equipped with a Shodex KW 803 column under previously described conditions (Kolarich et al, Transfusion 2006;46:1959-77). It was shown that the resulting preparation does not contain free interferon-gamma.

Method 3:

mg of interferon-gamma is dissolved in 8 ml of histidine buffer solution, pH 6.0 (20 mM Lhistidine, 150 mM NaCl). Then add 200 μl of an aqueous solution of sodium periodate (5 mM) and 2 ml of an aqueous solution of m-toluidine (50 mM). Next, the aminooxy-PSA reagent with an MW of 20 kDa (described above) is added until a 5-fold molar excess of the reagent is obtained. The mixture is incubated for 2 hours in the dark at room temperature with gentle stirring, and the reaction is stopped for 15 minutes at room temperature by adding 100 μl of a 1 M aqueous solution of cysteine.

Free interferon-gamma is removed using cation exchange chromatography (CEX). The reaction mixture is diluted with 20 ml of Buffer A (50 mM Gapes, pH 6.5) and applied to a 20 ml HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer A. The column is then eluted with Buffer B (50 mM Gapes, 1 M NaCl, pH 6.5). Free interferon-gamma is eluted by washing the column with 25% Buffer Solution B, and the conjugate with 50% Buffer Solution B. The conductivity of the conjugate-containing fractions is subsequently increased to ~190 mS/cm with Buffer Solution C (50 mM Hepes, 5 M NaCl, pH 6.9 ) and applied to a 20 mL HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer D (50 mM Gapes, 3 M NaCl, pH 6.9). The free PSA reagent is washed out with Buffer Solution D in an amount of 5 OK. Next, the conjugate is eluted with 100% Buffer Solution E (50 mM Gapes, pH 6.9). Conjugate-containing fractions were concentrated by UV/DP using a 10 kDa membrane made from regenerated cellulose (88 cm ² , 10 kDa cutoff/Millipore). The final stage of diafiltration is carried out against a histidine buffer solution with pH 6.9 containing 150 mM NaCl. The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field. For the PSA-interferon-gamma conjugate, a specific activity of >50% was determined compared to the activity of native interferon-gamma. The conjugate was further analyzed analytically by size exclusion HPLC using an Agilent 1200 HPLC system equipped with a Shodex KW 803 column under previously described conditions (Kolarich et al, Transfusion 2006;46:1959-77). It was shown that the resulting preparation does not contain free interferon-gamma.

Method 4:

Interferon-gamma is dissolved or transferred into a reaction buffer solution (for example, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve the final concentration

- 35 047322 protein 1.0±0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid (ΠΟΚ-ΟΝΗ ₂ ) reagent is added to this interferon-gamma solution in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting interferon-gamma conjugate is purified by ion exchange chromatography. The PSA-interferon-gamma-containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 28.

Polysialylation of G-CSF using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The initial concentration of granulocyte colony-stimulating factor (G-CSF) is transferred to a reaction buffer solution (for example, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. Oxidized G-CSF is eluted with Buffer Solution B (20 mM Gepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing G-CSF are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, the PSA-G-CSF containing fractions were collected and subjected to UV/DF using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. Granulocyte colony-stimulating factor (G-CSF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. Containing

The PSA-G-CSF eluate fractions were collected and subjected to UV/DF using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

Method 2:

G-CSF is transferred or dissolved in reaction buffer (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is carried out for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

Oxidized G-CSF is further purified by ion exchange chromatography. The eluate fractions containing oxidized G-CSF are collected and used in the conjugation reaction.

To the eluate containing purified oxidized G-CSF, add the reagent aminooxypolysialic acid (PSA-ONHj in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then, an aqueous solution of m-toluidine is added over 15 minutes. 50 mM) to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120 ± 10 min at pH 6.0 in the dark at a temperature (T) T = + 22 ± 2 ° C with gentle shaking (protein concentration: 1 mg/ml ).

The resulting PSA-G-CSF conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-G-CSF conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Granulocyte colony-stimulating factor (G-CSF) is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, the PSA-G-CSF containing fractions were collected and subjected to UV/DF using a membrane made from regenerated cellulose (Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 3 is performed as follows. Granulocyte colony-stimulating factor (G-CSF) is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxyPSA reagent (described above) is added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO4 (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. The eluate fractions containing PSA-G-CSF were collected and subjected to UV/DF using a membrane made from regenerated cellulose (Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

Method 4:

G-CSF is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25

- 37 047322 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, the aminooxy-polysialic acid (PSA-OCN ₂ ) reagent is added to this G-CSF solution in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting G-CSF conjugate is purified by ion exchange chromatography. The eluate fractions containing PSA-G-CSF were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 29.

Polysialylation of the Humira protein using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The original concentration of Humira protein is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. The oxidized Humira protein is eluted with Buffer Solution B (20 mM Hepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing Humira protein are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, Humira PSA protein-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. Humira protein is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. NaIO4 is added to this solution to obtain a final concentration of 200 µM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using ion exchange chromatography. Eluate fractions containing Humira PSC protein were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). After this, the drug is examined analytically.

- 38 047322 methods by measuring total protein (Coomassie, Bradford method) and biological activity in accordance with methods known in this field.

Method 2:

Humira protein is transferred or dissolved in a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is carried out for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of Lcysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

The oxidized Humira protein is further purified by ion exchange chromatography. Eluate fractions containing oxidized Humira protein are collected and used in the conjugation reaction.

To the eluate containing the purified oxidized Humira protein, add the aminohydroxypolysialic acid (ΠΟΚ-ΟΝΗ ₂ ) reagent in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-Humira protein conjugate is further purified by ion exchange chromatography. Fractions containing the PSA-Humira protein conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Humira protein is transferred to reaction buffer solution (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, Humira PSA protein-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

In an alternative embodiment of the invention, method 3 is performed as follows. Humira protein is transferred to a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT and the conjugate is purified by ion exchange chromatography. Eluate fractions containing Humira PSC protein were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art.

Method 4:

Humira protein is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid reagent (PSK-39 047322) is added to this Humira protein solution

ONH2) in 50-fold molar excess with gentle stirring for a maximum period of time (t) 15 min. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting Humira protein conjugate is purified by ion exchange chromatography. Humira PSA protein-containing eluate fractions were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Example 30.

Polysialylation of Prolia protein using aminooxy-PSA and m-toluidine as a nucleophilic catalyst.

Method 1:

The original concentration of Prolia protein is transferred to a reaction buffer solution (for example, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. NaIO ₄ is added to this solution to obtain a final concentration of 200 μM. Oxidation is carried out at RT for 30 minutes in the dark with gentle shaking. The reaction is then stopped with cysteine (final concentration: 10 mM) for 60 min at RT.

The solution is then subjected to UV/DF using Vivaspin centrifugal filtration devices to remove excess periodate, quencher and their by-products or alternatively transferred to a 20 ml IOC column (Merck EMD TMAE (M)) which is equilibrated with Buffer Solution A (20 mM Hepes, 5 mM CaCl ₂ , pH 7.0). The column is equilibrated with 5 OK Buffer Solution A. The oxidized Prolia protein is eluted with Buffer Solution B (20 mM Gepes, 5 mM CaCl ₂ , 1 M NaCl, pH 7.0). Fractions containing Prolia protein are collected. Determine the protein content (Coomassie, Bradford method), adjust it to 1 mg/ml using reaction buffer and adjust the pH to 6.0 by adding dropwise 0.5 M HCl.

A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. Excess aminooxy reagent is removed using CHB. The conductivity of the reaction mixture is adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column filled with 80 ml of phenylsepharose FF (GE Healthcare , Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.9. Next, the conjugate is eluted with a 50 mM Hepes buffer solution with pH 7.5 containing 5 mM CaCl ₂ . Finally, Prolia PSA protein-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is then examined by analytical methods by measuring total protein (Coomassie, Bradford method) and biological activity according to methods known in the art.

In an alternative embodiment of the invention, method 1 is performed as follows. 10 mg of Prolia protein is dissolved in 5 ml of histidine buffer solution, pH 6.0 (20 mM L-histidine, 150 mM NaCl). Then 100 μl of an aqueous solution of sodium periodate (5 mM) is added and the reaction mixture is incubated for 1 hour in the dark at 4°C with gentle stirring and the reaction is stopped for 15 minutes at room temperature by adding 50 μl of a 1 M aqueous solution of cysteine. The mixture is then subjected to UV/DF using Vivaspin 15R 10 kDa centrifugal filtration devices to remove excess periodate, the reaction quencher, and their by-products.

The concentrate (approx. 7 ml) containing oxidized Prolia protein is mixed with 2 ml of an aqueous solution of m-toluidine (50 mM) and incubated for 30 min at room temperature. Then the aminooxy-PSA reagent with an MW of 20 kDa (described above) is added until a 5-fold molar excess of the reagent is obtained. This mixture is incubated for 2.5 hours at RT in the dark with gentle stirring.

Free Prolia protein is removed using cation exchange chromatography (CEC). The reaction mixture is diluted with 20 ml of Buffer A (50 mM Gapes, pH 6.5) and applied to a 20 ml HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer A. The column is then eluted with Buffer B (50 mM Gapes, 1 M NaCl, pH 6.5). Free Prolia protein is eluted by washing the column with 25% Buffer Solution B, and the conjugate with 50% Buffer Solution B. The conductivity of the conjugate-containing fractions is subsequently increased to ~190 mS/cm with Buffer Solution C (50 mM Hepes, 5 M NaCl, pH 6.9) and applied to a 20 mL HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer D (50 mM Hepas, 3 M NaCl, pH 6.9). The free PSA reagent is washed out with Buffer Solution D in an amount of 5 OK. Next, the conjugate is eluted with 100% Buffer Solution E (50 mM Gapes, pH 6.9). Conjugate-containing fractions were concentrated by UV/DP using a 10 kDa membrane made from regenerated cellulose (88 cm ² , cutoff 10 kDa, Millipore). The final stage of diafiltration is carried out against a histidine buffer solution with pH 6.9 containing 150 mM NaCl. The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field. For the Prolia PSC protein conjugate, a specific activity of >50% was determined compared to the activity of the native Prolia protein. The conjugate was further analyzed analytically by size exclusion HPLC using an Agilent 1200 HPLC system equipped with a Shodex KW 803 column under previously described conditions (Kolarich et al, Transfusion 2006;46:1959-77). It was shown that the resulting preparation does not contain free Prolia protein.

Method 2:

Prolia protein is transferred or dissolved in a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution. Next, a 40 mM aqueous solution of sodium periodate is added over 10 minutes to obtain a concentration of 200 μM. The oxidation reaction is carried out for 30±5 minutes at temperature (T) T=+22±2°C. Then the reaction is stopped by adding an aqueous solution of Lcysteine (1 M) to the reaction mixture within 15 minutes at T=+22±2°C to obtain a final concentration of 10 mM and incubation for 60±5 minutes.

The oxidized Prolia protein is further purified by ion exchange chromatography. Eluate fractions containing oxidized Prolia protein are collected and used in the conjugation reaction.

To the eluate containing the purified oxidized Prolia protein, add the aminohydroxypolysialic acid (APS-OIN ₂ ) reagent in a 50-fold molar excess with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. The reaction mixture is incubated for 120±10 min at pH 6.0 in the dark at T=+22±2°C with gentle shaking (protein concentration: 1 mg/ml).

The resulting PSA-Prolia protein conjugate is further purified by ion exchange chromatography. Fractions containing Prolia protein conjugate are collected and concentrated by ultra/diafiltration (UV/DF) using a membrane made from regenerated cellulose with a suitable molecular weight cutoff (Millipore).

The conjugate obtained using this technique is examined analytically by measuring total protein, biological activity and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Method 3:

Prolia protein is transferred to the reaction buffer solution (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of the 20 kDa MW aminooxy-PSA reagent (described above) was added, followed by the addition of m-toluidine as a nucleophilic catalyst (final concentration: 10 mM) and NaIO ₄ (final concentration: 400 μM). The binding reaction is carried out for 2 hours in the dark with gentle shaking at room temperature. The reaction is then stopped with cysteine (cysteine concentration: 10 mM) for 60 min at RT. The conductivity of the reaction mixture is then adjusted by adding a buffer solution containing ammonium acetate (50 mM Hepas, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH 6.9) and the mixture is applied to a column packed with phenylsepharose FF (GE Healthcare, Fairfield, CT), pre-equilibrated with a solution of 50 mM Hepas, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9. Next, the conjugate is eluted with a solution of 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally, Prolia PSA protein-containing fractions were collected and subjected to UV/DP using a membrane made from regenerated cellulose (Millipore). The drug is examined by analytical methods by measuring total protein (Bradford method) and biological activity in accordance with methods known in this field.

In an alternative embodiment of the invention, method 3 is performed as follows. 10 mg of Prolia protein is dissolved in 8 ml of histidine buffer solution, pH 6.0 (20 mM L-histidine, 150 mM NaCl). Then add 200 μl of an aqueous solution of sodium periodate (5 mM) and 2 ml of an aqueous solution of m-toluidine (50 mM). Next, add the aminooxy-PSA reagent with an MW of 20 kDa (described above) until

- 41 047322 obtaining a 5-fold molar excess of the reagent. The mixture is incubated for 2 hours in the dark at room temperature with gentle stirring, and the reaction is stopped for 15 minutes at room temperature by adding 100 μl of a 1 M aqueous solution of cysteine.

Free Prolia protein is removed using cation exchange chromatography (CEC). The reaction mixture is diluted with 20 ml of Buffer A (50 mM Gapes, pH 6.5) and applied to a 20 ml HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer A. The column is then eluted with Buffer B (50 mM Gapes, 1 M NaCl, pH 6.5). Free Prolia protein is eluted by washing the column with 25% Buffer B and the conjugate with 50% Buffer B. The conductivity of the conjugate-containing fractions is subsequently increased to ~190 mS/cm with Buffer C (50 mM Gapes, 5 M NaCl, pH 6.9) and applied to a 20 mL HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, CT) pre-equilibrated with Buffer D (50 mM Hepas, 3 M NaCl, pH 6.9). The free PSA reagent is washed out with Buffer Solution D in an amount of 5 OK. Next, the conjugate is eluted with 100% Buffer Solution E (50 mM Gapes, pH 6.9). Conjugate-containing fractions were concentrated by UV/DP using a 10 kDa membrane made from regenerated cellulose (88 cm ² , 10 kDa cutoff/Millipore). The final stage of diafiltration is carried out against a histidine buffer solution with pH 6.9 containing 150 mM NaCl. The drug is tested analytically by measuring total protein (Bradford method) and biological activity according to methods known in the art. For the Prolia PSC protein conjugate, a specific activity of >50% was determined compared to the activity of the native Prolia protein. The conjugate was further analyzed analytically by size exclusion HPLC using an Agilent 1200 HPLC system equipped with a Shodex KW 803 column under previously described conditions (Kolarich et al, Transfusion 2006;46:1959-77). It was shown that the resulting preparation does not contain free Prolia protein.

Method 4:

Prolia protein is dissolved or transferred into a reaction buffer solution (eg, 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to achieve a final protein concentration of 1.0 ± 0.25 mg/ml. Then the pH of the solution is adjusted to 6.0 by adding dropwise 0.5 N. aqueous HCl solution.

Next, aminooxy-polysialic acid (PSCONH2) reagent in 50-fold molar excess is added to this Prolia protein solution with gentle stirring for a maximum period of time (t) of 15 minutes. Then an aqueous solution of m-toluidine (50 mM) is added over 15 min to achieve a final concentration of 10 mM. Finally, 40 mM aqueous sodium periodate solution is added to obtain a concentration of 400 μM.

The reaction mixture is incubated for 120±10 minutes in the dark at temperature (T) T=+22±2°C with gentle shaking. Then the reaction is stopped by adding an aqueous solution of L-cysteine (1 M) to the reaction mixture to obtain a final concentration in the reaction mixture of 10 mM and incubation for 60 ± 5 minutes.

The resulting Prolia protein conjugate is purified by ion exchange chromatography. Eluate fractions containing Prolia PSA protein were collected and concentrated by ultra/diafiltration (UV/DF) using a regenerated cellulose membrane (Millipore).

Conjugates obtained using this technique are examined analytically by measuring total protein, biological activity according to methods known in the art, and determining the degree of polysialylation by measuring PSA content (resorcinol assay).

Sequence Listing <110> BAXTER INTERNATIONAL INC. BAXTER HEALTHCARE SA <120> NUCLEOPHILIC CATALYSTS FOR OXIME BINDING <130> 31315/46928 <150> 13/488 043 <151> 2012-06-04 <150> 61/647 814 <151> 2012-05-16 < 160> 1

- 42 047322 <170> PatentIn version 3.5 <210>1 <211>422 <212> PRT <213> Homo sapiens <400>1

Leu Asn Arg Pro Lys Arg Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val

5 1015

Gln Gly Asn Leu Glu Arg Glu Cys Met Glu Glu Lys Cys Ser Phe Glu 20 2530

Glu Pro Arg Glu Val Phe Glu Asn Thr Glu Lys Thr Thr Glu Phe Trp 35 4045

Lys Gln Tyr Val Asp Gly Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn 50 5560

Gly Gly Ser Cys Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro 65 70 7580

Phe Gly Phe Glu Gly Lys Asn Cys Glu Leu Asp Val Thr Cys Asn Ile 85 9095

Lys Asn Gly Arg Cys Glu Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys

100 105110

Val Val Cys Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys

115 120125

Ser Cys Glu Pro Ala Val Pro Phe Pro Cys Gly Arg Val Ser Val Ser

130 135140

Gln Thr Ser Lys Leu Thr Arg Ala Glu Ala Val Phe Pro Asp Val Asp

145 150 155160

Tyr Val Asn Pro Thr Glu Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln

165 170175

Gly Thr Gln Ser Phe Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp

180 185190

Ala Lys Pro Gly Gln Phe Pro Trp Gln Val Val Leu Asn Gly Lys Val

195 200205

- 43 047322

Asp Ala Phe Cys Gly Gly Ser Ile Val

210 215

Ala Ala His Cys Val Glu Thr Gly Val 225 230

Glu His Asn Ile Glu Glu Thr Glu His

245

Ile Arg Ala Ile Ile Pro His His Asn

260 265

Tyr Asn His Asp Ile Ala Leu Leu Glu

275 280

Asn Ser Tyr Val Thr Pro Ile Cys Ile

290 295

Ile Phe Leu Lys Phe Gly Ser Gly Tyr

305 310

Phe His Lys Gly Arg Ser Ala Leu Val

325

Leu Val Asp Arg Ala Thr Cys Leu Arg

340 345

Asn Asn Met Phe Cys Ala Gly Phe His

355 360

Gln Gly Asp Ser Gly Gly Pro His Val

370 375

Phe Leu Thr Gly Ile Ile Ser Trp Gly 385 390

Lys Tyr Gly Ile Tyr Thr Lys Val Ser 405

Glu Lys Trp Ile Val Thr

220

Ile Thr Val Val Ala Gly

235 240

Glu Gln Lys Arg Asn Val

255

Asn Ala Ala Ile Asn Lys

270

Asp Glu Pro Leu Val Leu

285

Asp Lys Glu Tyr Thr Asn 300

Ser Gly Trp Ala Arg Val

315 320

Gln Tyr Leu Arg Val Pro

335

Thr Lys Phe Thr Ile Tyr

350

Gly Gly Arg Asp Ser Cys 365

Glu Val Glu Gly Thr Ser

380

Glu Cys Ala Met Lys Gly

395 400

Tyr Val Asn Trp Ile Lys

415

Glu Lys Thr Lys Leu Thr 420

-

Claims (18)

CLAIM 1) incubating a solution containing an unoxidized water-soluble polymer with an activated aminooxy linker containing an active aminooxy group under conditions that ensure the formation of a stable oxime bond between the unoxidized water-soluble polymer and the activated aminooxy linker, said conditions including incubation at a temperature of 22°C in for 2 hours; lack of light and mixing; due to which a water-soluble polymer containing an active aminooxy group is formed; And 1) incubating a solution containing an oxidized water-soluble polymer with an activated aminooxy linker containing an active aminooxy group, under conditions that ensure the formation of a stable oxime bond between the oxidized water-soluble polymer and the activated aminooxy linker, and these conditions include a period of time from 1 minute to 24 h; temperature from 2 to 8°C; in the presence or absence of light, presence or absence of stirring; due to which a water-soluble polymer containing an active aminooxy group is formed; And 1. A method for producing a modified therapeutic protein comprising an activated water-soluble polymer conjugated to one or more oxidized carbohydrate moieties of the therapeutic protein, said method comprising: a) the first stage, including adjusting the pH of the solution containing the therapeutic protein to a pH value of 6.0, wherein the concentration of the therapeutic protein is 1.0 mg/ml; b) a second step comprising adding an excess concentration of activated water-soluble polymer to the solution obtained in the first step over a maximum period of 15 minutes, the excess concentration being 50 molar excess, under conditions including a temperature of 22°C, no light and mixing; c) a third step comprising adding m-toluidine to the solution from the second step, wherein m-toluidine is added to provide a final concentration of 10 mM under conditions including a temperature of 22° C., no light and stirring; d) a fourth step comprising oxidizing one or more carbohydrate moieties of the therapeutic protein by adding the oxidizing agent sodium periodate (NaIO ₄ ) to the solution from the third step to provide a final concentration of 400 μM; e) a fifth step, wherein the therapeutic protein is incubated with the activated water-soluble polymer, m-toluidine and NaIO ₄ under conditions that allow the activated water-soluble polymer to be conjugated with one or more oxidized carbohydrate moieties of the therapeutic protein, said conditions including a time period of 2 hours, a temperature of 22 °C; in the absence of light and under agitation, where one or more carbohydrate moieties of the therapeutic protein are oxidized by an oxidizing agent; and wherein an oxime bond is formed between the oxidized carbohydrate moiety and an active aminooxy group on the water-soluble polymer and said formation of the oxime bond is catalyzed by m-toluidine; And f) a sixth step, wherein the conjugation of the water-soluble polymer to one or more oxidized carbohydrate moieties of the therapeutic protein is stopped in the fifth step by the addition of L-cysteine; wherein L-cysteine is added in an amount to provide a final concentration of 10 mM, under conditions including a time period of 60 minutes; temperature from 22°C; lack of light and mixing; wherein said water-soluble polymer is polysialic acid (PSA), wherein said activated water-soluble polymer contains an active aminooxy group and is prepared by a process comprising: 2) purification of the activated water-soluble polymer containing an active aminooxy group by a method selected from the group consisting of chromatography, filtration, dialysis and precipitation. 2. A method for producing a modified therapeutic protein comprising an activated water-soluble polymer conjugated to one or more oxidized carbohydrate moieties of the therapeutic protein, said method comprising: a) the first stage, including adjusting the pH of the solution containing the therapeutic protein to a pH value of 6.0, wherein the concentration of the therapeutic protein is 1.0 mg/ml; b) a second step comprising adding an excess concentration of activated water-soluble polymer to the solution from the first step for a maximum period of time of 15 minutes, the excess concentration being 50 molar excess, under conditions including a temperature of 22°C; lack of light and mixing; c) a third step comprising adding m-toluidine to the solution from the second step, wherein m-toluidine is added to provide a final concentration of 10 mM under conditions including a temperature of 22°C; lack of light and mixing; d) a fourth step comprising oxidizing one or more carbohydrate moieties of the therapeutic protein by adding the oxidizing agent sodium periodate (NaIO4) to the solution from the third step to provide a final concentration of 400 µM; e) a fifth step, where the therapeutic protein is incubated with the activated water-soluble polymer, m-toluidine and NaIO4 under conditions that allow the activated water-soluble polymer to conjugate with one or more oxidized carbohydrate moieties of the therapeutic protein, said conditions including a time period of 2 hours, a temperature of 22° C; absence of light and stirring, where one or more carbohydrate components of the therapeutic protein - 45 047322 are oxidized by an oxidizing agent; and wherein an oxime bond is formed between the oxidized carbohydrate moiety and an active aminooxy group on the water-soluble polymer, and said formation of the oxime bond is catalyzed by m-toluidine; And f) a sixth step, wherein the conjugation of the water-soluble polymer to one or more oxidized carbohydrate moieties of the therapeutic protein is stopped in the fifth step by the addition of L-cysteine; wherein L-cysteine is added in an amount to provide a final stopping agent concentration of 10 mM, under conditions including a time period of 60 minutes; temperature from 22°C; lack of light and mixing; wherein said activated water-soluble polymer is polysialic acid (pSA); wherein said activated water-soluble polymer contains an active aminooxy group and is prepared by a process comprising: 2) purification of the activated water-soluble polymer containing an active aminooxy group by a method selected from the group consisting of chromatography, filtration, dialysis and precipitation, at a temperature of 2 to 8°C. 3. The method according to claim 1 or 2, where the therapeutic protein is selected from the group consisting of: Factor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), von Willebrand factor (VWF), Factor FV (FV) , Factor X (FX), Factor XI (FXI), Factor XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1, tissue factor (TF), ADAMTS 13 protease, IL-1 alpha , IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL11, colony-stimulating factor-1 (CSF-1), M-CSF, CSF, GM-CSF, granulocyte colony-stimulating factor factor (G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8, IL-9, IL-10, IL -12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24 , IL-31, IL-32 alpha, IL-33, thrombopoietin (TPO), Ang-1, Ang-2, Ang-4, Ang-Y, angiopoietin-like polypeptide 1 (ANGPTL1), angiopoietin-like polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide 3 (ANGPTL3), angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin-like polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide 6 (ANGPTL6), angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin, vascular endothelial growth factor (VEGF), angiogenin, activin A, activin B, activin C, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein -8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA , bone morphogenetic protein receptor IB, bone morphogenetic protein receptor II, brain-derived neurotrophic factor, cardiotrophin-1, ciliary neurotrophic factor, ciliary neurotrophic factor receptor, crypto protein, cryptic protein, cytokine-inducible neutrophil chemotactic factor 1, cytokine-inducible neutrophil chemotactic factor 2α , cytokine-inducible neutrophil chemotactic factor 2β, endothelial cell growth factor β, endothelin 1, epidermal growth factor, epigen, epiregulin, neutrophil attractant of epithelial origin, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblast growth factor 13, fibroblast growth factor 16, fibroblast growth factor 17, fibroblast growth factor 19, fibroblast growth factor 20, fibroblast growth factor 21, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell line neurotrophic factor receptor α1, glial cell line neurotrophic factor receptor α2, growth-associated protein, growth-associated protein α, growth-associated protein β, growth-associated protein γ, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, hepatoma-derived growth factor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II , insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor α receptor, nerve growth factor, nerve growth factor receptor, neuropoietin, neurotrophin-3, neurotrophin-4, oncostatin M (OSM), placental growth factor , placental growth factor 2, platelet-derived growth factor - 46 047322 endothelial cells, platelet-derived growth factor, platelet-derived growth factor chain A, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor chain B, platelet-derived growth factor BB, platelet-derived growth factor receptor α, platelet-derived growth factor receptor β, stimulating B-cell progenitor growth factor, stem cell factor (SCF), stem cell factor receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor α, transforming growth factor β, transforming growth factor β 1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, thymic stromal lymphopoietin (TSLP), tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasmogen activator receptor, phospholipase activating protein (PUP), insulin, ricin lectin, prolactin, human chorionic gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue activator plasminogen, IgG, IgE, IgM, IgA and IgD, α-galactosidase, β-galactosidase, DNase, fetuin, luteinizing hormone, estrogen, insulin, albumin, lipoproteins, fetoprotein, transferrin, thrombopoietin, urokinase, integrin, thrombin, leptin, Humira (adalimumab), Prolia (denosumab), Enbrel (etanercept) and a protein from the table or a biologically active fragment thereof. 4. The method according to claim 1, where the solution containing the oxidized water-soluble polymer and the activated aminooxy linker is incubated at 4°C for 1 hour in the absence of light with stirring. 5. The method according to claim 4, in which the activated water-soluble polymer containing the active aminooxy group is purified using anion exchange chromatography at a temperature of 4°C. 6. The method of claim 5, wherein the water-soluble polymer is oxidized by incubation with NaIO ₄ to form the oxidized water-soluble polymer. 7. The method of claim 2, wherein the activated water-soluble polymer is produced by a method comprising the further step of adding an additional amount of activated aminooxy linker containing the active aminooxy group immediately before increasing the temperature. 8. The method according to claim 2 or 7, wherein the activated water-soluble polymer containing the active aminooxy group is purified by a method selected from the group consisting of dialysis, ultrafiltration/diafiltration (UV/DF) and chromatography at a temperature of 22°C. 9. The method of claim 8, wherein the activated water-soluble polymer containing the active aminooxy group is further purified by a method selected from the group consisting of dialysis, ultrafiltration/diafiltration (UV/DF) or chromatography at 4°C. 10. The method of claim 3, wherein the water-soluble polymer consists of 10-300 units of sialic acid. 11. The method according to claim 3, where the therapeutic protein is FIX or a biologically active fragment thereof. 12. The method according to claim 3, wherein the therapeutic protein is FVIIa or a biologically active fragment thereof. 13. The method according to claim 3, where the therapeutic protein is FVIII or a biologically active fragment thereof. 14. The method according to claim 1, where the activated aminooxy linker is selected from the group consisting of: a) 3-oxa-pentane-1,5-dioxyamine linker of the formula: b) 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula: and and c) 3,6,9,12,15-pentaoxa-heptadecane-1,17-dioxyamine linker of the formula: X /X XX Kk h ₂ n o o o nh ₂ where the water-soluble polymer is PSA, and PSA is oxidized by incubation with an oxidizing agent to form a terminal aldehyde group at the non-reducing end of PSA. 15. The method of claim 14, wherein the aminooxy linker is 3-oxa-pentane-1,5dioxyamine. 16. The method according to claim 1 or 2, further comprising the step of purifying the modified therapeutic protein. 17. The method of claim 16, wherein the modified therapeutic protein is purified by a method selected from the group consisting of chromatography, filtration and precipitation. 18. The method according to claim 16, in which the modified therapeutic protein is purified using chromatography; where an antichaotropic salt is used at the stage of loading the chromatographic medium and at the stage of washing the chromatographic medium; the method contains one or more stages -