( 12 ) United States Patent

US010414793B2 (12 ) United States Patent ( 10) Patent No. : US 10 ,414 , 793 B2 Haider et al. ( 45 ) Date of Patent: " Sep . 17, 2019 ( 54 ) NUCLEOPHILIC CATALYSTS FOR OXIME 9 /644 ; C12N 9/ 96 ; C12Y 304 /21021 ; LINKAGE C12Y 304 /21022 ; CO7K 14 /755 ; COOK 1/ 1075 ; CO7K 1/ 1077 ( 71) Applicants : BAXALTA INCORPORATED , See application file for complete search history . Bannockburn , IL (US ) ; BAXALTA GMBH , Zug (CH ) ( 56 ) References Cited (72 ) Inventors : Stefan Haider, Prinzersdorf (AT ) ; Andreas Ivens, Zurich (CH ) ; U . S . PATENT DOCUMENTS Hanspeter Rottensteiner , Vienna (AT ) ; 4 , 179 , 337 A 12 / 1979 Davis et al. Juergen Siekmann , Vienna (AT ) ; Peter 4 ,757 ,006 A 7 / 1988 Toole , Jr . et al . Turecek , Klosterneuburg (AT ) ; Oliver 4 , 966 , 999 A 10 / 1990 Coughlin et al. Zoechling , Vienna (AT ) 4 ,970 , 300 A 11/ 1990 Fulton et al . 5 , 122 ,614 A 6 / 1992 Zalipsky 5 , 153 , 265 A 10 / 1992 Shadle et al. ( 73 ) Assignees : Baxalta Incorporated , Bannockburn 5 , 198 , 349 A 3 / 1993 Kaufman ( IL ) ; Baxalta GmbH , Zug (CH ) 5 , 198 ,493 A 3 / 1993 Holmberg et al. 5 , 250 ,421 A 10 / 1993 Kaufman et al. ( * ) Notice : Subject to any disclaimer , the term of this 5 , 298 ,643 A 3 / 1994 Greenwald patent is extended or adjusted under 35 5 ,492 , 821 A 2 / 1996 Callstrom et al. 5 ,621 ,039 A 4 / 1997 Hallahan et al . U . S . C . 154 ( b ) by 0 days . 5 , 733 , 873 A 3 / 1998 Osterberg et al . This patent is subject to a terminal dis 5 ,919 , 766 A 7 / 1999 Osterberg et al. 5 , 969, 040 A 10 / 1999 Hallahan et al . claimer . 6 ,037 ,452 A 3 / 2000 Minamino et al . 6 ,048 , 720 A 4 / 2000 Dalborg et al. ( 21 ) Appl . No. : 15 / 281, 616 6 , 183 ,738 B1 2 /2001 Clark 6 ,586 ,398 B1 7 /2003 Kinstler et al . (22 ) Filed : Sep . 30, 2016 (Continued ) (65 ) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2017 /0240590 A1 Aug. 24 , 2017 CA 2647314 Al 11 / 2007 EP 0 306 968 A2 3 / 1989 Related U . S . Application Data (Continued ) (63 ) Continuation of application No. 14 / 136 , 233, filed on Dec . 20 , 2013 , now Pat. No. 9 , 492 , 555 , which is a OTHER PUBLICATIONS continuation of application No. 13 / 194 , 038 , filed on Abuchowski et al. , Cancer therapy with chemically modified enzymes . Jul. 29 , 2011 , now Pat. No . 8 ,642 ,737 , which is a I . Antitumor properties of polyethylene glycol- asparaginase conju continuation - in - part of application No. 12 /843 ,542 , gates. Cancer Biochem . Biophys. 7 : 175 - 86 ( 1984 ). filed on Jul . 26 , 2010 , now Pat. No . 8 ,637 ,640 . (Continued ) ( 60 ) Provisional application No . 61/ 369 , 186 , filed on Jul. 30 , 2010 , provisional application No . 61/ 347, 136 , Primary Examiner — Lisa J Hobbs filed on May 21, 2010 , provisional application No . (74 ) Attorney , Agent, or Firm — Morgan , Lewis & 61/ 228 ,828 , filed on Jul. 27 , 2009 . Bockius LLP Int. Cl. CO7K 14 /62 ( 2006 .01 ) (57 ) ABSTRACT COZK 1 / 107 ( 2006 . 01 ) A61K 47 /60 ( 2017 . 01 ) The invention relates to materials and methods of conjugat A61K 47 /61 ( 2017 .01 ) ing a water soluble polymer to an oxidized carbohydrate CO7K 1/ 20 ( 2006 . 01) moiety of a therapeutic protein comprising contacting the COZK 1 / 34 ( 2006 .01 ) oxidized carbohydrate moiety with an activated water CO7K 14 / 755 ( 2006 .01 ) soluble polymer under conditions that allow conjugation . (52 ) U . S . CI. More specifically , the present invention relates to the afore CPC ...... CO7K 1 / 1077 (2013 .01 ) ; A61K 47 /60 mentioned materials and methods wherein the water soluble (2017 . 08 ) ; A61K 47/ 61 (2017 .08 ) ; C07K 1/ 20 polymer contains an active aminooxy group and wherein an ( 2013. 01 ) ; C07K 1 / 34 ( 2013 .01 ) ; C07K 14 / 755 oxime or hydrazone linkage is formed between the oxidized (2013 .01 ) carbohydrate moiety and the active aminooxy group on the (58 ) Field of Classification Search water soluble polymer , and wherein the conjugation is CPC ...... A61K 47 /60 ; A61K 47 /61 ; A61K 38 /37 ; carried out in the presence of a nucleophilic catalyst . A61K 38 / 4846 ; A61K 47 / 54 ; A61K 47 /62 , A61K 47 /64 ; A61K 47 /642 ; A61K 47 /644 ; A61K 47 /6455 ; A61K 47 /65 ; 52 Claims, 6 Drawing Sheets C08B 37 /0006 ; C12N 9 /6437 ; C12N Specification includes a Sequence Listing . US 10 ,414 ,793 B2 Page 2

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WO WO 2005 /014035 A2 2 / 2005 Enjolras et al ., Two novel mutations in EGF- like domains of human WO WO 2005 /055950 A1 6 / 2005 factor IX dramatically impair intracellular processing and secretion . WO WO 2005 /070138 A2 8 / 2005 WO WO 2005 /092390 A2 10 / 2005 J . Thromb. Haemost. 2 : 1143 - 54 (2003 ) . WO WO 2006 /013202 A2 2 / 2006 Geoghegan et al ., Periodate inactivation of ovotransferrin and WO WO 2006 /016168 A2 2 / 2006 human serum transferrin . J. Biol . Chem . 255 (27 ): 11429 -34 ( 1980 ). WO WO 2006 /020372 A2 2 / 2006 Great Britain Search Report and Written Opinion , GB - 1012482 . 4 , WO WO 2006 /053299 A2 5 /2006 dated Nov . 24 , 2010 . wo WO 2006 /071801 A2 7 /2006 Gregoriadis et al . , Improving the therapeutic efficacy of peptides WO WO 2006 /074279 Al 7 /2006 and proteins: A role for polysialic acids. Int. J . Pharmaceut. , WO WO 2006 / 127896 A2 11/ 2006 300 ( 1- 2 ): 125 -30 (2005 ). US 10 ,Page 414 ,3793 B2

References Cited Mukherjee et al. , Structural analysis of factor IX protein variants to ( 56 ) predict functional aberration causing haemophilia B . Haemophilia , OTHER PUBLICATIONS 14 ( 5 ) : 1076 -81 (2008 ) . Nektar Advanced PEGylation Catalog 2005 - 2006 , p . 30 (2005 ) . Harris et al ., Effect of pegylation on pharmaceuticals. Nat Rev. Drug Nektar Advanced PEGylation Price List 2005 - 2006 , p . 11 (2005 ). Discovery . 2 : 214 -21 (2003 ). NOF Corporation DDS Catalogue, p . 58 (2005 ) . International Preliminary Report on Patentability , PCT/ GB2010 / Parti et al ., In vitro stability of recombinant human factor VIII 001422 , dated Jan . 31 , 2012 . ( Recombinate® ). Haemophilia , 6 : 513 - 22 ( 2000 ) . International Preliminary Report on Patentability , PCT/ US2007 / Roberts et a . , Chemistry for peptide and protein pegylation Adv. 007560 , dated Sep . 30 , 2008 . Drug Del . Rev. 54 : 459 -76 ( 2002 ). International Preliminary Report on Patentability , PCT/ US2009 / Rosen et al. , Assay of factor VIII : C with a chromogenic substrate . 052103 , dated Feb . 1 , 2011. Scand J. Haematol. 33 (Suppl . 40 ): 139 - 45 ( 1984 ). International Preliminary Report on Patentability, PCT/ US2010 / Rostin et al . , B -domain deleted recombinant coagulation factor VIII 043242, dated Jan . 31, 2012 . modified with monomethoxy polyethylene glycol. Bioconjugate International Preliminary Report on Patentability, PCT /US2011 / Chem . 11 : 387 - 96 ( 2000 ) . 045873 , dated Feb . 5 , 2013 . Saenko et al. , Strategies towards a longer acting factor VIII . International Search Report and Written Opinion of the Interna Haemophilia . 12 : 42 -51 (2006 ) . tional Searching Authority , PCT /US2011 / 045873 , European Patent Sakuragawa et al. , Studies on the stability of factor VIII modified by Office , dated Nov. 24 , 2011 . polyethylene glycol. Acta Med. Biol. 36 : 1 -5 ( 1988 ). International Search Report and Written Opinion of the Interna Schmidt et al. , Structure - function relationships in factor IX and tional Searching Authority , PCT/ US2007 /007560 , European Patent factor IXa. Trends Cardiovasc . Med . 13 ( 1 ) : 39 -45 (2003 ) . Office , dated Sep . 18 , 2007 . Seffernick et al. , Melamine deaminase and atrazine chlorohydrolase : International Search Report and Written Opinion of the Interna 98 % identical but functionally different. J . Bacteriology . 2405 - 10 tional Searching Authority , PCT/ US2009 /052103 , European Patent ( 2001) . Office , dated Feb . 12 , 2010 . Severs et al. , Characterization of PEGylated factor VIII molecules. International Search Report and Written Opinion of the Interna Blood . 108 : 11 - 12 ( 2006 ) . Abstract . tional Searching Authority , PCT/GB2010 /001422 , European Patent Study shows molecular size and structure of PEG interferon mol Office , dated Feb . 4 , 2011 . ecules , as used in pegintron ( R ) , affect antiviral activity in vitro . International Search Report and Written Opinion , PCT/ US2010 / Hispanic PR Wire , Oct. 28 , 2003 . 043242 , dated Feb . 10 , 2011 . Thygesen et al. , Nucleophilic catalysis of carbohydrate oxime Jain et al. , Polysialylation : The natural way to improve the stability formation by anilines. J. Org . Chem ., 75 : 1752 -5 (2010 ). and pharmacokinestics of protein and peptide drugs < < http : // www . Tsubery et al ., Prolonging the action of protein and peptide drugs by lipoxen .co .uk / media /48760 /dds % 20and % 20s % 20pp3 - 9 .pdf > > , dds a novel approach of reversible polyethylene glycol modification . J . & s, 4 ( 1) : 3 -9 (2004 ). Biol. Chem . 279 ( 37 ) : 38118 - 24 (2004 ) . Jenkins et al ., Mutations associated with hemophilia A in the Tsutsumi et al. , Site - specific chemical modification with polyeth 558 -565 loop of the factor VIIIa A2 subunit alter the catalytic ylene glycol of recombinant immunotoxin anti - Tac ( Fv )- PE38 (LMB activity of the factor Xase complex . Blood , 100 ( 2 ) : 501 -8 ( 2002) . 2 ) improves antitumor activity and reduces animal toxicity and Jiang et al. , Chemistry for pegylation of protein and peptide immunogenicity . Proc . Natl. Acad . Sci. USA . 97 : 8548 - 53 ( 2000 ). molecules , Chin . J . Organ . Chem . , 23 ( 12 ) : 1340 - 7 ( 2003 ) . — English Urrutigoity et al. , Biocatalysis in organic solvents with a polymer Abstract. bound horseradish peroxidase . Biocatalysis . 2 : 145 - 9 ( 1989 ) . Kohler , Aniline: A catalyst for sialic acid detection . ChemBioChem , Veronese et al. , Bioconjugation in pharmaceutical chemistry . IL 10 : 2147 -50 ( 2009 ) . Farmaco . 54 : 497 -516 ( 1999 ). Kozlowski et al ., Development of pegylated interferons for the Wells et al . , Additivity of mutational effects in proteins. Biochem treatment of chronic Hepatitis C . BioDrugs . 15 ( 7 ) : 419 - 29 (2001 ) . istry . 29 ( 37 ): 8509 - 17 ( 1990 ) . Lees et al. , Versatile and efficient synthesis of protein Wilchek et al. , Labeling glycoconjugates with hydrazide reagents . polysaccharide conjugate vaccines using aminooxy reagents and Methods Enzymol. 138 : 429 -42 ( 1987 ) . oxime chemistry . Vaccine , 24 ( 6 ) : 716 - 29 ( 2006 ) . Yang et al. , Expression , purification and characterization of factor Lenting et al. , Factor VIII and von Willebrand factor — too sweet for IX derivatives using a novel vector system . Protein Expr. Purif. their own good . Haemophilia , 16 (Suppl . 5 ) : 194 - 9 ( 2010 ) . 50 ( 2 ): 196 - 202 (2006 ) . Lenting et al. , The life cycle of coagulation factor VIII in view of Zalipsky et al. , Hydrazide derivatives of polyethylene glycol) and its structure and function . Blood , 92( 11) : 3983 - 96 ( 1998 ). their bioconjugates. Poly ( ethylene glycol ) Chemistry and Biological Mazsaroff et al. , Quantitative comparison of global carbohydrate Applications . Chapter 21 , pp . 318 - 341 ( 1997 ) . structures of glycoproteins using LC -MS and in - source fragmenta Zeng et al ., High -efficency labeling of sialylated glycoproteins on tion . Anal. Chem . 69 ( 13 ) : 2517 -24 ( 1997 ) . living cells . Nat. Methods, 6 ( 3 ) : 207 - 9 ( 2009 ) . atent Sep . 17 , 2019 Sheet 1 of 6 US 10 ,414 ,793 B2

Figure 1

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2000000000 1000000 COMO - linked Glycan Gla Domain Niisked Glycan 3 . FXla cleavage site atent Sep . 17 , 2019 Sheet 2 of 6 US 10 ,414 ,793 B2

Figure 2

COOH ? pamama - -NHAC

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COOH O ONRYFIX

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N R- FIX wym -NHAC OH atent Sep . 17 , 2019 Sheet 3 of 6 US 10 ,414 ,793 B2

Figure 3

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Figure 4

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Figure 5

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Figure 6

$ 0 .0 % 20 . 0 %

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nocatalyst 10mManiiine 10MM10mMom -toluidine- aminobenzioc 10mm m - aminobenzioc 10mMpacid -aminobenzioc acid10mMo -arninobenzamideacid 10MMsulfanilic acid US 10 ,414 ,793 B2 NUCLEOPHILIC CATALYSTS FOR OXIME 06 /071801 which teaches the oxidation of carbohydrate LINKAGE moieties in Von Willebrand factor and subsequent coupling to PEG using hydrazide chemistry ; US Publication No . FIELD OF THE INVENTION 2009 /0076237 which teaches the oxidation of rFVIII and subsequent coupling to PEG and other water soluble poly The present invention relates to materials and methods for mers ( e . g . PSA , HES , dextran ) using hydrazide chemistry ; conjugating a water soluble polymer to a protein . WO 2008 /025856 which teaches oxidation of different coagulation factors , e . g . rFIX , FVIII and FVIIa and subse BACKGROUND OF THE INVENTION quent coupling to e . g ., PEG , using aminooxy chemistry by forming an oxime linkage; and U . S . Pat . No . 5 , 621, 039 The preparation of conjugates by forming a covalent which teaches the oxidation of FIX and subsequent coupling linkage between the water soluble polymer and the thera to PEG using hydrazide chemistry . peutic protein can be carried out by a variety of chemical Recently, an improved method was described comprising methods . PEGylation of polypeptide drugs protects them in mild periodate oxidation of sialic acids to generate alde circulation and improves their pharmacodynamic and phar - 15 hydes followed by reaction with an aminooxy group con macokinetic profiles (Harris and Chess , Nat Rev Drug taining reagent in the presence of catalytic amounts of Discov. 2003 ; 2 : 214 - 21 ). The PEGylation process attaches aniline (Dirksen A ., and Dawson P E , Bioconjugate Chem . repeating units of ethylene glycol (polyethylene glycol 2008 ; 19, 2543 - 8 ; and Zeng Y et al. , Nature Methods 2009 ; (PEG ) ) to a polypeptide drug. PEG molecules have a large 6 :207 - 9 ) . The aniline catalysis dramatically accelerates the hydrodynamic volume ( 5 - 10 times the size of globular 20 oxime ligation , allowing the use of very low concentrations proteins ), are highly water soluble and hydrated , non - toxic , of the reagent. The use of nucelophilic catalysts are also non - immunogenic and rapidly cleared from the body . PEGY - described in Dirksen , A ., et al ., J Am Chem Soc ., 128 : lation of molecules can lead to increased resistance of drugs 15602 - 3 (2006 ) ; Dirksen , A . , et al. , Angew chem . Int Ed . , to enzymatic degradation , increased half- life in vivo , 45: 7581 -4 (2006 ) ; Kohler, J . J ., ChemBioChem ., 10 :2147 reduced dosing frequency , decreased immunogenicity , 25 50 (2009 ) ; Giuseppone, N ., et al ., J Am Chem Soc. , 127 : increased physical and thermal stability, increased solubility , 5528 -39 (2005 ) ; and Thygesen , M . B ., et al. , J Org Chem ., increased liquid stability , and reduced aggregation . The first 75 : 1752 -5 ( 2010 ) . PEGylated drugs were approved by the FDA in the early Although aniline catalysis can accelerate the oxime liga 1990s. Since then , the FDA has approved several PEGylated tion allowing short reaction times and the use of low drugs for oral, injectable , and topical administration . 30 concentrations of the aminooxy reagent, aniline has toxic Polysialic acid (PSA ) , also referred to as colominic acid properties that must be considered when , for example , the ( CA ) , is a naturally occurring polysaccharide. It is a conjugated therapeutic protein to form the basis of a phar homopolymer of N -acetylneuraminic acid with a 2( 8 ) maceutical. For example, aniline has been shown to induce ketosidic linkage and contains vicinal diol groups at its methemoglobinemia (Harrison , J . H . , and Jollow , D . J. , non - reducing end . It is negatively charged and a natural 35 Molecular Pharmacology, 32 (3 ) 423 -431 , 1987 ) . Long - term constituent of the human body . It can easily be produced dietary treatment of rats has been shown to induce tumors in from bacteria in large quantities and with pre - determined the spleen (Goodman , D G ., et al. , J Natl Cancer Inst . , physical characteristics ( U . S . Pat. No . 5 ,846 , 951 ) . Because 73 ( 1 ) : 265 -73 , 1984 ) . In vitro studies have also shown that the bacterially -produced PSA is chemically and immuno - aniline has the potential to induce chromosome mutations logically identical to PSA produced in the human body, 40 and has the potentially genotoxic activity (Bombhard E . M . bacterial PSA is non - immunogenic , even when coupled to et Herbold B , Critical Reviews in Toxicology 35 ,783 - 835 , proteins. Unlike some polymers , PSA acid is biodegradable . 2005 ) . Covalent coupling of colominic acid to catalase and aspara Considering the potentially dangerous properties of ani ginase has been shown to increase enzyme stability in the line and notwithstanding the methods available of conjugat presence of proteolytic enzymes or blood plasma. Compara - 45 ing water soluble polymers to therapeutic proteins, there tive studies in vivo with polysialylated and unmodified remains a need to develop materials and methods for con asparaginase revealed that polysialylation increased the jugating water soluble polymers to proteins that improves half- life of the enzyme (Fernandes and Gregoriadis , Int J the protein ' s pharmacodynamic and /or pharmacokinetic Pharm . 2001; 217 :215 - 24 ) . properties while minimizing the costs associated with the Coupling of PEG - derivatives to peptides or proteins is 50 various reagents and minimizing the health risks to the reviewed by Roberts et al. ( Adv Drug Deliv Rev 2002 ; patient recipient. 54 :459 - 76 ) . One approach for coupling water soluble poly mers to therapeutic proteins is the conjugation of the poly SUMMARY OF THE INVENTION mers via the carbohydrate moieties of the protein . Vicinal hydroxyl (OH ) groups of carbohydrates in proteins can be 55 The present invention provides materials and methods for easily oxidized with sodium periodate (Nal04 ) to form conjugating polymers to proteins that improves the protein ' s active aldehyde groups ( Rothfus et Smith , J Biol Chem pharmacodynamic and /or pharmacokinetic properties while 1963; 238 : 1402 - 10 ; van Lenten et Ashwell, J Biol Chem minimizing the costs associated with the various reagents 1971 ; 246 : 1889 - 94 ) . Subsequently the polymer can be and the health risks to the patient recipients when the coupled to the aldehyde groups of the carbohydrate by use 60 conjugation reaction is catalyzed by a nucleophilic catalyst. of reagents containing , for example , an active hydrazide In various embodiments of the invention , alternative cata group (Wilchek M and Bayer E A , Methods Enzymol 1987 ; lysts to substitute for aniline are provided . 138 :429 - 42 ) . A more recent technology is the use of reagents In one embodiment, a method of conjugating a water containing aminooxy groups which react with aldehydes to soluble polymer to an oxidized carbohydrate moiety of a form oxime linkages (WO 96 /40662 , WO2008 /025856 ). 65 therapeutic protein is provided comprising contacting the Additional examples describing conjugation of a water oxidized carbohydrate moiety with an activated water soluble polymer to a therapeutic protein are described in WO soluble polymer under conditions that allow conjugation ; US 10 , 414 , 793 B2 said water soluble polymer containing an active aminooxy endothelial cell growth factor , endothelin 1 , epidermal group and is selected from the group consisting of polyeth - growth factor, epigen , epiregulin , epithelial -derived neutro ylene glycol (PEG ) , branched PEG , PolyPEG® (Warwick phil attractant, fibroblast growth factor 4 , fibroblast growth Effect Polymers; Coventry , UK ) , polysialic acid (PSA ) , factor 5 , fibroblast growth factor 6 , fibroblast growth factor starch , hydroxyalkyl starch (HAS ) , hydroxylethyl starch 5 7 , fibroblast growth factor 8 , fibroblast growth factor 8b , (HES ) , carbohydrate , polysaccharides , pullulane, chitosan , fibroblast growth factor 8c , fibroblast growth factor 9 , hyaluronic acid , chondroitin sulfate , dermatan sulfate , fibroblast growth factor 10 , fibroblast growth factor 11 , starch , dextran , carboxymethyl- dextran , polyalkylene oxide fibroblast growth factor 12 , fibroblast growth factor 13 , ( PAO ) , polyalkylene glycol (PAG ) , polypropylene glycol fibroblast growth factor 16 , fibroblast growth factor 17 , (PPG ), polyoxazoline, polyacryloylmorpholine, polyvinyl 10 fibroblast growth factor 19 , fibroblast growth factor 20 , alcohol (PVA ) , polycarboxylate , polyvinylpyrrolidone , fibroblast growth factor 21 , fibroblast growth factor acidic , polyphosphazene, polyoxazoline , polyethylene- co -maleic fibroblast growth factor basic , glial cell line -derived neutro acid anhydride , polystyrene - co -maleic acid anhydride , poly p hic factor receptor al , glial cell line - derived neutrophic ( 1 -hydroxymethylethylene hydroxymethylformal) ( PHF ) , factor receptor a2, growth related protein , growth related 2 -methacryloyloxy - 2' - ethyltrimethylammoniumphosphate 15 protein a , growth related protein ß , growth related protein y , (MPC ) ; and said carbohydrate moiety oxidized by incuba- heparin binding epidermal growth factor, hepatocyte growth tion with a buffer comprising an oxidizing agent selected factor, hepatocyte growth factor receptor , hepatoma- derived from the group consisting of sodium periodate (NalO4 ) , lead growth factor, insulin - like growth factor I, insulin -like tetraacetate (Pb (OAc ) 4 ) and potassium perruthenate growth factor receptor, insulin - like growth factor II , insulin (KRu04 ) ; wherein an oxime linkage is formed between the 20 like growth factor binding protein , keratinocyte growth oxidized carbohydrate moiety and the active aminooxy factor, leukemia inhibitory factor, leukemia inhibitory factor group on the water soluble polymer , and wherein said oxime receptor a , nerve growth factor nerve growth factor recep linkage formation is catalyzed by a nucleophilic catalyst tor, neuropoietin , neurotrophin -3 , neurotrophin -4 , oncosta selected from the group consisting of o -amino benzoic acid , tin M (OSM ) , placenta growth factor, placenta growth factor m - amino benzoic acid , p - amino benzoic acid , sulfanilic 25 2 , plateletderived endothelial cell growth factor, platelet acid , o - aminobenzamide , o - toluidine , m - toluidine , p -tolui - derived growth factor, platelet derived growth factor A dine , o - anisidine , m - anisidine , and p - anisidine . chain , platelet derived growth factor AA , platelet derived In another embodiment, a method of conjugating a water growth factor AB , platelet derived growth factor B chain , soluble polymer to an oxidized carbohydrate moiety of a platelet derived growth factor BB , platelet derived growth therapeutic protein is provided comprising contacting the 30 factor receptor a , platelet derived growth factor receptor B , oxidized carbohydrate moiety with an activated water pre - B cell growth stimulating factor, stem cell factor (SCF ) , soluble polymer under conditions that allow conjugation ; stem cell factor receptor, TNF, TNFO , TNF1, TNF2 , trans said therapeutic protein selected from the group consisting forming growth factor a , transforming growth factor B , of Factor IX ( FIX ) , Factor VIII (FVIII ) , Factor VIIa (FVIIa ) , transforming growth factor B1, transforming growth factor Von Willebrand Factor (VWF ) , Factor FV (FV ), Factor X 35 31. 2 , transforming growth factor B2, transforming growth (FX ) , Factor XI (FXI ) , Factor XII (FXII ), thrombin ( FII ), factor B3, transforming growth factor B5 , latent transform protein C , protein S , DPA , PAI - 1 , tissue factor ( TF ) , ing growth factor B1, transforming growth factor ß binding ADAMTS 13 protease, IL - 1 alpha , IL - 1 beta , IL - 2 , IL - 3 , protein I , transforming growth factor ß binding protein II , IL - 4 , IL - 5 , IL - 6 , IL - 11, colony stimulating factor- 1 (CSF - 1 ) , transforming growth factor ß binding protein III , thymic M -CSF , SCF, GM -CSF , granulocyte colony stimulating fac - 40 stromal lymphopoietin ( TSLP ), tumor necrosis factor recep tor (G - CSF ) , EPO , interferon - alpha (IFN -alpha ), consensus tor type I , tumor necrosis factor receptor type II , urokinase interferon , IFN - beta , IFN -gamma , IFN - omega, IL - 7 , IL - 8 , type plasminogen activator receptor , phospholipase- activat IL - 9, IL -10 , IL - 12 , IL - 13 , IL - 14 , IL - 15 , IL - 16 , IL - 17 , IL - 18 , ing protein ( PUP ), insulin , lectin ricin , prolactin , chorionic IL - 19 , IL - 20 , IL -21 , IL -22 , IL - 23 , IL - 24 , IL - 31, IL - 32 alpha , gonadotropin , follicle - stimulating hormone , thyroid - stimu IL -33 , thrombopoietin ( TPO ) , Ang - 1, Ang- 2 , Ang- 4 , Ang - Y , 45 lating hormone , tissue plasminogen activator , IgG , IgE , angiopoietin - like polypeptide 1 ( ANGPTL1 ) , angiopoietin - IgM , IgA , and IgD , a - galactosidase , B - galactosidase , like polypeptide 2 (ANGPTL2 ) , angiopoietin - like polypep - DNAse , fetuin , leutinizing hormone , estrogen , insulin , albu tide 3 (ANGPTL3 ) , angiopoietin - like polypeptide 4 ( AN - min , lipoproteins, fetoprotein , transferrin , thrombopoietin , GPTL4 ), angiopoietin - like polypeptide 5 (ANGPTL5 ) , urokinase , integrin , thrombin , leptin , Humira (adalimumab ) , angiopoietin - like polypeptide 6 ( ANGPTL6 ), angiopoietin - 50 Prolia ( denosumab ) , Enbrel ( etanercept) , a protein in Table like polypeptide 7 (ANGPTL7 ) , vitronectin , vascular 1 , or a biologically active fragment, derivative or variant endothelial growth factor (VEGF ) , angiogenin , activin A , thereof; said water soluble polymer containing an active activin B , activin C , bone morphogenic protein - 1 , bone aminooxy group and is selected from the group consisting of morphogenic protein - 2 , bone morphogenic protein - 3 , bone polyethylene glycol (PEG ) , branched PEG , PolyPEG® morphogenic protein - 4 , bone morphogenic protein - 5 , bone 55 (Warwick Effect Polymers ; Coventry, UK ), polysialic acid morphogenic protein - 6 , bone morphogenic protein - 7 , bone (PSA ) , starch , hydroxyalkyl starch (HAS ) , hydroxylethyl morphogenic protein - 8 , bone morphogenic protein - 9 , bone starch (HES ) , carbohydrate , polysaccharides , pullulane , chi morphogenic protein - 10 , bone morphogenic protein - 11, tosan , hyaluronic acid , chondroitin sulfate , dermatan sulfate , bone morphogenic protein - 12 , bone morphogenic protein - starch , dextran , carboxymethyl -dextran , polyalkylene oxide 13 , bone morphogenic protein - 14 , bone morphogenic pro - 60 (PAO ) , polyalkylene glycol (PAG ) , polypropylene glycol tein - 15 , bone morphogenic protein receptor IA , bone mor - (PPG ) , polyoxazoline , polyacryloylmorpholine , polyvinyl phogenic protein receptor IB , bone morphogenic protein alcohol (PVA ) , polycarboxylate , polyvinylpyrrolidone , receptor II , brain derived neurotrophic factor, cardiotrophin - polyphosphazene , polyoxazoline , polyethylene - co - maleic 1 , ciliary neutrophic factor, ciliary neutrophic factor recep - acid anhydride, polystyrene -co -maleic acid anhydride , poly tor, cripto , cryptic , cytokine - induced neutrophil chemotactic 65 ( 1 -hydroxymethylethylene hydroxymethylformal) (PHF ) , factor 1 , cytokine - induced neutrophil , chemotactic factor 2 -methacryloyloxy - 2 ' -ethyltrimethylammoniumphosphate 2a , cytokine - induced neutrophil chemotactic factor 26 , B (MPC ) ; and said carbohydrate moiety oxidized by incuba US 10 , 414 , 793 B2 tion with a buffer comprising an oxidizing agent selected C .; in the presence or absence of light ; and with or without from the group consisting of sodium periodate (Nal04 ) , lead stirring. In another embodiment, the final concentration of tetraacetate (Pb (OAc ) 4 ) and potassium perruthenate oxidizing agent is about 400 uM , and the conditions com (KRu04 ) ; wherein an oxime linkage is formed between the prise a time period of about 10 minutes, a temperature of oxidized carbohydrate moiety and the active aminooxy 5 about 22° C . , the absence of light and with stirring . group on the water soluble polymer ; and wherein in said In yet another embodiment, an aforementioned method is oxime linkage formation is catalyzed by a nucleophilic provided wherein the conjugating the water soluble polymer catalyst selected from the group consisting of o - amino to the oxidized carbohydrate moiety of the therapeutic benzoic acid , m - amino benzoic acid , p -amino benzoic acid , protein is stopped by the addition of a quenching agent sulfanilic acid , o - aminobenzamide , o - toluidine, m - toluidine , 10 selected from the group consisting of L - cysteine , methio p -toluidine , o -anisidine , m - anisidine , and p -anisidine . nine, glutathione , glycerol, sodium meta bisulfite In still another embodiment, an aforementioned method is (Na2S205 ) , tryptophane, tyrosine , histidine or derivatives provided wherein a solution comprising an initial concen - thereof , kresol, imidazol, and combinations thereof; wherein tration of the therapeutic protein between about 0 .3 mg/ml the quenching agent is added in an amount to result in a final and about 3 . 0 mg/ ml is adjusted to a pH value between about 15 concentration between about 1 mM and about 100 mm 5 . 0 and about 8 . 0 prior to contacting with the activated water quenching agent, under conditions comprising a time period soluble polymer. between about 5 minutes and about 120 minutes ; a tempera As used herein , the term “ about” means a value above or ture between about 2° C . and about 37° C .; in the presence below a stated value . In various embodiments , the term or absence of light; and with or without stirring . In another “ about” includes the stated value plus or minus 0 . 1 , 0 . 2 , 0 . 3 , 20 embodiment, the quenching agent is L - cysteine . In still 0 . 4 , 0 . 5 , 0 . 6 , 0 .7 , 0 . 8 , 0 . 9 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 or 10 % of another embodiment, the L -cysteine is added to result in a the stated value . final concentration of about 10 mM and the conditions In yet another embodiment, an aforementioned method is comprise a time period of about 60 minutes , a temperature provided wherein the initial concentration of the therapeutic of about 22° C . , the absence of light and with stirring . protein is about 1 . 0 mg/ ml and the pH is about 6 . 0 . In a 25 In another embodiment, an aforementioned method is related embodiment, the initial concentration of the thera - provided comprising : a ) a first step comprising adjusting the peutic protein is about 0 . 75 mg/ml and the pH is about 6 . 0 . pH value of a solution comprising the therapeutic protein to In still another related embodiment, the initial concentration a pH value between about 5 . 0 and about 8 . 0 , wherein the of the therapeutic protein is about 1 . 25 mg/ ml and the pH is therapeutic protein concentration is between about 0 . 3 about 6 . 0 . 30 mg/ ml and about 3 . 0 mg/ ml; b ) a second step comprising In another embodiment, an aforementioned method is oxidizing one or more carbohydrates on the therapeutic provided wherein the therapeutic protein is contacted by a protein , wherein the oxidizing agent is added to the solution desired excess concentration of activated water soluble in the first step to result in a final concentration between polymer , wherein the excess concentration is between about about 50 uM and about 1000 UM , under conditions com 1 -molar and about 300 -molar excess . In another embodi - 35 prising a time period between about 0 . 1 minutes and about ment, the excess concentration is about 50 - fold molar 120 minutes ; a temperature between about 2° C . and about excess . 37° C . ; in the presence or absence of light, and with or In still another embodiment, an aforementioned method is without stirring ; c ) a third step comprising contacting the provided wherein the therapeutic protein is incubated with therapeutic protein with a desired excess concentration of the activated water soluble polymer under conditions com - 40 activated water soluble polymer , wherein the excess con prising a time period between about 0 . 5 hours and about 24 centration is between about 1 -molar excess and about 300 hours ; a temperature between about 2° C . and about 37° C . ; molar excess , under conditions comprising a time period in the presence or absence of light; and with or without between about 0 . 5 hours and about 24 hours, a temperature stirring . In another embodiment, the conditions comprise a between about 2° C . and about 37° C . ; in the presence or time period of about 120 minutes , a temperature of about 22° 45 absence of light ; and with or without stirring ; d ) a fourth step C ., the absence of light ; and with stirring . As used herein , the comprising adding a nucleophilic catalyst to the solution of term “ stirring" is meant to include stirring at various speeds the third step , wherein the nucleophilic catalyst is added to and intensities ( e . g ., gentle stirring ) by commonly used result in a final concentration between about 1 mM and laboratory or manufacturing equipment and products . about 50 mm , under conditions comprising a time period In another embodiment, an aforementioned method is 50 between about 0 . 1 minutes and about 30 minutes ; a tem provided wherein the nucleophilic catalyst is added in an perature between about 2° C . and about 37° C . ; in the amount to result in a final concentration between about 1 . 0 presence or absence of light, and with or without stirring; e ) mM and about 50 mM nucleophilic catalyst, under condi- a fifth step wherein the therapeutic protein is incubated with tions comprising a time period between about 0 . 1 minutes the activated water soluble polymer and nucleophilic cata and about 30 minutes; a temperature between about 2° C . 55 lyst under conditions that allow conjugation of the activated and about 37° C . ; in the presence or absence of light; and water - soluble polymer to one or more oxidized carbohy with or without stirring . In another embodiment, the final drates on the therapeutic protein , said conditions comprising concentration of the nucleophilic catalyst is about 10 mm , a time period between about 0 . 5 hours and about 24 hours , and the conditions comprise a time period of up to about 15 a temperature between about 2° C . and about 37° C . ; in the minutes , a temperature of about 22° C . , the absence of light ; 60 presence or absence of light, and with or without stirring ; and with stirring . and f ) a sixth step wherein the conjugating the water soluble In still another embodiment, an aforementioned method is polymer to the one or more oxidized carbohydrates of the provided wherein the oxidizing agent is added in an amount therapeutic protein in the fifth step is stopped by the addition to result in a final concentration between about 50 uM and of a quenching agent selected from the group consisting of about 1000 uM oxidizing agent, under conditions compris - 65 L -cysteine , methionine , glutathione, glycerol, Na2S205 (so ing a time period between about 0 . 1 minutes and 120 dium meta bisulfite ), tryptophane , tyrosine , histidine or minutes ; a temperature between about 2° C . and about 37° derivatives thereof, kresol, imidazol, and combinations US 10 , 414 ,793 B2 thereof; wherein the quenching agent is added to result in a final concentration of about 1 mM and about 100 mM , under H2N - NH2 conditions comprising a time period between about 5 min utes and about 120 minutes ; a temperature between about 2° C . and about 37° C . ; in the presence or absence of light, and 5 with or without stirring . In another embodiment, the initial ind concentration of the therapeutic protein in the first step is c ) a 3 , 6 , 9 , 12 , 15 -penatoxa -heptadecane - 1 , 17 - dioxyamine about 1 mg/ ml and the pH is about 6 . 0 ; wherein the final linker of the formula :

HON NH , concentration of oxidizing agent in the second step is about 15 wherein the PSA is oxidized by incubation with a oxidiz 400 uM , and the conditions in the fifth step comprise a time ing agent to form a terminal aldehyde group at the non period of about 10 minutes , a temperature of about 22° C ., reducing end of the PSA . In a related embodiment, the the absence of light and with stirring ; wherein the excess aminooxy linker is 3 -oxa -pentane - 1 , 5 - dioxyamine . concentration in the third step is about 50 molar excess ; In still another embodiment , an aforementioned method is wherein the conditions in the third step comprise a time 20 provided wherein the oxidizing agent is Nal04 . period of about 15 minutes , a temperature of about 22° C . , In another embodiment, an aforementioned method is the absence of light and with stirring ; wherein the final provided wherein the nucleophilic catalyst is provided at a concentration of the nucleophilic catalyst in the fourth step concentration between about 1 mM and about 50 mM . is about 10 mM , and the conditions in the fourth step In one embodiment, the nucleophilic catalyst is m - tolui comprise a time period of about 15 minutes, a temperature 25 dine . In still another embodiment, the m -toluidine is present of about 22° C . , the absence of light and with stirring ; in the conjugation reaction at a concentration of about 10 wherein the conditions of incubating the therapeutic protein mM . with the activated water soluble polymer and nucleophilic In yet another embodiment, an aforementioned method is catalyst in the fifth step comprise a time period of about 2 provided further comprising the step of reducing an oxime hours ; a temperature of about 22° C .; the absence of light; " linkage in the conjugated therapeutic protein by incubating and with stirring ; and wherein the quenching agent in the the conjugated therapeutic protein in a buffer comprising a sixth step is L - cysteine ; and wherein the L - cysteine is added reducing compound selected from the group consisting of to result in a final concentration of about 10 mM and the sodium cyanoborohydride (NaCNBH3 ) , ascorbic acid ( vita conditions in the sixth step comprise a time period of about 35 min C ) and NaBH3. In one embodiment, the reducing 60 minutes , a temperature of about 22° C ., the absence of compound is sodium cyanoborohydride (NaCNBH3 ) . light and with stirring . In still another embodiment, an aforementioned method is In another embodiment, an aforementioned method is provided further comprising the step of purifying the con provided wherein the water soluble polymer is PSA . In jugated therapeutic protein . In another embodiment, the another embodiment the PSA is comprised of about 10 -300 40 conjugated therapeutic protein is purified by a method sialic acid units . In another embodiment, the water soluble selected from the group consisting of chromatography , fil polymer is PEG . In another embodiment, the water soluble tration and precipitation . In another embodiment, the chro polymer is HES . In still another embodiment, the water m atography is selected from the group consisting of Hydro soluble polymer is HAS . phobic Interaction Chromatography (HIC ) , Ion Exchange In still another embodiment, an aforementioned method is 45 chromatography ( IEC ), Size exclusion chromatography provided wherein the therapeutic protein is FIX . In another (SEC ) , Affinity chromatography, and Reversed -phase chro embodiment, the therapeutic protein is FVIIa . In another matography. In still another embodiment, an anti -chaotropic embodiment, the therapeutic protein is FVIII . salt is used in a chromotagraphy loading step and in a In yet another embodiment, an aforementioned method is chromatography washing step . In yet another embodiment, provided wherein the oxidizing agent is sodium periodate 50 the chromatography takes place in a column . In another (NalO4 ) . embodiment , the column comprises a chromatography resin In another embodiment, an aforementioned method is selected from the group consisting of Phenyl - Sepharose FF provided wherein the oxidized carbohydrate moiety of the and Butyl- Sepharose FF . In another embodiment , the resin is therapeutic protein is located in the activation peptide of the present in the column at a bed height of between about 5 cm blood coagulation protein . 55 and about 20 cm . In one embodiment, thebed height is about In one embodiment, an aforementioned method is pro - 10 cm . vided wherein PSA is prepared by reacting an activated In another embodiment, an aforementioned method is aminooxy linker with oxidized PSA ; wherein the aminooxy provided comprising one or more washing steps wherein linker is selected from the group consisting of: flow direction is set to up - flow and wherein the flow rate is a ) a 3 -oxa - pentane - 1 , 5 - dioxyamine linker of the formula : 60 between about 0 . 2 cm /min and about 6 . 7 cm /min . As used herein , the term " down- flow ” refers to a flow direction from the top of the chromatographic column to the bottom of the HN NH2, chromatographic column (normal flow direction /standard mode ) . As used herein , the term " up - flow ” refers to a flow 65 direction from the bottom to the top of the column ( reversed b ) a 3 , 6 , 9 - trioxa - undecane - 1 , 11 -dioxyamine linker of the flow direction ) . In one embodiment , the flow rate is about 2 formula : cm /min . US 10 ,414 ,793 B2 In another embodiment, an aforementioned method is In yet another embodiment, a method of forming an provided comprising one ormore elution steps wherein flow oxime linkage between an oxidized carbohydrate moiety on direction is set to down - flow and wherein the flow rate is a therapeutic protein and an activated water soluble polymer between about 0 . 1 cm /min and about 6 .7 cm /min . In a containing an active aminooxy group is provided comprising related embodiment, the flow rate is about 1 cm /min . 5 the steps of: a ) oxidizing a carbohydrate moiety on a In still another embodiment , an aforementioned method is therapeutic protein by incubating said protein with an oxid provided comprising concentrating the conjugated therapeu inzing agent selected from the group consisting of sodium periodate ( NalO4) , lead tetraacetate (Pb (OAc ) 4 ) and potas tic protein by ultra- /diafiltration (UF /DF ). In another sium perruthenate (KRu04 ) ; and b ) forming an oxime embodiment, the final concentration of therapeutic protein *is 10 linkage between the oxidized carbohydrate moiety of the between about 0 . 5 and about 3 mg/ ml . therapeutic protein and the activated water soluble polymer In another embodiment, an aforementioned method is containing an active aminooxy group in the presence of a provided wherein the therapeutic protein comprises between nuclephilic catalyst under conditions allowing formation of about 5 and about 11 water- soluble polymer moieties . In said oxime linkage; wherein the therapeutic protein is another embodiment, the therapeutic protein comprises 15 selected from the group consisting of Factor IX (FIX ) . between about 1 and about 3 water - soluble polymers . Factor VIII (FVIII ) , Factor VIIa (FVIIa ), Von Willebrand In still another embodiment, an aforementioned method is Factor (WWF ) , Factor FV (FV ) , Factor X (FX ) , Factor XI provided wherein the conjugated therapeutic protein is puri - (FXI ) , Factor XII (FXII ), thrombin (FII ) , protein C , protein fied using chromatography ; wherein an anti -chaotropic salt S , DPA , PAI- 1, tissue factor ( TF ) , ADAMTS 13 protease , is used for a loading step and for a washing step ; themethod 20 IL - 1 alpha, IL - 1 beta , IL - 2 , IL -3 , IL - 4 , IL - 5 , IL -6 , IL - 11 , comprising one or more washing steps wherein flow direc - colony stimulating factor- 1 (CSF - 1 ) , M - CSF, SCF , GM tion is set to up - flow and wherein the flow rate is between CSF , granulocyte colony stimulating factor (G -CSF ) , EPO , about 0 . 2 cm /min and about 6 .7 cm /min and one or more interferon - alpha (IFN -alpha ), consensus interferon , IFN elution steps wherein flow direction is set to down - flow and beta , IFN - gamma, IFN -omega , IL - 7 , IL - 8 , IL - 9 , IL - 10 , wherein the flow rate is between about 0 . 2 cm /min and about 25 IL - 12 , IL - 13, IL - 14 , IL - 15 , IL - 16 , IL - 17 , IL - 18, IL - 19 , 6 . 7 cm /min ; further comprising concentrating the conju IL - 20 , IL - 21 , IL -22 , IL - 23 , IL - 24 , IL - 31 , IL - 32 alpha , IL - 33 , gated therapeutic protein by ultra - / diafiltration (UF /DF ) . In thrombopoietin ( TPO ) , Ang - 1 , Ang - 2 , Ang - 4 , Ang - Y , another embodiment, the chromatography is hydrophobic angiopoietin - like polypeptide 1 (ANGPTL1 ) , angiopoietin interaction chromatography (HIC ) ; wherein the one or more like polypeptide 2 (ANGPTL2 ) , angiopoietin - like polypep washing steps flow rate is about 2 cm /min ; and wherein the 30 tide 3 (ANGPTL3 ), angiopoietin - like polypeptide 4 (AN one or more elution steps flow rate is about 1 cm /min . GPTL4 ) , angiopoietin - like polypeptide 5 ( ANGPTL5 ) , In another embodiment, a modified therapeutic protein angiopoietin - like polypeptide 6 ( ANGPTL6 ), angiopoietin produced by any of the aforementioned methods is provided . like polypeptide 7 (ANGPTL7 ) , vitronectin , vascular In still another embodiment, a method of forming an endothelial growth factor (VEGF ) , angiogenin , activin A , oxime linkage between an oxidized carbohydrate moiety on 35 activin B , activin C , bone morphogenic protein - 1 , bone a therapeutic protein and an activated water soluble polymer morphogenic protein -2 , bone morphogenic protein -3 , bone containing an active aminooxy group is provided comprising morphogenic protein - 4 , bone morphogenic protein - 5 , bone the steps of: a ) oxidizing a carbohydrate moiety on a morphogenic protein - 6 , bone morphogenic protein - 7 , bone therapeutic protein by incubating said protein with an oxi morphogenic protein - 8 , bone morphogenic protein - 9 , bone dizing agent selected from the group consisting of sodium 40 morphogenic protein - 10 , bone morphogenic protein - 11 , periodate (NalO4 ), lead tetraacetate ( Pb (OAc )4 ) and potas - bone morphogenic protein - 12 , bone morphogenic protein sium perruthenate (KRu04 ) ; and b ) forming an oxime 13 , bone morphogenic protein - 14 , bone morphogenic pro linkage between the oxidized carbohydrate moiety of the tein - 15 , bone morphogenic protein receptor IA , bone mor therapeutic protein and the activated water soluble polymer phogenic protein receptor IB , bone morphogenic protein containing an active aminooxy group in the presence of a 45 receptor II, brain derived neurotrophic factor, cardiotrophin nuclephilic catalyst under conditions allowing formation of 1 , ciliary neutrophic factor, ciliary neutrophic factor recep said oxime linkage ; wherein said water soluble polymer tor, cripto , cryptic , cytokine - induced neutrophil chemotactic containing an active aminooxy group is selected from the factor 1, cytokine - induced neutrophil, chemotactic factor group consisting polyethylene glycol (PEG ) , branched PEG , 2a , cytokine - induced neutrophil chemotactic factor 2B , B PolyPEG® (Warwick Effect Polymers; Coventry, UK ) , 50 endothelial cell growth factor, endothelin 1 , epidermal polysialic acid (PSA ) , starch , hydroxyalkyl starch (HAS ) , growth factor , epigen , epiregulin , epithelial - derived neutro hydroxylethyl starch (HES ) , carbohydrate , polysaccharides , phil attractant, fibroblast growth factor 4 , fibroblast growth pullulane , chitosan , hyaluronic acid , chondroitin sulfate , factor 5 , fibroblast growth factor 6 , fibroblast growth factor dermatan sulfate , starch , dextran , carboxymethyl- dextran , 7 , fibroblast growth factor 8 , fibroblast growth factor 8b , polyalkylene oxide (PAO ) , polyalkylene glycol (PAG ), 55 fibroblast growth factor 8c , fibroblast growth factor 9 , polypropylene glycol (PPG ) , polyoxazoline, polyacryloyl fibroblast growth factor 10 , fibroblast growth factor 11 , morpholine , polyvinyl alcohol (PVA ) , polycarboxylate , fibroblast growth factor 12 , fibroblast growth factor 13 , polyvinylpyrrolidone , polyphosphazene, polyoxazoline , fibroblast growth factor 16 , fibroblast growth factor 17 , polyethylene - co -maleic acid anhydride , polystyrene - co -ma - fibroblast growth factor 19 , fibroblast growth factor 20 , leic acid anhydride , poly ( 1 -hydroxymethylethylene 60 fibroblast growth factor 21 , fibroblast growth factor acidic , hydroxymethylformal) (PHF ) , 2 -methacryloyloxy - 2 ' - ethylt - fibroblast growth factor basic , glial cell line- derived neutro rimethylammoniumphosphate (MPC ) ; wherein the nucleo - phic factor receptor al , glial cell line -derived neutrophic philic catalyst is selected from the group consisting of factor receptor a2, growth related protein , growth related 0 -amino benzoic acid , m - amino benzoic acid , p -amino ben protein a , growth related protein ß , growth related protein y , zoic acid , sulfanilic acid , o -aminobenzamide , o - toluidine , 65 heparin binding epidermal growth factor, hepatocyte growth m - toluidine , p -toluidine , o -anisidine , m - anisidine, and factor, hepatocyte growth factor receptor, hepatoma -derived p -anisidine . growth factor, insulin - like growth factor i , insulin - like US 10 ,414 ,793 B2 growth factor receptor, insulin - like growth factor II, insulin - formation of said hydrazone linkage ; wherein said water like growth factor binding protein , keratinocyte growth soluble polymer containing an active hydrazide group is factor, leukemia inhibitory factor, leukemia inhibitory factor selected from the group consisting of polyethylene glycol receptor a , nerve growth factor nerve growth factor recep (PEG ) , branched PEG , PolyPEG® (Warwick Effect Poly tor, neuropoietin , neurotrophin - 3 , neurotrophin - 4 , oncosta - 5 mers ; Coventry, UK ) , polysialic acid (PSA ) , starch , tin M ( OSM ), placenta growth factor, placenta growth factor hydroxyalkyl starch (HAS ), hydroxylethyl starch (HES ) , 2 , platelet - derived endothelial cell growth factor, platelet carbohydrate , polysaccharides , pullulane , chitosan , derived growth factor , platelet derived growth factor A hyaluronic acid , chondroitin sulfate , dermatan sulfate , chain , platelet derived growth factor AA , platelet derived starch , dextran , carboxymethyl- dextran , polyalkylene oxide growth factor AB , platelet derived growth factor B chain , 10 (PAO ), polyalkylene glycol (PAG ), polypropylene glycol platelet derived growth factor BB , platelet derived growth (PPG ) , polyoxazoline , polyacryloylmorpholine, polyvinyl factor receptor a , platelet derived growth factor receptor ß , alcohol (PVA ), polycarboxylate , polyvinylpyrrolidone , pre - B cell growth stimulating factor, stem cell factor ( SCF ) , polyphosphazene , polyoxazoline, polyethylene - co -maleic stem cell factor receptor , TNF, TNFO , TNF1, TNF2 , trans - acid anhydride , polystyrene - co -maleic acid anhydride , poly forming growth factor a , transforming growth factor B , 15 ( 1 - hydroxymethylethylene hydroxymethylformal) (PHF ) , transforming growth factor B1, transforming growth factor 2 -methacryloyloxy - 2 ' - ethyltrimethylammoniumphosphate B1. 2 , transforming growth factor B2, transforming growth (MPC ) ; wherein the nucleophilic catalyst is selected from factor B3 , transforming growth factor B5 , latent transform the group consisting of o - amino benzoic acid , m - amino ing growth factor B1, transforming growth factor ß binding benzoic acid , p - amino benzoic acid , sulfanilic acid , o - amin protein I , transforming growth factor ß binding protein II , 20 obenzamide , o - toluidine, m - toluidine , p - toluidine, o - ani transforming growth factor B binding protein III , thymic sidine , m - anisidine , and p -anisidine . stromal lymphopoietin ( TSLP ) , tumor necrosis factor recep In another embodiment, a method of forming a hydrazone tor type I, tumor necrosis factor receptor type II , urokinase - linkage between an oxidized carbohydrate moiety on a type plasminogen activator receptor, phospholipase - activat - therapeutic protein and an activated water soluble polymer ing protein (PUP ) , insulin , lectin ricin , prolactin , chorionic 25 containing an active hydrazide group comprising the steps gonadotropin , follicle - stimulating hormone, thyroid -stimu - of: a ) oxidizing a carbohydrate moiety on a therapeutic lating hormone , tissue plasminogen activator, IgG , IgE , protein by incubating said protein with an oxidinzing agent IgM , IgA , and IgD , A - galactosidase, B - galactosidase , selected from the group consisting of sodium periodate DNAse, fetuin , leutinizing hormone , estrogen , insulin , albu - (NalO4 ) , lead tetraacetate (Pb (OAC ) 4 ) and potassium per min , lipoproteins, fetoprotein , transferrin , thrombopoietin , 30 ruthenate (KRu04 ); and b ) forming a hydrazone linkage urokinase , integrin , thrombin , leptin , Humira ( adalimumab ) , between the oxidized carbohydrate moiety of the therapeutic Prolia (denosumab ) , Enbrel ( etanercept) , a protein from protein and the activated water soluble polymer containing Table 1 , or a biologically active fragment, derivative or an active hydrazide group in the presence of a nuclephilic variant thereof; wherein said water soluble polymer con catalyst under conditions allowing formation of said hydra taining an active aminooxy group is selected from the group 35 zone linkage ; wherein the therapeutic protein is selected consisting of polyethylene glycol ( PEG ) , branched PEG , from the group consisting of Factor IX ( FIX ), Factor VIII PolyPEG® (Warwick Effect Polymers ; Coventry , UK ) , (FVIII ) , Factor VIIa (FVIIa ) , Von Willebrand Factor (VWF ) , polysialic acid (PSA ) , starch , hydroxyalkyl starch (HAS ) , Factor FV (FV ) , Factor X (FX ) , Factor XI (FXI ) , Factor XII hydroxylethyl starch (HES ) , carbohydrate , polysaccharides , (FXII ) , thrombin ( FII ) , protein C , protein S , DPA , PAI- 1 , pullulane , chitosan , hyaluronic acid , chondroitin sulfate , 40 tissue factor ( TF ), ADAMTS 13 protease , IL - 1 alpha, IL - 1 dermatan sulfate , starch , dextran , carboxymethyl - dextran , beta , IL - 2 , IL - 3 , IL - 4 , IL - 5 , IL - 6 , IL - 11 , colony stimulating polyalkylene oxide (PAO ) , polyalkylene glycol ( PAG ), factor- 1 ( CSF - 1 ) , M - CSF , SCF, GM - CSF , granulocyte polypropylene glycol ( PPG ) , polyoxazoline , polyacryloyl colony stimulating factor ( G - CSF ) , EPO , interferon - alpha morpholine , polyvinyl alcohol (PVA ) , polycarboxylate , (IFN - alpha ), consensus interferon , IFN -beta , IFN - gamma, polyvinylpyrrolidone , polyphosphazene, polyoxazoline, 45 IFN -omega , IL -7 , IL - 8, IL - 9 , IL - 10, IL - 12 , IL - 13, IL -14 , polyethylene - co -maleic acid anhydride, polystyrene- co -ma - IL - 15 , IL - 16 , IL - 17 , IL -18 , IL - 19, IL -20 , IL - 21, IL -22 , leic acid anhydride , poly ( 1 -hydroxymethylethylene IL - 23 , IL - 24 , IL - 31, IL - 32 alpha , IL - 33 , thrombopoietin hydroxymethylformal) (PHF ), 2 - methacryloyloxy - 2 '- ethylt - ( TPO ), Ang - 1 , Ang - 2 , Ang - 4 , Ang - Y , angiopoietin - like rimethylammoniumphosphate (MPC ); wherein the nucleo - polypeptide 1 (ANGPTL1 ) , angiopoietin - like polypeptide 2 philic catalyst is selected from the group consisting of 50 (ANGPTL2 ), angiopoietin - like polypeptide 3 ( ANGPTL3 ), 0 - amino benzoic acid , m - amino benzoic acid , p -amino ben - angiopoietin - like polypeptide 4 ( ANGPTL4 ) , angiopoietin zoic acid , sulfanilic acid , o - aminobenzamide, o -toluidine , like polypeptide 5 ( ANGPTL5 ) , angiopoietin - like polypep m -toluidine , p -toluidine , o -anisidine , m -anisidine , and tide 6 (ANGPTL6 ) , angiopoietin - like polypeptide 7 ( AN p - anisidine. GPTL7 ) , vitronectin , vascular endothelial growth factor In yet another embodiment, a method of forming a 55 (VEGF ), angiogenin , activin A , activin B , activin C , bone hydrazone linkage between an oxidized carbohydrate moi - morphogenic protein - 1 , bone morphogenic protein - 2 , bone ety on a therapeutic protein and an activated water soluble morphogenic protein - 3 , bone morphogenic protein - 4 , bone polymer containing an active hydrazide group is provided morphogenic protein - 5 , bone morphogenic protein -6 , bone comprising the steps of: a ) oxidizing a carbohydrate moiety morphogenic protein - 7 , bone morphogenic protein - 8 , bone on a therapeutic protein by incubating said protein with an 60 morphogenic protein - 9 , bone morphogenic protein - 10 , bone oxidinzing agent selected from the group consisting of morphogenic protein - 11 , bone morphogenic protein - 12 , sodium periodate (NaI04 ), lead tetraacetate (Pb (OAc ) 4 ) and bone morphogenic protein - 13 , bone morphogenic protein potassium perruthenate (KRu04 ) ; and b ) forming a hydra - 14 , bone morphogenic protein - 15 , bone morphogenic pro zone linkage between the oxidized carbohydrate moiety of tein receptor IA , bone morphogenic protein receptor IB , the therapeutic protein and the activated water soluble 65 bone morphogenic protein receptor II , brain derived neuro polymer containing an active hydrazide group in the pres - trophic factor, cardiotrophin - 1 , ciliary neutrophic factor, ence of a nuclephilic catalyst under conditions allowing ciliary neutrophic factor receptor, cripto , cryptic , cytokine US 10 ,414 ,793 B2 13 14 induced neutrophil chemotactic factor 1 , cytokine- induced ( 1 - hydroxymethylethylene hydroxymethylformal ) ( PHF) , neutrophil, chemotactic factor 2a , cytokine - induced neutro 2 -methacryloyloxy - 2 '- ethyltrimethylammoniumphosphate phil chemotactic factor 2ß , ß endothelial cell growth factor, (MPC ) ; wherein the nucleophilic catalyst is selected from endothelin 1 , epidermal growth factor, epigen , epiregulin , the group consisting of o - amino benzoic acid , m - amino epithelial- derived neutrophil attractant, fibroblast growth 5 benzoic acid , p -amino benzoic acid , sulfanilic acid , o - amin factor 4 , fibroblast growth factor 5 , fibroblast growth factor obenzamide , o - toluidine , m -toluidine , p - toluidine , o - ani 6 , fibroblast growth factor 7 , fibroblast growth factor 8 , sidine , m - anisidine , and p - anisidine . fibroblast growth factor 8b , fibroblast growth factor 8c , In another embodiment, an aforementioned method is fibroblast growth factor 9 , fibroblast growth factor 10 , provided wherein the water soluble polymer containing an fibroblast growth factor 11, fibroblast growth factor 12 , 10 active aminooxy group is prepared by a method comprising : fibroblast growth factor 13 , fibroblast growth factor 16 , incubating a solution comprising an oxidized water - soluble fibroblast growth factor 17 , fibroblast growth factor 19 , polymer with an activated aminooxy linker comprising an fibroblast growth factor 20 , fibroblast growth factor 21 , active aminooxy group under conditions that allow the fibroblast growth factor acidic , fibroblast growth factor formation of a stable oxime linkage between the oxidized basic , glial cell line - derived neutrophic factor receptor al, 15 water - soluble polymer and the activated aminooxy linker, glial cell line -derived neutrophic factor receptor a2 , growth said conditions comprising a time period between about 1 related protein , growth related protein a , growth related minute and about 24 hours ; a temperature between about 2° protein ß , growth related protein y , heparin binding epider C . and about 37° C .; in the presence or absence of light, and mal growth factor, hepatocyte growth factor, hepatocyte with or without stirring ; thereby forming a water soluble growth factor receptor , hepatoma - derived growth factor, 20 polymer containing an active aminooxy group ; and b ) puri insulin - like growth factor I , insulin - like growth factor recep fying the water soluble polymer containing an active ami tor, insulin - like growth factor II , insulin -like growth factor nooxy group by a method selected from the group consisting binding protein , keratinocyte growth factor, leukemia inhibi of chromatography, filtration and precipitation . The term tory factor , leukemia inhibitory factor receptor a , nerve " activated water -soluble polymer ” referes , in one embodi growth factor nerve growth factor receptor, neuropoietin , 25 ment, to a water- soluble polyer containing an aldehyde neurotrophin - 3 , neurotrophin - 4 , oncostatin M (OSM ) , pla - group. centa growth factor, placenta growth factor 2 , platelet In yet another embodiment , an aforementioned method is derived endothelial cell growth factor, platelet derived provided wherein the water soluble polymer containing an growth factor , platelet derived growth factor A chain , plate - active aminooxy group is prepared by a method comprising : let derived growth factor AA , platelet derived growth factor 30 a ) incubating a solution comprising an oxidized water AB , platelet derived growth factor B chain , platelet derived soluble polymer with an activated aminooxy linker com growth factor BB , platelet derived growth factor receptor a , prising an active aminooxy group under conditions that platelet derived growth factor receptor B , pre - B cell growth allow the formation of a stable oxime linkage between the stimulating factor, stem cell factor (SCF ) , stem cell factor oxidized water -soluble polymer and the activated aminooxy receptor, TNF, TNFO , TNF1, TNF2 , transforming growth 35 linker, said conditions comprising a time period between factor a , transforming growth factor B , transforming growth about 1 minute and about 24 hours ; a temperature between factor B1, transforming growth factor B1 . 2 , transforming about 2° C . and about 37° C . ; in the presence or absence of growth factor B2 , transforming growth factor B3 , transform - light, and with or without stirring ; thereby forming a water ing growth factor B5 , latent transforming growth factor B1, soluble polymer containing an active aminooxy group ; b ) transforming growth factor ß binding protein 1 , transforming 40 incubating a solution comprising the water soluble polymer growth factor B binding protein II , transforming growth containing an active aminooxy group of step a ) with a factor ß binding protein III , thymic stromal lymphopoietin reducing agent under conditions that allow the formation of ( TSLP ) , tumor necrosis factor receptor type I , tumor necro - a stable alkoxamine linkage between the oxidized water sis factor receptor type II, urokinase - type plasminogen acti soluble polymer and the activated aminooxy linker, said vator receptor, phospholipase - activating protein (PUP ) , 45 conditions comprising a time period between about 1 minute insulin , lectin ricin , prolactin , chorionic gonadotropin , fol- and about 24 hours; a temperature between about 2° C . and licle -stimulating hormone, thyroid - stimulating hormone, tis - about 37° C .; in the presence or absence of light; and with sue plasminogen activator, IgG , IgE , IgM , IgA , and IgD , or without stirring; and c ) purifying the water soluble a - galactosidase , B - galactosidase , DNAse , fetuin , leutinizing polymer containing an active aminooxy group by a method hormone , estrogen , insulin , albumin , lipoproteins , fetopro - 50 selected from the group consisting of chromatography , fil tein , transferrin , thrombopoietin , urokinase , integrin , throm - tration and precipitation . bin , leptin , Humira (adalimumab ) , Prolia (denosumab ) , In still another embodiment, an aforementioned method is Enbrel ( etanercept) , a protein from Table 1 , or a biologically provided wherein the water soluble polymer containing an active fragment, derivative or variant thereof; wherein said active aminooxy group is prepared by a method comprising : water soluble polymer containing an active hydrazide group 55 a ) incubating a solution comprising an oxidized water is selected from the group consisting of polyethylene glycol soluble polymer with an activated aminooxy linker com (PEG ) , branched PEG , PolyPEG® ( Warwick Effect Poly - prising an active aminooxy group under conditions that mers ; Coventry , UK ), polysialic acid ( PSA ) , starch , allow the formation of a stable oxime linkage between the hydroxyalkyl starch (HAS ) , hydroxylethyl starch (HES ) , oxidized water -soluble polymer and the activated aminooxy carbohydrate , polysaccharides , pullulane , chitosan , 60 linker, said conditions comprising a time period between hyaluronic acid , chondroitin sulfate , dermatan sulfate , about 1 minute and about 24 hours ; a temperature between starch , dextran , carboxymethyl- dextran , polyalkylene oxide about 2° C . and about 37° C . ; in the presence or absence of ( PAO ), polyalkylene glycol (PAG ) , polypropylene glycol light, and with or without stirring ; thereby forming a water ( PPG ) , polyoxazoline, polyacryloylmorpholine , polyvinyl soluble polymer containing an active aminooxy group ; b ) alcohol ( PVA ) , polycarboxylate, polyvinylpyrrolidone , 65 incubating a solution comprising the water soluble polymer polyphosphazene, polyoxazoline , polyethylene - co -maleic containing an active aminooxy group of step a ) with a acid anhydride, polystyrene - co -maleic acid anhydride, poly n ucleophilic catalyst under conditions comprising a time US 10 ,414 ,793 B2 15 16 period between 1 minute and 24 hours ; a temperature a ) a 3 -oxa - pentane - 1 , 3 - dioxyamine linker of the formula : between 2° C . and 37° C .; in the presence or absence of light; and with or without stirring ; and c ) purifying the water soluble polymer containing an active aminooxy group by a HN NH2, method selected from the group consisting of chromatogra - 5 phy, filtration and precipitation . In yet another embodiment, an aforementioned method is b ) a 3 , 6 , 9 -trioxa - undecane - 1 , 11- dioxyamine linker of the provided wherein the water soluble polymer containing an formula : active aminooxy group is prepared by a method comprising: a ) incubating a solution comprising an oxidized water - 10 soluble polymer with an activated aminooxy linker com H?N prising an active aminooxy group under conditions that on NH2 allow the formation of a stable oxime linkage between the oxidized water -soluble polymer and the activated aminooxy and linker , said conditions comprising a time period between 15 c ) a 3 ,6 , 9 ,12 , 15 - penatoxa -heptadecane - 1 , 17 -dioxyamine about 1 minute and about 24 hours ; a temperature between linker of the formula :

H?N NH2

about 2° C . and about 37° C . ; in the presence or absence of In yet another embodiment , an aforementioned method is light, and with or without stirring ; thereby forming a water provided wherein the reducing agent is selected from the soluble polymer containing an active aminooxy group ; b ) 25 group consisting of sodium cyanoborohydride (NaCNBH3 ) , incubating a solution comprising the water soluble polymer containing an active aminooxy group of step a ) with a ascorbic acid ( vitamin C ) and NaBH3. In one embodiment, nucleophilic catalyst under conditions comprising a time the reducing agent is sodium cyanoborohydride period between 1 minute and 24 hours ; a temperature (NaCNBH3 ) . between 2° C . and 37° C . ; in the presence or absence of In another embodiment, an aforementioned method is light ; and with or without stirring ; c ) incubating a solution 3030 provided wherein the nucleophilic catalyst is selected from comprising the water soluble polymer containing an active the group consisting of o -amino benzoic acid , m -amino aminooxy group of step b ) with a reducing agent under benzoic acid , p - amino benzoic acid , sulfanilic acid , c -amin conditions that allow the formation of a stable alkoxamine obenzamide , o - toluidine , m - toluidine , p - toluidine , o - ani linkage between the oxidized water - soluble polymer and the sidine , m -anisidine , and p -anisidine . In one embodiment, the activated aminooxy linker, said conditions comprising a 35 nucleophilic catalyst is m -toluidine . In another embodiment , time period between about 1 minute and about 24 hours ; a the nucleophilic catalyst is added in an amount to result in temperature between about 2° C . and about 37° C . ; in the a final concentration between about 1 .0 mM and about 50 presence or absence of light; and with or without stirring ; mM nucleophilic catalyst . and d ) purifying the water soluble polymer containing an In another embodiment, an aforementioned method is active aminooxy group by a method selected from the group 40 provided further comprising concentrating the conjugated consisting of chromatography , filtration and precipitation . therapeutic protein by ultra - /diafiltration (UF /DF ) . In another embodiment, an aforementioned method is In another embodiment, a method of conjugating a water provided wherein the oxidized water soluble polymer is soluble polymer to an oxidized carbohydrate moiety of a selected from the group consisting of polyethylene glycol blood coagulation protein is provided comprising contacting ( PEG ) , branched PEG , PolyPEG® (Warwick Effect Poly - 45 the oxidized carbohydrate moiety with an activated water mers ; Coventry , UK ) , polysialic acid ( PSA ) , starch , soluble polymer under conditions that allow conjugation ; hydroxyalkyl starch ( HAS ), hydroxylethyl starch (HES ) , said blood coagulation protein selected from the group carbohydrate , polysaccharides , pullulane , chitosan , consisting of Factor IX (FIX ) , Factor VIII (FVIII ) , Factor hyaluronic acid , chondroitin sulfate , dermatan sulfate , VIIa ( FVIIa ) , Von Willebrand Factor (VWF ) , Factor FV starch , dextran , carboxymethyl -dextran , polyalkylene oxide 50 (FV ) , Factor X (FX ) , Factor XI (FXI ) , Factor XII (FXII ), (PAO ) , polyalkylene glycol (PAG ) , polypropylene glycol thrombin (FII ), protein C , protein S , PA , PAI- 1 , tissue factor (PPG ), polyoxazoline , polyacryloylmorpholine, polyvinyl ( TF ) and ADAMTS 13 protease or a biologically active alcohol (PVA ) , polycarboxylate, polyvinylpyrrolidone , fragment, derivative or variant thereof; polyphosphazene , polyoxazoline , polyethylene - co - maleic said water soluble polymer containing an active aminooxy acid anhydride , polystyrene - co -maleic acid anhydride , poly 55 group and is selected from the group consisting of polyeth ( 1 -hydroxymethylethylene hydroxymethylformal) (PHF ), ylene glycol (PEG ) , branched PEG , polysialic acid ( PSA ) , 2 -methacryloyloxy - 2 '- ethyltrimethylammoniumphosphate carbohydrate , polysaccharides, pullulane , chitosan , (MPC ), and wherein said water - soluble polymer is oxidized hyaluronic acid , chondroitin sulfate , dermatan sulfate , by incubation with a oxidizing agent to form a terminal starch , dextran , carboxymethyl- dextran , polyalkylene oxide aldehyde group at the non -reducing end of the water- soluble 60 (PAO ) , polyalkylene glycol ( PAG ) , polypropylene glycol polymer. In one embodiment, the water - soluble polymer is (PPG ) , polyoxazoline , polyacryloylmorpholine , polyvinyl PSA . alcohol (PVA ) , polycarboxylate , polyvinylpyrrolidone , In another embodiment, an aforementioned method is polyphosphazene , polyoxazoline, polyethylene- co -maleic provided wherein the oxidizing agent is NalO4 . acid anhydride, polystyrene - co -maleic acid anhydride , poly In still another embodiment, an aforementioned method is 65 ( 1- hydroxymethylethylene hydroxymethylformal ) (PHF ), provided wherein the aminooxy linker is selected from the 2 -methacryloyloxy - 2 ' - ethyltrimethylammoniumphosphate group consisting of: (MPC ); and US 10 ,414 ,793 B2 17 18 said carbohydrate moiety oxidized by incubation with a bodies, receptors , blood coagulation proteins, growth fac buffer comprising an oxidizing agent selected from the tors , hormones , and ligands . In certain embodiments , the group consisting of sodium periodate (NalO4 ) , lead tetraac - therapeutic protein is a blood coagulation protein such as etate (Pb (OAC ) 4 ) and potassium perruthenate (KRu04 ) ; Factor IX ( FIX ) , Factor VIII (FVIII ), Factor VIIa ( FVIIa ) , wherein an oxime linkage is formed between the oxidized 5 Von Willebrand Factor (VWF ) , Factor FV (FV ), Factor X carbohydrate moiety and the active aminooxy group on the (FX ) , Factor XI ( FXI ), Factor XII (FXII ) , thrombin (FII ) , water soluble polymer. protein C , protein S , PA , PAI- 1 , tissue factor ( TF ) or ADAMTS 13 protease . In one embodiment, a therapeutic FIGURES protein according to the invention is a glycoprotein or, in 10 various embodiments , a protein that is not naturally glyco FIG . 1 shows the primary structure of coagulation Factor sylated in vivo ( i . e ., a protein that does not contain a natural IX (SEQ ID NO : 1 ) . glycosylation site or a protein that is not glycosylated in a FIG . 2 shows the coupling of oxidized rFIX to aminooxy host cell prior to purification ) . PSA . In certain embodiments , the therapeutic protein is immu FIG . 3 shows the synthesis of the water soluble di- 15 noglobulins, cytokines such IL - 1 alpha , IL - 1 beta , IL - 2 , aminoxy linkers 3 - oxa -pentane - 1 , 5 -dioxyamine and 3 , 6 , 9 . IL - 3 , IL - 4 , IL - 5 , IL - 6 , IL - 11 , colony stimulating factor- 1 trioxa -undecane - 1 , 11 -dioxyamine . (CSF - 1) , M -CSF , SCF , GM - CSF , granulocyte colony stimu FIG . 4 shows the preparation of aminooxy -PSA . lating factor ( G - CSF ) , EPO , interferon - alpha ( IFN - alpha ), FIG . 5 shows the visualization of PSA - FIX conjugates consensus interferon , IFN -beta , IFN - gamma, IFN -omega , prepared in the presence of different catalysts by SDS 20 IL - 7 , IL - 8 , IL - 1 , IL - 10 , IL - 12 , IL - 13 , IL - 14 , IL - 15 , IL - 16 , PAGE . a ) Comparison of aniline with m - toluidine using IL - 17 , IL - 18 , IL - 19 , IL -20 , IL -21 , IL -22 , IL - 23 , IL -24 , different concentrations ; b ) Comparison of aniline with ?L - 31 , IL - 32 alpha , IL - 33 , thrombopoietin ( TPO ) , angiopoi 0 -aminobenzoic acid , m -aminobenzoic acid , p -aminoben etins, for example Ang - 1 , Ang -2 , Ang - 4 , Ang - Y, the human zoic acid , p -aminobenzamide and sulfanilic acid ; c ) Com - angiopoietin -like polypeptides ANGPTL1 through 7 , vit parison of aniline and m - toluidine with 0 -anisidine and 25 ronectin , vascular endothelial growth factor (VEGF ) , angio m - anisidine. genin , activin A , activin B , activin C , bone morphogenic FIG . 6 shows percent of polysialylation with various protein - 1 , bone morphogenic protein - 2 , bone morphogenic nucleophilic catalysts . protein - 3 , bone morphogenic protein - 4 , bone morphogenic protein - 5 , bone morphogenic protein - 6 , bone morphogenic DETAILED DESCRIPTION OF THE 30 protein - 7 , bone morphogenic protein - 8 , bone morphogenic INVENTION protein - 9 , bone morphogenic protein - 10 , bone morphogenic protein - 11 , bone morphogenic protein - 12, bone morpho The pharmacological and immunological properties of genic protein - 13 , bone morphogenic protein - 14 , bone mor therapeutic proteins can be improved by chemical modifi- phogenic protein - 15 , bone morphogenic protein receptor IA , cation and conjugation with polymeric compounds such as 35 bone morphogenic protein receptor IB , bone morphogenic polyethylene glycol ( PEG ), branched PEG , polysialic acid protein receptor II , brain derived neurotrophic factor, car ( PSA ) , hydroxyalkyl starch (HAS ) , hydroxylethyl starch diotrophin -1 , ciliary neutrophic factor , ciliary neutrophic (HES ) , carbohydrate , polysaccharides, pullulane , chitosan , factor receptor, cripto , cryptic , cytokine - induced neutrophil hyaluronic acid , chondroitin sulfate , dermatan sulfate , chemotactic factor 1 , cytokine - induced neutrophil, chemot starch , dextran , carboxymethyl -dextran , polyalkylene oxide 40 actic factor 20 , cytokine -induced neutrophil chemotactic ( PAO ) , polyalkylene glycol (PAG ), polypropylene glycol factor 26 , ß endothelial cell growth factor , endothelin 1 , (PPG ) , polyoxazoline, polyacryloylmorpholine , polyvinyl epidermal growth factor, epigen , epiregulin , epithelial -de alcohol ( PVA ) , polycarboxylate , polyvinylpyrrolidone , rived neutrophil attractant, fibroblast growth factor 4 , fibro polyphosphazene , polyoxazoline , polyethylene - co -maleic blast growth factor 5 , fibroblast growth factor 6 , fibroblast acid anhydride , polystyrene - co -maleic acid anhydride , poly 45 growth factor 7 , fibroblast growth factor 8 , fibroblast growth ( 1 -hydroxymethylethylene hydroxymethylformal) (PHF ) , factor 8b , fibroblast growth factor 8c, fibroblast growth 2 -methacryloyloxy - 2 ' - ethyltrimethylammoniumphosphate factor 9 , fibroblast growth factor 10 , fibroblast growth factor (MPC ). The properties of the resulting conjugates generally 11 , fibroblast growth factor 12 , fibroblast growth factor 13 , strongly depend on the structure and the size of the polymer. fibroblast growth factor 16 , fibroblast growth factor 17 , Thus , polymers with a defined and narrow size distribution 50 fibroblast growth factor 19 , fibroblast growth factor 20 , are usually preferred in the art . Synthetic polymers like PEG fibroblast growth factor 21 , fibroblast growth factor acidic , can be manufactured easily with a narrow size distribution , fibroblast growth factor basic , glial cell line -derived neutro while PSA can be purified in such a manner that results in phic factor receptor al, glial cell line - derived neutrophic a final PSA preparation with a narrow size distribution . In factor receptor a2 , growth related protein , growth related addition PEGylation reagents with defined polymer chains 55 protein a , growth related protein ß , growth related protein y , and narrow size distribution are on the market and commer - heparin binding epidermal growth factor, hepatocyte growth cially available for a reasonable price . factor, hepatocyte growth factor receptor , hepatoma- derived The addition of a soluble polymer , such as through growth factor, insulin - like growth factor I , insulin - like polysialylation , is one approach to improve the properties of growth factor receptor, insulin - like growth factor II, insulin therapeutic proteins such as the blood coagulation protein 60 like growth factor binding protein , keratinocyte growth FIX , as well as other coagulation proteins ( e . g . , VWF, FVIIa factor, leukemia inhibitory factor , leukemia inhibitory factor ( see, e. g ., US 2008 /0221032A1 , incorporated herein by receptor a , nerve growth factor nerve growth factor recep reference ) and FVIII ). tor, neuropoietin , neurotrophin - 3 , neurotrophin - 4 , oncosta Therapeutic Proteins tin M (OSM ) , placenta growth factor, placenta growth factor In certain embodiments of the invention , the aforemen - 65 2 , plateletderived endothelial cell growth factor, platelet tioned polypeptides and polynucleotides are exemplified by derived growth factor, platelet derived growth factor A the following therapeutic proteins: enzymes , antigens, anti chain , platelet derived growth factor AA , platelet derived US 10 , 414 , 793 B2 20 growth factor AB , platelet derived growth factor B chain , growth factors , platelet derived growth factors, phospholi platelet derived growth factor BB , platelet derived growth pase - activating protein (PUP ) , insulin , plant proteins such as factor receptor a , platelet derived growth factor receptor ß , lectins and ricins , tumor necrosis factors and related alleles, soluble forms of tumor necrosis factor receptors , interleukin pre -B cell growth stimulating factor, stem cell factor (SCF 1 ), 5 receptors and soluble forms of interleukin receptors , growth stem cell factor receptor , TNF, including TNFO , TNF1 , 5 factors such as tissue growth factors , such as TGFas or TNF2 , transforming growth factor a , transforming growth TGFBs and epidermal growth factors , hormones , somato factor B , transforming growth factor B1, transforming medins , pigmentary hormones , hypothalamic releasing fac growth factor $ 1 . 2 , transforming growth factor B2 , trans tors , antidiuretic hormones, prolactin , chorionic gonadotro forming growth factor B3 , transforming growth factor B5, 10 pin , follicle -stimulating hormone , thyroid - stimulating latent transforming growth factor B1 , transforming growth hormone , tissue plasminogen activator, and immunoglobu factor ß binding protein I, transforming growth factor B lins such as IgG , IgE , IgM , IgA , and IgD , a galactosidase , binding protein II, transforming growth factor ß binding a - galactosidase , B - galactosidase , DNAse , fetuin , leutinizing protein III, thymic stromal lymphopoietin ( TSLP ) , tumor hormone , estrogen , corticosteroids, insulin , albumin , lipo necrosis factor receptor type I , tumor necrosis factor recep - 15 proteins, fetoprotein , transferrin , thrombopoietin , urokinase , tor type II , urokinase - type plasminogen activator receptor, DNase , integrins, thrombin , hematopoietic growth actors , vascular endothelial growth factor, and chimeric proteins leptin , glycosidases, Humira (adalimumab ), Prolia (deno and biologically or immunologically active fragments sumab ) , Enbrel ( etanercept ) , and fragments thereof , or any thereof. fusion proteins comprising any of the above mentioned In certain embodiments , the therapeutic protin is alpha - , 30 proteins or fragments thereof. In addition to the aforemen beta - , and gamma - interferons , colony stimulating factors tioned proteins , the following Table 1 provides therapeutic including granulocyte colony stimulating factors , fibroblast proteins contemplated by the present invention : US 10 ,414 ,793 B2

Secretedphosphoprotein2,24kDa Arylacetamidedeacetylase-like2 Bovineseminalplasmaproteinhomolog1 Plateletderived-growthfactorreceptorlike Leucine-richrepeatcontainingprotein28 Ly6/PLAURdomain-containingprotein2 Myelinproteinzero-like3 lectin14bindinglikeSialicIg-acid Epididymal-specificlipocalin10 Epididymal-specificlipocalin8Basicproline-richpeptidePE Epididymal-specificlipocalin12 ComplementC1q-likeprotein3 LRRN4C-terminallikeprotein Transmembraneprotein81 Protocadherinalpha-1 Transmembraneprotein161A Transmembraneprotein161B Transmembraneprotein182 Transmembraneprotein52 Odontogenicameloblast-associated NeurosecretoryproteinVGF Growthdifferentiationfactor9 domain-containingprotein2 C-typelectindomaincontainingprotein Adisintegrinandmetalloproteinasewith thrombospondinmotifs8 C10orf99proteinPutativeuncharacterized UncharacterizedproteinC17orf77 UncharacterizedproteinC7orf69 UDP-glucuronosyltransferase3A2 Retinoldehydrogenase10 containingprotein4 UDP-glucuronosyltransferase2A3 Clusterin-likeprotein1 Transmembraneandimmunoglobulin Bmelanomaantigen2 UPF0565proteinC2orf69 UPF0669proteinC6orf120 Colipase-likeproteinCorf127 Proteinnotumhomolog PhospholipaseD4 ProteinFAM24B FAM150BProtein PRO19627UNQ5810/ Herstatin protein Bmelanom protein

Mammalianependymin-relatedprotein1 protein2expressedProstatetestisand Contactin-associatedproteinlike3 Glycoproteinhormonebeta-5 Interleukin-1familymember6 GroupXIIAsecretoryphospholipaseA2Collagenalpha-3(V)chain Alpha-2macroglobulinlikeprotein1 Extracellularmatrixprotein2 Bonemorphogeneticprotein2Bonemorphogeneticprotein6 Carbohydratesulfotransferase8 facilitatorsuperfamilydomainsecretoryphospholipaseA2-likeproteinMajorreceptorexpressedonmyeloidcells2GroupXIIBTriggering Adisintegrinandmetalloproteinasewith Adisintegrinandmetalloproteinasewith Adisintegrinandmetalloproteinasewith UncharacterizedproteinKIAA0564 thrombospondinmotifs19 thrombospondinmotifs18 thrombospondinmotifs3 Dickkopf-relatedprotein4 thrombospondinmotifs5 Fibroblastgrowthfactor6 Keratinocytegrowthfactor Growth/differentiationfactor8 Interferonalpha-4 Interferonalpha-5 Interferonalpha-7 thrombospondinmotifs7 Cartilage-associatedprotein UPF0556proteinC19orf10 C—Xmotifchemokine3 Gastrin-releasingpeptide Gastricintrinsicfactor Interleukin-33 Cystatin-M Defensin-5 Defensin-6 GranzymeM SerineproteaseHTRA1 Dermatopontin Cerberus Corticoliberin Fibrillin-3 Fetuin-B TABLE1 AdisintegrinandmetalloproteinasewithDisintegrinmetalloproteinasedomain ComplementClqsubcomponentsubunitBDeserthedgehogprotein Transforminggrowthfactor-betainducedAdisintegrinandmetalloproteinasewith GastricinhibitorypolypeptideComplementcomponentC8gammachain ComplementcomponentC8betachain Granulocytecolony-stimulatingfactor Collagenalpha-4(IV)chain Keratinocytedifferentiation-associated Collagenalpha-2(V)chain Collagenalpha-1(VII)chain chainXValpha)Collagen-1(Collagenalpha-1(XVI)chain Collagenalpha-1(XVIII)chain Collagenalpha-1(XIX)chain Cartilageoligomericmatrixprotein Angiotensin-convertingenzyme 100ApolipoproteinB- protein7morphogeneticBone C4b-bindingproteinalphachain Corticosteroid-bindingglobulin Collagenalpha-1(I)chain LCCLproteinsecretoryCysteine-rich Complement-BC4 ComplementcomponentC7 Granulocyte-macrophagecolony Antithrombin-III Beta-1,4galactosyltransferase CarboxypeptidaseA1 CarboxypeptidaseA2 ComplementfactorDChromogranin-A containingprotein18 domain-containing1 ApolipoproteinDApolipoproteinE C-motifchemokine13 C-motifchemokine18 C-motifchemokine20 C-motifchemokine2 proteinig-h3 ComplementC5 stimulatingfactor Calreticulin Eotaxin CD40ligand Corneodesmosin protein C-reactiveprotein

ligandsuperfamilymember15Tumornecrosisfactor vonWillebrandfactorAdomain-containing Deletedinmalignantbraintumors1protein Folliculardendriticcellsecretedpeptide Secretedfrizzled-relatedprotein1 Secretedfrizzled-relatedprotein2 Secretedfrizzled-relatedprotein5 Secretedfrizzled-relatedprotein4 Secretedfrizzled-relatedprotein3 Pmel17Melanocyteprotein SecretedLy-6/uPARrelatedprotein1 Tumornecrosisfactorligandsuperfamily Anteriorgradientprotein2homolog SPARC-relatedmodularcalciumbinding C-typelectindomainfamily11memberA SecretedLy-6/uPARrelatedprotein2 Secretedandtransmembraneprotein1 Testis-expressedsequence264protein 39SribosomalproteinL55mitochondrial, Fibroblastgrowthfactorreceptor2 SPARC-relatedmodularcalciumbinding Ectodysplasin-A Secretedphosphoprotein24 Proteincanopyhomolog2 WNT1-induciblesignalingpathway ProteinNipSnaphomolog3A Carbonicanhydrase6 C-motifchemokine4 Resistin-likebeta C-motifchemokine1 Serineprotease23 Dermokine Resistin Osteopontin Glypican-6 Beta-microseminoproteinGlypican-4 member12 SPARC Glypican-5 Glypican-1 protein2 protein1 protein2 Glypican-3 Glypican-2 Fibronectin Neudesin protein1 US 10 ,414 ,793 B2 24

Epididymalsperm-bindingprotein1 Putativepregnancy-specificbeta1 proteinexpressed1Prostatetestisand PutativephospholipaseB-like1 Chondroadherin-likeprotein DANdomainfamilymember5 Insulin-likegrowthfactorbindingprotein Growthdifferentiationfactor6 Plasmakallikrein-likeprotein4 Epididymal-specificlipocalin9 cDNAFLJ60957,highlysimilartorelatedprotein4frizzled-Secreted PutativeinactivegroupIICsecretory NHLrepeat-containingprotein3Olfactomedin-likeprotein2B Putativeuncharacterizedprotein Putativeuncharacterizedprotein Putativeuncharacterizedprotein R3HDMLinhibitorPeptidase Glucose-fructoseoxidoreductasedomain containingprotein1 Glutathioneperoxidase6 Ovochymase-2 Putativeuncharacterizedprotein Ovochymase-1 Ovostatinhomolog2 OrexigenicneuropeptideQRFP Lymphocyteantigen6K Putativeuncharacterizedprotein UNQ6490/PRO21339 PRO21345UNQ6493/ UNQ5815/PRO19632 Cystatin-A Cystatin-9 Uncharacterizedprotein Beta-defensin128 Interleukin-31 Interleukin-34 LipasememberM phospholipaseA2SerineproteaseMPN2 UNQ3029/PRO9830 glycoprotein7 FAM55AProtein UNQ511/PRO1026 Netrin-5 like1 Elafin Erythropoietin CLECSF12 FLJ42147 Otogelin

Immunoglobulinsuperfamilymember10 SerineproteaseinhibitorKazal-type2 relatedPMP-22effectortoapoptosisp53 Protease-associateddomaincontaining Abhydrolasedomain-containingprotein Keratinocyte-associatedprotein2 Leukocytecell-derivedchemotaxin2 homologydomain-containingprotein3 Pancreaticlipase-relatedprotein2 Epididymis-specificalphamannosidase FibronectintypeIIIdomain-containing Microfibrillar-associatedprotein5 phosphotransferasesubunitgamma ProteinkinaseC-bindingproteinNELL1ProteinkinaseC-bindingproteinNELL2 Group10secretoryphospholipaseA2GroupIIDsecretoryphospholipase A2 Tuberoinfundibularpeptideof Adisintegrinmetalloproteinaseandwith thrombospondinmotifs9 BMP-bindingendothelialregulator alphasubunit-1Laminin Gastrictriacylglycerollipase Leucine-richrepeatandcalponin Muellerian-inhibitingfactor Matrixmetalloproteinase-21Matrixmetalloproteinase-17 Matrixmetalloproteinase-20 FRAS1-relatedextracellularmatrix Epididymalsecretoryglutathione Placenta-specificprotein1 kDa21ofprotein Interleukin-9receptor Interleukin-9 InhibinbetaBchain N-acetylglucosamine1 Multimerin-2 Neurotrophin-4 FAM108A1 protein protein7 Promotilin protein3 FAM108B1 peroxidase Lactoperoxidase 39residues Prolargin TABLE1-continued Abhydrolasedomain-containingproteinTumornecrosisfactorreceptorsuperfamily

Insulin-likegrowthfactorbindingprotein4Nidogen2 Complementcomponentreceptor1-like phosphotransferasesubunitsalpha/beta Insulin-likegrowthfactorbindingprotein1Neurotrypsin Insulin-likegrowthfactorbindingprotein2Neuroserpin galactosamide-alpha2,3sialyltransferase Epidermis-specificserineproteaselike Glycosyltransferase1domain-containing ComplementClqtumornecrosisfactor Hepatocytegrowthfactor-likeprotein lymphocytegrowthfactor;cellStem epitheliumnasalLongpalatelungand, carcinoma-associatedprotein1 Glycoproteinhormonesalphachain Interferon-inducedhelicaseCdomain CMP-Nacetylneuraminatebeta Dipeptidylpeptidase4 Ephrintype-Areceptor3 Ephrintype-Breceptor6 CoagulationfactorX CoagulationfactorVIII Fibroblastgrowthfactor10 Interferonalpha-1/13 containingprotein1Interferonalpha-2 Insulin-likegrowthfactorIB secretedC-typelectin Endothelin-1 relatedprotein7 Alpha-2HSglycoprotein Fibrinogenalphachain Fibrinogenbetachain N-acetylglucosamine1 IndianhedgehogproteinNeuralcelladhesionmolecule Interleukin-13 B1Ephrin- EMILIN-1 protein1 Fibrillin-2 GranzymeA Interferonbeta Interferongamma ProteinCYR61 protein Dentinsialophosphoprotein protein Endoplasmin Gastrin member10D L1-likeprotein

Membranemetallo-endopeptidaselike1 Gammaaminobutyric-acidtypeBreceptor Carcinoembryonicantigen-relatedcell Pulmonarysurfactant-associatedproteinA1 CMP-Nacetylneuraminatebeta1,4 Glycoproteinhormonealpha-2 Epithelialdiscoidindomain-containing VascularendothelialgrowthfactorA kexinsubtilisinconvertase/Proprotein stimulatingfactorreceptorsubunitalpha C-typelectindomainfamily4memberMGranulocytecolony-stimulatingfactor C-typelectindomainfamily7memberA Cholinetransporter-likeprotein4 galactosidealpha-2,3sialyltransferase Tumornecrosisfactorreceptorsuperfamily Pro-neuregulin1,membranebound chainXI)-2(alphaCollagenGranulocytemacrophage -colony Interleukin-15receptorsubunitalpha Peptidyl-glycinealphaamidating Apolipoprotein-IA Tumornecrosisfactorreceptorsuperfamily AmyloidbetaA4protein C-motifchemokine4like Integrinalpha-7 Brain-derivedneurotrophicfactor Insulin-likegrowthfactorII CMRF35-likemolecule1 likeAreceptor-Fc Proteoglycan4 Prostate-associatedmicroseminoprotein Alpha-amylase1 adhesionmolecule1 Kallikrein-8 member6 subunit1 receptor1 Mucin-1 Fibulin-1 Prolactinreceptor CD209antigen Mucin-4 member25 Spermineoxidase isoform type6 Elastin Midkine monooxygenase Attractin receptor US 10 ,414 ,793 B2 25 26

Histidinetriadnucleotide-bindingprotein2 Abhydrolasedomain-containingprotein15 Nuclearporecomplex-interactingprotein like1Proactivatorpolypeptide- Methyltransferase-likeprotein7B AlkylatedDNArepairproteinalkB Insulingrowthfactor-likefamilymember3 Nuclearporecomplex-interactingprotein Hepatocellularcarcinoma-associated Regulatedendocrine-specificprotein18 ComplementClqtumornecrosisfactor Proteinspinsterhomolog2 vonWillebrandfactorCdomain containingprotein2-like UPF0514membraneproteinFAM159A Transmembraneemp24domain domain-containingprotein7 Collagenalpha-5(VI)chain WAPfour-disulfidecoredomainprotein C10orf31proteinPutativeuncharacterized PutativeuncharacterizedproteinC11orf45 UncharacterizedproteinC12orf28 Bonemorphogeneticprotein8A Ribonuclease-likeprotein9 UncharacterizedproteinC2orf66 UncharacterizedproteinC17orf99 containingprotein6 PutativeV-setandimmunoglobulin Secretedphosphoprotein1 Bmelanomaantigen5 UPF0369proteinC6orf57 UncharacterizedproteinC17orf67 Placenta-specificprotein9 Ig-likedomaincontainingprotein Ribonuclease8 Urotensin-2B Tetraspanin-18 homolog7 XK-relatedprotein5 Beta-defensin121 Beta-defensin130 TD26protein relatedprotein8 ProteinWnt-8a like2 ProteinTEX261 like1 Persephin ProteinWFDC13 Latherin 10A Apelin ENSP00000270642

Endonucleasedomain-containing1 Dehydrogenase/reductaseSDRfamily Calcium-bindingandcoiledcoildomain UDP-GlcNAc:betaGalbeta1,3N Oncoprotein-inducedtranscript3protein FCHanddoubleSH3domainsprotein1 Hepatoma-derivedgrowthfactorrelated ComplementClqtumornecrosisfactor Prolyl4-hydroxylasesubunitalpha3 Poliovirusreceptor-relatedprotein4 acetylglucosaminyltransferase4 Trypsin-X3(EC3.421) G1NCU-proteinLysosomal Solutecarrierfamily22member15 lactonase3paraoxonase/Serum EMIdomain-containingprotein1 Long-chainfattyacidCoAligase Peptidyl-prolylcistransisomerase Secretogranin-2 Transcobalamin-1 RINGfingerandSPRYdomaincontainingprotein1 containingprotein1 diphosphohydrolase8 UncharacterizedproteinCorf15 Proline-richacidicprotein1 Interleukin-12subunitalpha Peptidaseinhibitor16 Semaphorin-3B like2-member4 Trefoilfactor2 Tolloid-likeprotein2 2ProteinWnt-Ectonucleosidetriphosphate ProteinWnt-8b 15Peptidaseinhibitor HHIP-likeprotein2 ProteinWnt-11 ProteinWnt-7a UPF0577proteinKIAA1324 relatedprotein9 GPIinositol-deacylase protein Somatostatin Testican-1 Trypsin-2 Collectin-10 ACSBG2 Urocortin Fractalkine protein2 Mucin-17 SDCCAG10 TABLE1-continued

Transmembraneandubiquitin-likedomain Inter-alphatrypsininhibitorheavychainH1 heavychainH2trypsininhibitor-alphaInterInter-alphatrypsininhibitorheavychainH3 Lymphocyteantigen6complexlocus Plateletderived-growthfactorsubunitA Plasminogenactivatorinhibitor2 ProcollagenC-endopeptidaseenhancer1 Chymotrypsin-likeelastasefamily Pancreaticsecretorytrypsininhibitor Tumornecrosisfactorreceptorsuperfamily Calcium-activatedchloridechannel Bactericidal/permeability-increasing Adisintegrinandmetalloproteinasewith thrombospondinmotifs4 Microfibrillar-associatedprotein1 derivedMesencephalicastrocyte- Tumornecrosisfactorligandsuperfamily Plasminogenactivatorinhibitor1 factorderivedepithelium-Pigment InhibinbetaAchain Prostate-specificantigen Hepatictriacylglycerollipase Lutropinsubunitbeta 72kDatypeIVcollagenase Stromelysin-1 Oxytocinneurophysin-1 Neurotrophin-3 containingprotein2 Proteindisulfide-isomerase Interleukin-2 Kallikrein-4 regulator4 protein-like1 Eosinophillysophospholipase neurotrophicfactorMatrixGlaprotein Neutrophilcollagenase Beta-nervegrowthfactor member2A member21 Plasmakallikrein Leptin G6cprotein Mucin-5AC Mucin-6 member18 Phosphopantothenoylcysteine decarboxylase PepsinA Mesothelin Norrin Gastricsin

HLAclassIhistocompatibilityantigen,A-2 Macrophagecolony-stimulatingfactor1 GPItransamidasecomponentPIG-T Interleukin-22receptorsubunitalpha2 Deformedepidermalautoregulatoryfactor1 Autophagy-relatedprotein161 Low-densitylipoproteinreceptorrelated interactingmultifunctionalprotein1 PeroxisomalN(1)-acetyl Cholesterylestertransferprotein Collagenalpha-1(II)chain Stromalcell-derivedfactor1 Disintegrinandmetalloproteinasedomain Adisintegrinandmetalloproteinasewiththrombospondinmotifs13 chainglycoproteinCD8alphasurfacecellT- Breastcanceranti-estrogenresistance Neuralcelladhesionmolecule1 Tumornecrosisfactorligandsuperfamily EGF-likedomaincontainingprotein8AminoacyltRNAsynthetasecomplex Prostaglandin-H2Disomerase Tissue-typeplasminogenactivator spermine/spermidineoxidaseProbablepalmitoyltransferaseZDHHC4 12proteincontaining EGFR-coamplifiedandoverexpressed E3ubiquitin-proteinligaseLRSAM1 linkedNeuroliginX-4, SLAMfamilymember7 ADAMTS-likeprotein4 CoagulationfactorXI Pro-interleukin16 Folatereceptoralpha Seizure6-likeprotein Tumornecrosisfactor Acyl-CoAbindingprotein Leptinreceptor protein3 Cadherin-23 protein8 Netrin-G1 Alpha-1antitrypsinAlpha-1antichymotrypsin alphachain Decorin Tenascin protein Kitligand Uromodulin member13 ProteinCREG1 homolog US 10 ,414 ,793 B2 27LT 28

Putativeneurofibromin1-likeprotein4/6 Histidine-richcarboxylterminusprotein1 Leucine-richrepeatcontainingprotein70 ComplementC1q-likeprotein4 Abhydrolasedomain-containingprotein Transmembraneprotease,serine11E2 Submaxillaryglandandrogen-regulated Putativemacrophage-stimulatingprotein Placenta-specific1likeprotein C-typelectindomainfamily2memberA BTB/POZdomaincontaining-protein17 C-typelectindomainfamily9memberA Transmembraneprotein14E Transmembraneprotein207 regulatorfamilymember3 UncharacterizedproteinC18orf20 UncharacterizedproteinC12orf53 CMRF35-likemolecule4 TOMM20-likeprotein1UncharacterizedproteinC3orf41 Bmelanomaantigen1 Inactivecarboxylesterase4 Four-jointedboxprotein1 Kielin/chordin-likeprotein Peroxidasin-likeprotein Putativeuncharacterizedprotein Calcium-activatedchloridechannel UncharacterizedproteinC4orf26 UncharacterizedproteinC4orf40 C5orf55proteinUncharacterized UncharacterizedproteinC15orf61 B2Chymotrypsinogen PutativeV-setandimmunoglobulin domain-containingprotein6 Beta-defensin110Neuritin -likeprotein FAM108A2/A3 UPF0624proteinCoorf186 UNQ9165/PRO28630 Probableserineprotease UNQ9391/PRO34284 Beta-defensin111 ProteinFAM150A SerpinA13 ProteinFAM151B Osteocrin protein3A ProteinHSN2 Humanin SCO-spondin MSTP9 Beta-defensin108A

Carboxypeptidase-likeproteinX2 Dehydrogenase/reductaseSDRfamily EpididymalsecretoryproteinE3-beta Fibroblastgrowthfactor-bindingprotein2 SerineproteaseinhibitorKazal-type6 Vitellinemembraneouterlayerprotein1 Secretoglobinfamily3Amember2 Angiopoietin-relatedprotein2Angiopoietin-relatedprotein6 DBH-likemonooxygenaseprotein1 discoidin-likeIrepeatandEGFlike Fibroblastgrowthfactor17 Growthdifferentiationfactor3 Hyaluronanandproteoglycanlink subunitbetaChoriogonadotropin Matrixmetalloproteinase-28 Olfactomedin-likeprotein1 Olfactomedin-likeprotein2A Adisintegrinandmetalloproteinasewith thrombospondinmotifs2 Transmembraneprotein43 Brain-specificserineprotease4 UncharacterizedproteinClorf56 Colipase-likeproteinCorf126 UncharacterizedproteinCllorf83 UncharacterizedproteinC16orf89 Cytokine-likeprotein1 Dickkopf-relatedprotein2Dickkopf-likeprotein1 domain-containingprotein3 Fibroblastgrowthfactor22 GLIPR1-likeprotein1 Lysozyme-likeprotein1 WAPfour-disulfidecoredomain ArylsulfataseK Cerebellin-3 Cerebellin-4 Cystatin-9like Interleukin-17B Interleukin-170 Interleukin-17D Serineprotease27 Augurin member13 Beta-defensin123Beta-defensin132 ProteinFAM55D protein3 homolog variant1 Nephronectin protein12 TABLE1-continued

PeptidoglycanrecognitionproteinI-alpha Extracellularsuperoxidedismutase[Cu-Zn] Pulmonarysurfactant-associatedproteinC Transforminggrowthfactorbeta-1 Submaxillaryglandandrogen-regulated C-typelectindomainfamily11,memberA Serumparaoxonase/arylesterase1Alkalinephosphatase,placentaltype Peptidyl-prolylcistransisomeraseB Basicsalivaryproline-richprotein1 Basementmembrane-specificheparan Lymphocyteantigen6complexlocus SerumamyloidP-component sulfateproteoglycancoreprotein Structuralmaintenanceofchromosomes Metalloproteinaseinhibitor1inhibitor2Metalloproteinase inhibitor3 Metalloproteinase Urokinase-typeplasminogen activator Tumornecrosisfactorreceptorsuperfamily Vascularendothelialgrowthfactor UncharacterizedproteinC14orf93 Prolactin-inducibleprotein Secretogranin-1 VitaminD-bindingprotein Zinc-alpha2glycoprotein Alpha-1,3mannosyltransferaseALG2 isoformCRA_b Sonichedgehogprotein Plateletfactor4 Bonemarrowproteoglycan Parathyroidhormone Spondin-1 Syntaxin-1A Trypsin-1 vonWillebrandfactor Biglycan Plasminogen Antileukoproteinase Stabilin-1 Somatotropin SerpinB5 protein3 Tetranectin Thyroglobulin Lactotransferrin 3Bprotein member1A receptor1 Vitronectin proteinG50 Retinoschisin

CUBandsushidomain-containingprotein1 Transmembraneprotease,serine6 Choriogonadotropinsubunitbeta Parathyroidhormone-relatedprotein Prolyl4-hydroxylasesubunitalpha2 Cysteine-richwithEGFlikedomain proteincontaining8ArepeatLeucine-rich Urokinaseplasminogenactivatorsurface Dehydrogenase/reductaseSDRfamily EpidermalgrowthfactorreceptorEcto-NOXdisulfidethiolexchanger2 Interleukin-1receptorantagonistprotein Interleukin-6receptorsubunitbeta Interleukin-1receptorlike JunctionaladhesionmoleculeA SLITandNTRK-likeprotein2 Sulfatase-modifyingfactor1 Sulfatase-modifyingfactor2 receptorsuperfamilyfactorTumornecrosis V-setdomaincontainingTcellactivation N-acetylmuramoylLalanineamidase Interleukin-18bindingprotein Myeloid-associateddifferentiationmarker ComplementfactorB Hyaluronidase-1 Fcreceptor-likeprotein5 Lymphotoxin-alpha Sulfhydryloxidase1 KinofIRRE-likeprotein2 phosphateglycerol3-acylsn1 Versicancoreprotein Multipleinositolpolyphosphate phosphatase1 Neuropilin-1 ProteinRIC-3 acyltransferasegamma CD276antigen protein1 Ficolin-2 Plexin-A4 Plexin-B1 member10B inhibitor1 member4 Insulin Glycodelin Nurim ProteinGPR89 Periostin receptor Glucagon Chordin US 10 ,414 ,793 B2 6029 30

SerineproteaseinhibitorKazal-type SerineproteaseinhibitorKazal-type9 PutativeserineproteaseinhibitorKazal Dehydrogenase/reductaseSDRfamily Pancreaticlipase-relatedprotein3 Testis,prostateandplacenta-expressed Neuronalpentraxin-likeproteinC16orf38 Iron/zincpurpleacidphosphatase-like Plasminogen-relatedproteinA Ribonuclease-likeprotein10 Ribonuclease-likeprotein12 Ribonuclease-likeprotein13 Putativetestisserineprotease2 Secretedfrizzled-relatedprotein4ComplementClq-likeprotein2 PutativeuncharacterizedproteinC17orf69 PeptidaseS1domain-containingprotein Putativelipocalin1-likeprotein PutativepeptideYY-2PutativepeptideYY-3 inhibitor4proteasetypeKunitz- UncharacterizedproteinFLJ37543 Putativecystatin-13 Growth/differentiationfactor7 Putativeserineprotease29 LipasememberN Neuromedin-S Ovostatinhomolog1 Polyserase-3 Meteorin-likeprotein IgA-inducingproteinhomolog type5-like2 Beta-defensin131 Beta-defensin134 Beta-defensin136 Beta-defensin116 Beta-defensin115 Beta-defensin114 NeuropeptideS Beta-defensin112 Beta-defensin109 Beta-defensin113 Beta-defensin135 member7C Otolin-1 SerpinA11 ProteinFAM24A 5-like3 ProteinFAM132A ProteinFAM132B protein protein LOC136242

SerineproteaseinhibitorKazal-type7 Zymogengranuleprotein16homologB Transmembraneprotease,serine11D ComplementClqtumornecrosisfactor DDRGKdomain-containingprotein1 SerineproteaseinhibitorKazal-type4 Bactericidal/permeability-increasing PutativeannexinA2-likeproteinprotein10morphogeneticBone Keratinocyte-associatedprotein3 Collagenalpha-1(VIII)chain Interleukin-1familymember8 Liver-expressedantimicrobialpeptide2 Longpalate,lungandnasalepithelium carcinoma-associatedprotein4 domain-containingprotein28 Neurexophilin-4 Scrapie-responsiveprotein1 UncharacterizedproteinClorf54 UncharacterizedproteinC7orf34 UncharacterizedproteinC18orf54 Fibroblastgrowthfactor5 ProbableserineproteaseHTRA3 Lysyloxidasehomolog1 Lysyloxidasehomolog2 Lysozymeg-likeprotein2 Disintegrinandmetalloproteinase phosphodiesterase3b ProteinWnt-9b Semaphorin-3D Secretogranin-3 relatedprotein4 CarboxypeptidaseA6 NeuropeptideB Kinesin-likeproteinKIF7 protein-like2 C-motifchemokine25Chymotrypsin-likeelastasefamily Glycerophosphodiesterphosphodiesterase member2B ProteinCEI ProteinFAM55C ProteinFAM19A2 ProteinFAM5B Otospiralin Endomucin TABLE1-continued Acidsphingomyelinase-likeNADHdehydrogenase?ubiquinone]1beta Ly6/PLAURCollagenalpha-1(XXVI)chaindomaincontainingprotein C-motifchemokine19Leucine-richrepeatLGIfamilymember3 PotassiumchannelsubfamilyKmember17UncharacterizedproteinCorf47 Leucine-richrepeatcontainingprotein8DC2domaincontainingprotein2 ApolipoproteinL4Chondroitinsulfateglucuronyltransferase UncharacterizedproteinCorfiProbableG-proteincoupledreceptor114 Cystatin-like1Leucinerichrepeatcontainingprotein8B Carbonicanhydrase-relatedprotein10 Chitinasedomain-containingprotein1 ComplementfactorH-relatedprotein4 glycoprotein2specific-Pregnancybeta1 subcomplexsubunit11,mitochondrial Dehydrogenase/reductaseSDRfamily ProbableG-proteincoupledreceptor113 Glycerol-3phosphateacyltransferase4 KDELmotif-containingprotein2 Sialicacid-bindingIglikelectin6 Ly6/PLAURdomain-containingprotein5 Macrophagemigrationinhibitoryfactor Mitochondrialcarrierhomolog1 Voltage-gatedhydrogenchannel1 Long-chainfattyacidtransportprotein3 Majorfacilitatorsuperfamilydomain UPF0546membraneproteinClorf91 UncharacterizedproteinCorf89 TransmembraneproteinC9orf7 Crumbsproteinhomolog3 domain-containingprotein5 MLN64N-terminaldomainhomolog 2-acylglycerolOacyltransferase3 Protocadherinalpha-6 Protocadheringamma-A12 All-transretinol13,14reductase containingprotein7Leucine-richrepeattransmembrane neuronalprotein1 CMRF35-likemolecule9 CytochromeP450251 ApolipoproteinL6 Regulatorofmicrotubuledynamics Vesicle-traffickingproteinSEC22c Cholecystokinin Codanin-1 member7 ProteinENED Gliomedin Gremlin-1 Layilin protein2R-spondin4 Claudin-1

antigen-likeTubulointerstitialnephritis ComplementClqsubcomponentsubunitAComplementClqsubcomponentsubunitC Inter-alphatrypsininhibitorheavychainH5 Sialicacid-bindingIglikelectin10 Leucine-richrepeatcontainingprotein20 Lymphocyteantigen6complexlocus Solublecalcium-activatednucleotidase1 Fibroblastgrowthfactorreceptor3 Fibroblastgrowthfactorreceptor4 Advancedglycosylationendproduct NLRfamilyCARDdomain-containing Interleukin-7receptorsubunitalpha Tumornecrosisfactorligandsuperfamily Long-chainfattyacidCOAligase5 Collagenalpha-6(IV)chainCollagenalpha-2(VI)chain chainalpha(XI)Collagen-1 Growtharrest-specificprotein6 Pro-neuregulin2,membranebound Sperm-associatedantigen11A Oocyte-secretedprotein1homolog PlasmaproteaseC1inhibitor Platelet-derivedgrowthfactorD Interleukin-family1member7 Amelogenin,Xisoform Anthraxtoxinreceptor2 ApolipoproteinC-III Neutrophildefensin1 Lowaffinityimmunoglobulinepsilon ProteinS100-A7 L1Apolipoprotein CD97antigen( Crumbshomolog1 Growthhormonereceptor specificreceptor Claudin-14 C-motifchemokine15 Contactin-4 ComplementC2 Cystatin-C Endothelin-3 protein4 isoform Serumalbumin Cochlin member13B proteinG5b Acetylcholinesterase Angiogenin A2Annexin Calcitonin Fcreceptor US 10 ,414 ,793 B2 32

Putativescavengerreceptorcysteine-rich memberfamily2factorInsulingrowth-like Putativezinc-alpha2glycoproteinlike1 Insulingrowthfactor-likefamilymember4 SerineproteaseinhibitorKazal-type8 Putativezinc-alpha2glycoproteinlike Replicationinitiation-likeproteinProstateandtestisexpressedprotein3 Secretedfrizzled-relatedprotein2 Basicproline-richpeptideIB1 UDP-GlcNAc:betaGalbeta1,3N Putativeabhydrolasedomain-containing Putativestereocilin-likeprotein UncharacterizedproteinC2orf72 PutativeuncharacterizedproteinClorf191 Fibroblastgrowthfactor16 Plateletbasicprotein-like2 Stressinducedsecretedprotein1 PutativeuncharacterizedproteinClorf134 acetylglucosaminyltransferase9 UncharacterizedproteinC1lorf44UncharacterizedproteinC12orf73 Putativecystatin-9like2 Putativetestis-specificprionprotein PutativeuncharacterizedproteinFP248 domain-containingproteinLOC619207Secretoglobin-likeprotein Beta-defensin108BlikeproteinFLJ90687 Uncharacterized UncharacterizedproteinKIAA0495 Secretedphosphoprotein1 ProteinWnt(Fragment) PutativeserineproteaseLOC138652 Putativeuncharacterizedprotein Probablefolatereceptordelta Proline-richprotein1 Bmelanomaantigen4 NPIP-likeproteinENSP00000346774 UPF0670proteinC8orf55 Otopetrin-1 KIR2DL4 SerpinE3 CR1receptor ProteinWnt TOM1 FLJ46089 FAM108A5protein Beta-defensin133 Fibrosin-1 RPE-spondin SPARCprotein

Adipocyteenhancer-bindingprotein1 Interphotoreceptormatrixproteoglycan1 Proapoptoticcaspaseadapterprotein WAPfour-disulfidecoredomainprotein8 Zonapellucida-bindingprotein2SH3domain-bindingprotein5like ApolipoproteinA-Ibindingprotein Collagenalpha-1(XXVIII)chain GRAMdomaincontaining-protein1B Phosphatidylinositolglycananchor Leucine-richrepeatneuronalprotein3 NMDAreceptor-regulatedprotein2 C-typelectindomainfamily12memberB Solutecarrierfamily35memberF5lectin12bindinglikeSialicIg-acid Coiled-coildomaincontainingprotein80 Calcium-dependentphospholipaseA2 Integrinbeta-likeprotein1 Kunitz-typeproteaseinhibitor3 UncharacterizedproteinC12orf59 UncharacterizedproteinC7orf58 proteinC16orf48Uncharacterized biosynthesisclassUprotein NADH-cytochromeb5reductase1 WDrepeat-containingprotein82 Ecto-NOXdisulfidethiolexchanger1 Neuronalgrowthregulator1 Tolloid-likeprotein1 Adipocyteadhesionmolecule Inactivecaspase-12 Dentinmatrixprotein4 Carboxylesterase3 Interleukin-27subunitalpha FK506-bindingprotein11 ADAMTS-likeprotein3 likereceptor2 ProteinWnt-5b ProteinWnt-7b GPN-loopGTPase3 ProteinTMEM155 Prosalusin Proteinamnionless ProteinWFDC10B Claudin-17 ProteinFAM20B isoform protein1 ProteinFAM19A3 TABLE1-continued EGFcontaining-fibulinlikeextracellularPro-neuregulin4,membranebound TumornecrosisfactorligandsuperfamilyParkinsondisease7domain-containing Leukocyte-associatedimmunoglobulinLeucinerichrepeatsandimmunoglobulin

Platelet-derivedgrowthfactorsubunitB Tetratricopeptiderepeatprotein14 Zonapellucidasperm-bindingprotein3 Leucine-richrepeatcontainingprotein39 Lipopolysaccharide-bindingprotein kinase32BproteinSerinethreonine-/ XTP3-transactivatedgeneBprotein Transmembraneprotein139 Carbohydratesulfotransferase9 Cysteine-richmotorneuron1protein Connectivetissuegrowthfactor Fibroblastgrowthfactor19Follistatin-relatedprotein3 FXYDdomaincontaining-iontransport Group3secretoryphospholipaseA2 Kunitz-typeproteaseinhibitor1 likedomainsprotein3SLAMfamilymember9 Tryptasealpha-1 PalmitoyltransferaseZDHHC15 Pancreatictriacylglycerollipase Leukemiainhibitoryfactor Proteineyesshuthomolog Hedgehoginteracting-protein Interleukin-17receptorB GroupXVphospholipaseA2 InactiveserineproteasePAMR1 Xylosyltransferase1 CD5antigen-like Mucin-likeprotein1 Prokineticin-1 Galectin-1 C-motifchemokine21 regulator5 Endotheliallipase matrixprotein2 Ribonuclease7 Spondin-2 Testican-2 Vasohibin-1 Transthyretin Noggin Otoraplin 14member Plexin-A2 Papilin Torsin-2A Vasorin

Immunoglobulinsuperfamilymember8 Pulmonarysurfactant-associatedproteinB Multipleepidermalgrowthfactor-like BaculoviralIAPrepeat-containingprotein7 BifunctionalUDP-Nacetylglucosamine2 Mannan-bindinglectinserineprotease1Mannan-bindinglectinserineprotease2 Neutrophilgelatinase-associatedlipocalin Poliovirusreceptor-relatedprotein1 EpididymalsecretoryproteinE1 acetylgalactosaminyltransferase1 galactosamide-alpha2,3sialyltransferase WAPfour-disulfidecoredomainprotein2 Gamma-secretasesubunitAPH1A Signalinglymphocyticactivationmolecule EGF-likedomaincontainingprotein7 Coxsackievirusandadenovirusreceptor epimerase/N-acetylmannosaminekinase Interleukin-4receptoralphachain Lamininsubunitbeta-3 Fibroblastgrowthfactor23 Transmembraneprotein25 UDP-GalNAc:beta1,3N Interleukin-15(IL) Mucinandcadherin-likeprotein SH2domain-containingprotein3C Transmembraneprotein9 Chorionicsomatomammotropinhormone Leucyl-cystinylaminopeptidase NeuropeptideYprotein Aggrecancore Semenogelin-111 Tissuefactorpathwayinhibitor Interleukin-23subunitalpha ADAMTS-likeprotein1 Chemokine-likefactor CMP-Nacetylneuraminatebeta AdenosineA3receptor ComplementfactorH Kallikrein-14 Kallikrein-6 Ribonucleasepancreatic Tectonic-1 domains11 Ribonuclease4 Alpha-S1casein Cyclin-L1 Renin Usherin Basigin Calumenin US 10 ,414 ,793 B2 34

FibroblastgrowthfactorreceptorFGFR-1 Secretedproteinacidicandrichin Scavengerreceptorcysteine-richdomain COBW-likeplacentalprotein(Fragment) Glycosyltransferase54domain-containing cDNAFLJ55667,highlysimilarto memberfamilyCdomain18typelectin- Prostateandtestisexpressedprotein4Collagenalpha-1(XXII)chain PutativeuncharacterizedproteinC13orf28 Putativecateyesyndromecriticalregion Plexindomain-containingprotein1MC51L-53L54Lhomolog(Fragment ) Cytokinereceptor-likefactor2 Interleukin-28receptoralphachain secretedformprotein(Fragment) Collagenalpha-2(IV)chain Odorant-bindingprotein2a Kidneyandrogen-regulatedprotein UNQ5830/PRO19650PRO19816 Secretedphosphoprotein1 ADAMTS-likeprotein2 LipasememberK Putativeuncharacterizedprotein ComplementC3 Uncharacterizedprotein HMSDproteinSerpinlike- Putativeuncharacterizedprotein Putativeuncharacterizedprotein Tachykinin-3 LOC284297proteincontaining Tryptasebeta-1 Hyaluronidase-3 Chordin-likeprotein1 Putativeuncharacterizedprotein NetrinreceptorUNC5B Uncharacterizedprotein UNQ6125/PRO20090 UNQ6126/PRO20091 Cystatin-S R-spondin1 UNQ6975/PRO21958 Tryptasedelta protein9 Beta-defensin103 Beta-defensin106 UNQ9370/PRO34162 cysteine C&orf2 Opiorphin Sclerostin protein ENSP00000244321

Zonapellucidasperm-bindingprotein4 TRACH2015180,highlysimilarto Secretedfrizzled-relatedprotein2 Psoriasissusceptibility1candidategene Signalpeptidasecomplexsubunit3 Spermequatorialsegmentprotein1 Acyl-CoAsynthetasefamilymember2, C-typelectindomainfamily1memberA C-typelectindomainfamily3memberA C-typelectindomainfamily4memberEC-typelectindomainfamily4memberG Endoplasmicreticulumresidentprotein DnajhomologsubfamilyBmember9 Adenosinemonophosphate-protein cDNAFLJ36603fis,clone Integralmembraneprotein2A vonWillebrandfactorAdomain CD164sialomucin-like2protein TransmembraneproteinC3orfl UncharacterizedproteinC6orf72 UncharacterizedproteinCilorf24 ProbableUDP-sugartransporterprotein TransmembraneproteinC16orf54 Thioredoxindomain-containing Prenylcysteineoxidase-like Phytanoyl-CoAhydroxylaseinteracting FXYDdomain-containingiontransport Mucin-19(MUC) VesicletransportproteinSFT2B containingprotein3A Proteinshisa-2homolog Probablecation-transporting CytochromeP4504F12 CytochromeP4504X1421P450 Cytochrome LipasememberH Cerebellin-2 UPF0480proteinC15orf24 Dipeptidase3FAM174AproteinMembrane Cadherin-16 Cadherin-19 ATPase13A4 ProteinCREG2 15protein ProteinFAM19A4 transferaseFICD protein-like regulator4 protein2 mitochondrial SLC35A5 ERp27 TABLE1-continued

Hyaluronanandproteoglycanlinkprotein1 Transmembraneprotease,serine11E Leukocyteimmunoglobulin-likereceptor SerineproteaseinhibitorKazal-type5 Pregnancy-specificbeta1glycoprotein4 Thrombospondintype-1domaincontaining AngiogenicfactorwithGpatchandFHA pyrophosphatase/phosphodiesterasefamily HERV-FRD_6p24provirus.1ancestralEnv HLAclassIhistocompatibilityantigen, Adisintegrinandmetalloproteinasewith Interleukin-1receptoraccessoryprotein Matrixmetalloproteinase-16 Pituitaryadenylatecyclase-activating Latent-transforminggrowthfactorbeta Wntinhibitoryfactor1 Deoxyribonucleasegamma thrombospondinmotifs6 FRAS1-relatedextracellularmatrix subfamilyAmember3 Interferonalpha-14 Angiopoietin-2 CarboxypeptidaseA5 C—Xmotifchemokine2 C—Xmotifchemokine5 N-acetylgalactosaminyltransferase1 bindingprotein3 Oncostatin-M Cw-16alphachain C-typenatriureticpeptide C-motifchemokine14 Interleukin-5 Interleukin-10 Interleukin-17F Kallikrein-15 Collagenase3 Prokineticin-2 Ectonucleotide member6 Derlin-1 polyprotein Prostasin Polypeptide Fibulin-2 Ficolin-1 SLcytokine Follistatin protein1 Enamelin polypeptide Somatoliberin protein1 domains1

Leucine-richrepeatcontainingGprotein ProteinO-linkedmannosebeta1,2N Sarcolemmalmembrane-associatedproteinSushi,vonWillebrandfactortypeAEGF andpentraxindomain-containingprotein1 Transmembraneandcoiled-coildomain Transmembraneprotease,serine3 pyrophosphatase/phosphodiesterasefamily LowaffinityimmunoglobulingammaFc Leucine-richrepeattransmembraneprotein TransmembraneglycoproteinNMB JunctionaladhesionmoleculeB growthfactorbetaLatenttransforming- Myelinproteinzero-like1 Neurobeachin-likeprotein2 acetylglucosaminyltransferase1 Tumornecrosisfactorreceptorsuperfamily Tumornecrosisfactorreceptorsuperfamily CoagulationfactorIX regionreceptorIII-B Fcreceptor-likeprotein2 Interleukin-31receptorA ADP-ribosepyrophosphatase, SerumamyloidAprotein Sexhormone-bindingglobulinSLAMfamilymember6 Thyroxine-bindingglobulin containingprotein1 ERO1-likeproteinalpha IgmuchainCregion Interleukin-1alpha Lipocalin-1 coupledreceptor6 bindingprotein1 Protocadherin-15 Placentagrowthfactor ProbablehydrolasePNKD Reticulon-4receptor Ficolin-3 Matrilin-3 Poliovirusreceptor Ectonucleotide member2 FLRT3 Gelsolin Granulysin Granulins Heparanase Nicastrin mitochondrial Pleiotrophin member10C member11B US 10 ,414 ,793 B2 35 36

Putativesolubleinterleukin18receptor1Putativeabhydrolasedomain-containing Methylthioribose-1phosphateisomerase Methyltransferase-likeprotein7A WNT1inducedsecretedprotein1splice Chemokine-likefactorsuperfamily2 domain-containinglikeprotein Bcellmaturationantigentranscriptvariant superfamilymember17) 17-betahydroxysteroiddehydrogenase13 Interleukin-1familymember10 Olfactomedin-likeprotein3 Extracellularglycoproteinlacritin Keratinocytesassociatedtransmembrane Leucine-richrepeatandimmunoglobulinlikedomain-containingnogo receptor PutativeV-setandimmunoglobulin 4(Tumornecrosisfactorreceptor variantx(Fragment) Retinoldehydrogenase13 Neutrophildefensin3 C17/TNF-relatedprotein8KIR2DL4 (Fragment) RTFV9368(SLEdependent-upregulation1) interactingprotein4 UPF0672proteinC3orf58 AminopeptidaseB N-acetyltransferase15 Kallikrein-11 Proteoglycan3INSL5peptideInsulinlike - transcriptvariant2 proteinFAM108A6 Ephrin-A4 ProteinPlunc protein1 POM121-like ECE2 EPA6 ENSP00000303034 Dermcidin Meteorin NL3 PLA2G2D GLGQ5807 TUFT1 DRLV8200 IDLW5808 UBAP2 GKGM353 MATL2963 NINP6167

Polycystickidneydiseaseprotein1-like WAP,kazalimmunoglobulinkunitzand Leucine-richrepeatcontainingprotein33MANSCdomain-containingprotein1 Mesodermdevelopmentcandidate2 FibronectintypeIIIdomain-containing Solutecarrierfamily35memberE3 Abhydrolasedomain-containingprotein associatedprotein2-like Growthdifferentiationfactor11 Cerebraldopamineneurotrophicfactor NTRdomaincontaining-protein1 KDELmotif-containingprotein1 Collagenalpha-6(VI)chain Dickkopf-relatedprotein1 PDZdomain-containingprotein2 Matrixmetalloproteinase-26 Ankyrinrepeatdomain-containing Growthhormone-inducible Chondromodulin-1 Olfactoryreceptor10W1 Seizure6-likeprotein2 Synaptogyrin-2 GPN-loopGTPase2 transmembraneprotein Lactase-likeprotein Lipocalin-15 ArylsulfataseI ProteinPARM-1 Semaphorin-3A4CSemaphorin- Proteinshisa-4 Neuromedin-U protein2 protein1 Noelin-2 protein36 Nodalhomolog 104protein Glycerophosphodiester Adipophilin Podocan Neurotrimin Proepiregulin WLPL514 14A Brain-specificangiogenesisinhibitor1Immunoglobulinsuperfamilycontaining TABLE1-continued pyrophosphatase/phosphodiesterasefamilyphosphodiesterasedomain-containing RELT-likeprotein2ProcollagenCendopeptidaseenhancer Coiled-coildomaincontainingLeucinerichrepeatandimmunoglobulin Leucine-richrepeatLGIfamilymember4ZinctransporterZIP9 acetylgalactosaminyltransferase-like Carbonicanhydrase-relatedprotein11 Secretoglobinfamily3Amember1 proteinrelated2factorComplementH- ComplementClqtumornecrosis Angiopoietin-relatedprotein3 Angiopoietin-relatedprotein7 LCCLproteinsecretoryCysteine-rich Spermacrosomemembrane-associated likedomain-containingnogoreceptor TGF-betareceptortypeIII ThyrotropinsubunitbetaUncharacterizedproteinC19orf36 ProbablecarboxypeptidaseX1 Fibroblastgrowthfactor21 Plasmaalpha-Lfucosidase Glutathioneperoxidase7 ApolipoproteinC-I Left-rightdeterminationfactor1 BRCA1-AcomplexsubunitAbraxas Leucinezipperprotein2 Kazal-typeserineproteaseinhibitordomain-containingprotein1 factor-relatedprotein2 Slithomolog1protein Ecto-ADPribosyltransferase5 Probableribonuclease11 C—Xmotifchemokine14 domain-containing2 Neurexophilin-3 Claudin-2(SP82) leucine-richrepeatprotein interactingprotein1 PolypeptideN Growthhormonevariant Beta-defensin127 Beta-defensin129 Gastrokine-1 Gastrokine-2 HHIP-likeprotein1 Interferonkappa Ectonucleotide member5 protein2 ProteinFAM3D Osteomodulin protein3 Tsukushin

cDNA,FLJ96669highlysimilartoHomo sapienssecretedprotein,acidiccysteine DNAFLJ77519,highlysimilartoHomo Programmedcelldeath1ligand2 cDNAFLJ75759,highlysimilartoHomo rich(osteonectin)SPARC,mRNA sapienssecretedfrizzledrelatedprotein Tumornecrosisfactorreceptorsuperfamily UPF0414transmembraneproteinC20orf30 C-typelectindomainfamily4memberUPF0317proteinC14orf159,mitochondrial Sushidomain-containingprotein4 T-cellsurfaceglycoproteinCD8betachain ComplementClqtumornecrosisfactor EGF-likemodulecontainingmucin sapiensfollistatin-like3(secretedmRNA FSTL3),glycoprotein( Vesicle-associatedmembraneproteinCassociatedproteinB/ Fibrinogen-likeprotein1 C-motifchemokine3like1 Fibroblastgrowthfactor8 Sialomucincoreprotein24 Secretedandtransmembrane1 hormonereceptor-like3 Odorant-bindingprotein2b WNT1inducible-signalingpathway Tryptasebeta-2 T-celldifferentiationantigenCD6 Sperm-associatedantigen11BCoagulationfactorXII Tomoregulin-2 Chordin-likeprotein2 C4b-bindingproteinbetachain Interleukin-32 MetalloreductaseSTEAP2 ApolipoproteinM relatedprotein6 Urotensin-2 ProteinYIPF5 Matrilin-4 member6B Netrin-G2 ProteinYIF1B Noelin-3 protein3 Serotransferrin mRNA Pikachurin Hepcidin Klotho Serglycin Vitrin US 10 ,414 ,793 B2 37 38

S100calciumbindingproteinA7-like3 CR1C3b/C4breceptorSCR9(or16)C GTWW5826(LP5085protein) carcinoma-associatedprotein3(Ligand term.exonSCR=shortconsensusrepeat HCG2020043)(KTIS8219 Hyaluronanandproteoglycanlink Longpalate,lungandnasalepithelium bindingproteinRYA3) KCNQ2 ELCV5929 KVVM3106 ISPF6484 LKHP9428 VNFT9373 ACAH3104 RVLA1944 Wpep3002 ZDHHC11 AGLW2560 TSSP3028 RFVG5814 SHSS3124 MMP19 GSQS6193 VGPW2523 LMNE6487 ALLA2487 GALI1870 FRSS1829 MRSS6228 GRPR5811 AVLL5809 PIKR2786 protein4 Micronovel SAMK3000 VFLL3057 CVWG5837 VGSA5840 GHPS3125 GRTR3118 PAMP6501 LTLL9335 VCEW9374 AHPA9419 MDHV1887 HSAL5836 LHLC1946 LPPA601 PINK1

Transmembrane4L6familymember20 Mitochondrialuncouplingprotein4 proteinbinding1pellucidasperm-Zona Zonapellucidasperm-bindingprotein2 Heparansulfateglucosamine3-0 Spermatogenesis-associatedprotein6 Twistedgastrulationproteinhomolog1 ConservedoligomericGolgicomplex Adiponectinreceptorprotein2 PhospholipaseA1memberABasicsalivaryproline-richprotein2 Sushirepeat-containingproteinSRPX2 memberBfamilydomain18typelectinC- Transmembraneprotein107Transmembraneprotein143 Transmembraneprotein178Transmembraneprotein205 Transmembraneprotein41A Transmembraneprotein50A Transmembraneprotein50B Neuronalpentraxin-2 Transmembraneprotein92Transmembraneprotein95 Transmembraneprotein9B ProbablecarboxypeptidasePM20D1 UPF0513transmembraneprotein ProbablepalmitoyltransferaseZDHHC24 Leptinreceptoroverlappingtranscript ProteinHEGhomolog1FibrinogenCdomain-containing Lysosomal-associatedtransmembrane Interleukin-28B 12Tetraspanin-13Tetraspanin- Tetraspanin-15 Polyserase-2 InhibinbetaCchain Semaphorin-3C sulfotransferase2 SPARC-likeprotein1 5aProteinWnt- Acrosin-bindingprotein 3ESemaphorin- subunit7 Fibulin-7 protein1 Torsin-1B 4Aprotein Collectrin Brorin like1 Ameloblastin TABLE1-continued

Melanomainhibitoryactivityprotein3 Endothelin-convertingenzymelike1 domainEGF-likeSignalpeptideCUBand, ComplementfactorH-relatedprotein3 Proheparin-bindingEGFlikegrowthfactor Leucine-richrepeatcontainingprotein4 Leucine-richrepeatneuronalprotein1 3-hydroxybutyratedehydrogenasetype2 CarboxypeptidaseNcatalyticchain Short-chaindehydrogenase/reductase3 Insulin-likegrowthfactorbindingprotein5 acetylgalactosaminyltransferase1 Insulin-likegrowthfactorbindingprotein7 Hematopoieticcellsignaltransducer CIGALT1-specificchaperone1 Chondroitinsulfatesynthase1Chondroitinsulfatesynthase2 DeltaandNotch-likeepidermalgrowth Globosidealpha-1,3N Glycerol-3phosphateacyltransferase Hyaluronan-bindingprotein2 Carbohydratesulfotransferase14 Interleukin-20receptorbetachain KinofIRRE-likeprotein3 SerumamyloidA-4protein TransmembraneproteinC2orf18 CMRF35-likemolecule7 Proteincanopyhomolog3 Integralmembraneprotein2B Endothelialcell-selectiveadhesion containingprotein1 Follistatin-relatedprotein1 G-proteincoupledreceptor56 pyrophosphatase/phosphodiesterase Follitropinsubunitbeta Zinctransporter5 Apicalendosomalglycoprotein Beta-1,4galactosyltransferase7 Delta-likeprotein4 Gamma-glutamylhydrolase Histidine-richglycoprotein familymember3 Kappa-casein CD320antigen factor-relatedreceptor Dolicholkinase ProrelaxinH1 Cadherin-24 Probetacellulin Beta-casein molecule Ectonucleotide Kallistatin

factor-likeinflammatory1Allograft Glycosyltransferase8domain-containing Pulmonarysurfactant-associatedproteinA2serinerich16factorarginine/-Splicing, Angiotensin-convertingenzyme2 Bactericidalpermeability-increasingprotein Cartilageintermediatelayerprotein1 DnajhomologsubfamilyCmember10 CoagulationfactorXIIIAchain Leucine-richgliomainactivatedprotein1 kexinsubtilisinconvertase/Proprotein Armadillorepeat-containingXlinked acetylgalactosaminyltransferase1 Claudindomaincontaining-protein1 ProbableG-proteincoupledreceptor125 GPIethanolaminephosphatetransferase2 GPIethanolaminephosphatetransferase3 proteinSCAMC-2(Smallcalciumbinding mitochondrialcarrierprotein2) Angiopoietin-relatedprotein4 CUBdomaincontaining-protein1 Collagenalpha-1(XXV)chain EGF-likedomaincontainingprotein6 Interleukin-20receptoralphachain Apolipoprotein-VA Collagenalpha-1(V)chain Estradiol17-betadehydrogenase11 Interleukin-12subunitbeta Lymphocyteantigen96 PeptidoglycanrecognitionproteinInterferon-induced17kDaprotein ChondroitinsulfateN Golgimembraneprotein1 Calcium-bindingmitochondrialcarrier Beta-AlaHisdipeptidase Glucose-6phosphateisomerase Appetite-regulatinghormone Interleukin-22 ProteinWnt-4 Chitotriosidase-1 NKG2Dligand4 Prolyl3-hydroxylase1 Intelectin-1 Mucin-20 protein3 protein1 Galectin-7 L-aminoacidoxidase Adiponectin Asporin Matrilysin type9 Erlin-2 US 10 ,414 ,793 B2 39 40

Stromalcell-derivedfactor2likeprotein1 cDNAFLJ53955,highlysimilarto Secretedfrizzled-relatedprotein4. Deoxyribonuclease-1like2 Endothelialcell-specificmolecule1 Butyrophilinsubfamily1memberA1 ImmunoglobulinalphaFcreceptor Interleukin-27subunitbeta Keratinocyte-associatedtransmembrane Ephrintype-Areceptor10 Follistatin-relatedprotein4 Stanniocalcin-2 Carboxylesterase7 ProteinNOVhomologUPF0528proteinFAM172A Exostosin-like2 Beta-defensin108B Beta-defensin118 Beta-defensin124 Beta-defensin125 Beta-defensin126 ProteinFAM3C protein2 EMILIN-2 SERH2790 FLFF9364 APELIN GLSH6409 SFVP2550 RRLF9220 PTML5838 VLGN1945 AVPC1948 AWQG2491 PSVL6168 LCII3035 PPRR6495 RLSC6348 CSRP2BP GLLV3061 GWSI6489 PPIF VSSW1971 KLIA6249 ALLW1950 GVEI466 ESFI5812 GNNC2999 AAGG6488 HHSL751

EpididymalsecretoryproteinE3-alpha Fibroblastgrowthfactor-bindingprotein3 Multipleepidermalgrowthfactor-like WAPfour-disulfidecoredomainprotein1 Cartilageintermediatelayerprotein2 Phosphatidylethanolamine-binding Wntprotein-1Protooncogene Myelinproteinzero-like2 protein1likeSerineprotease- Coiled-coildomaincontainingprotein70 CUBdomain-containingprotein2 Cardiotrophin-1(CT) Majorfacilitatorsuperfamilydomain Acidsphingomyelinase-like ADAMTS-likeprotein5 Bonemorphogeneticprotein3bBonemorphogeneticprotein5 Bonemorphogeneticprotein8B (EpithelialV-likeantigen1) UncharacterizedproteinC4orf29 UncharacterizedproteinCorf58 UncharacterizedproteinC10orf25 Fibroblastgrowthfactor20 Transmembraneprotein204 containingprotein5Angiopoietin-1 Angiopoietin-4 phosphodiesterase3a Putativetrypsin-6 Carboxypeptidaseo Trem-liketranscript4protein ChymotrypsinogenB C—Xmotifchemokine9 C—Xmotifchemokine13 factorVCoagulationCoagulationfactorVII Cerebellin-1 C-motifchemokine28 Cystatin-8 EMILIN-3 Folatereceptorgamma domains9 ProteinFAM26D Clq-relatedfactor Isthmin-1 ProteinFAM5C protein4 Mucin-7 Spexin Chondroadherin Secretagogin Epiphycan Pro-MCH TABLE1-continued

protein5Transmembranechannel-like Thioredoxindomain-containingprotein12 FibronectintypeIIIdomain-containing Neuropilinandtolloid-likeprotein2 Peptidyl-prolylcistransisomeraselike1 Transmembraneprotease,serine4 VascularendothelialgrowthfactorB VascularendothelialgrowthfactorC Neuropilinandtolloid-likeprotein1glycoprotein11specificbeta-1Pregnancy UDP-GICNAc:betaGalbeta1,3N Cardiotrophin-likecytokinefactor1 Disintegrinandmetalloproteinasedomain Ly6/PLAURdomain-containingprotein3 C-typelectindomainfamily14memberA Chitobiosyldiphosphodolicholbeta Sialicacid-bindingIglikelectin9 Metastasis-suppressorKiSS1 Calcitoningene-relatedpeptide2 chainVIIIalpha)Collagen-2( Leukemiainhibitoryfactorreceptor Protocadherinbeta-13 Prenylcysteineoxidase1 Proteinpatchedhomolog2 SLITandNTRK-likeprotein1 acetylglucosaminyltransferase3 Thioredoxin-relatedtransmembrane containingprotein32 FK506-bindingprotein14 Prostatestemcellantigen Proteinsel-1homolog Trem-liketranscript2protein Reticulocalbin-3 CarboxypeptidaseE Crumbshomolog2 Lin-7homologB Proteincornichonhomolog Isletamyloidpolypeptide ADP-dependentglucokinaseAlpha-amylase 2B protein3B 1protein ProteinFAM151A Fibrillin-1 ProteinFAM3A ProteinG7c SerpinB4 ADAMDEC1 Peflin mannosyltransferase ProSAAS Statherin Testisin

Scavengerreceptorcysteine-richtype1 -2,6acetylgalactosaminidealphaAlphaN Complementreceptortype1 Tumornecrosisfactorligandsuperfamily Tumornecrosisfactorreceptorsuperfamily ProteinZ-dependentproteaseinhibitor Bonemorphogeneticprotein4 Collagenalpha-2(I)chain Collagenalpha-1(III)chainchain alpha(IV)Collagen-1Collagenalpha-3(IV)chain Collagenalpha-5(IV)chainCollagenalpha-3(VI)chain ComplementcomponentC6Collagenalpha-1(IX)chain Collagenalpha-1(X)chain chainXVIIalpha)Collagen-1( Collagenalpha-1(XXI)chain Deoxyribonuclease-1 Extracellularmatrixprotein1 Heparin-bindinggrowthfactor2 Glialcellline-derivedneurotrophicfactor SingleIgIL-1relatedreceptor PalmitoyltransferaseZDHHC9 Pancreaticalpha-amylaseNatriureticpeptidesB CarboxypeptidaseB2 CarboxypeptidaseZ ComplementfactorI Coatomersubunitalpha Lowaffinityimmunoglobulingamma FcregionreceptorIII-A FibrinogengammachainGrowth/differentiationfactor5 sialyltransferase6 Alpha-2macroglobulin Agouti-relatedprotein Atrialnatriureticfactor C-motifchemokine5 C-motifchemokine7 C-motifchemokine8 Tectonic-3 member11 member19 Fibulin-5 Neutralceramidase Beta-2microglobulin Biotinidase proteinM130 CD59glycoprotein Clusterin Cystatin-SN Alpha-fetoprotein US 10 ,414 ,793 B2 41

Immunoglobulinsuperfamilymember21 GDNFfamilyreceptoralpha-4 ComplementClqtumornecrosisfactor Pregnancy-specificbeta1glycoprotein5 Triggeringreceptorexpressedonmyeloid Growthdifferentiationfactor2 GRAMdomain-containingprotein10 VitaminK-dependentproteinS Butyrophilin-likeprotein8 Lymphaticvesselendothelialhyaluronic protein2Apolipoproteinlike(a)- Tubulointerstitialnephritisantigen Alpha-1,3mannosylglycoprotein4beta GroupIIEsecretoryphospholipaseA2 Cytokinereceptor-likefactor1 Follistatin-relatedprotein5 Transmembraneprotein66 Iggamma-4chainCregion Lymphocyteantigen86 Lamininsubunitbeta-4 Transmembraneprotein59 Lysozyme-likeprotein2Lysozyme-likeprotein4 Retinol-bindingprotein4 Transmembraneemp24domain Left-rightdeterminationfactor2 InhibinbetaEchain Interferonalpha-10 Interferonalpha-16Interferon alpha-6 Kelch-likeprotein11 1-acylsnglycerol3phosphate Carbonicanhydrase14 N-acetylglucosaminyltransferaseB containingprotein5 Podocan-likeprotein1 NKG2Dligand2 ProteinWnt-16 Kallikrein-13 acyltransferasedeltaKallikrein-9 acidreceptor1 Cystatin-SA NeuropeptideW relatedprotein3 Macrophagemetalloelastase Agrin Prolactin Properdin Reelin Keratocan cells1 Secretin

factor1growthdifferentiationEmbryonic glycoprotein1specific-betaPregnancy Melanomainhibitoryactivityprotein2 ProbableG-proteincoupledreceptor171 Microfibril-associatedglycoprotein4 Spermacrosomemembrane-associated MAMdomain-containingprotein2Microfibrillar-associatedprotein2 Matrixmetalloproteinase-19 containingprotein3 Lamininsubunitgamma-3 Neurotensin/neuromedinN Matrixmetalloproteinase-24Matrixmetalloproteinase-25 Alpha-Nacetylgalactosaminidealpha FMRFamide-relatedpeptides containingprotein1 Stromelysin-2 Pappalysin-2 Neuromedin-B Insulin-likepeptideINSL6 ProteinWnt-2b Neurexophilin-2 Plateletfactor4variant Prolactin-releasingpeptide Galanin-likepeptide Hemicentin-1 Interleukin-6 Interleukin-8 Interleukin-11 Interleukin-17A Interleukin-18 Interleukin-26 Interleukin-29 2,6-sialyltransferase1 2,6-sialyltransferase3 Otoconin-90 V-setandtransmembranedomain Gremlin-2 Mimecan protein4 Netrin-1 Netrin-3 protein Nociceptin TABLE1-continued Caspaserecruitmentdomain-containingAlphaNacetylgalactosaminidealpha Melanoma-derivedgrowthregulatoryThyrotropinreleasinghormonedegrading Neurexophilin-1Immunoglobulinlambdalikepolypeptide Leucine-richrepeatLGIfamilymember2Prolineprotein4 Interleukin-28APregnancyspecificbeta1glycoprotein10 Leucine-richrepeattransmembraneproteinTransmembraneemp24domain Leucine-richrepeatcontainingprotein31Lysyloxidasehomolog3 Immunoglobulinsuperfamilymember1 Heparansulfateglucosamine3-0 Dentinmatrixacidicphosphoprotein1 Pancreaticlipase-relatedprotein1 Matrix-remodelingassociatedprotein5 Basicsalivaryproline-richprotein3 Collagenandcalcium-bindingEGF Germcell-specificgene1likeprotein Gamma-secretasesubunitAPH1B Cholinetransporter-likeprotein3 17-betahydroxysteroiddehydrogenase14Neurturin DnajhomologsubfamilyBmember14 Methionine-RsulfoxidereductaseB3, Downsyndromecelladhesionmolecule Interleukin-6receptorsubunitalpha Leucine-richalpha2glycoprotein Hepatocytegrowthfactorreceptor domain-containingprotein1 defensin4NeutrophilAmphoterin-inducedprotein3 ApolipoproteinC-IV GOTIBproteinVesicletransport Interleukin-4 Interleukin-24 LipasememberI C-motifchemokine22 Dipeptidase2 Apolipoprotein0 ArylsulfataseG Glia-activatingfactor sulfotransferase3A1 F-boxonlyprotein8 Ladinin-1 Netrin-4 NyctalopinOsteocalcin FLRT2 spondin-3R Sialoadhesin Trypsin-3 Dystroglycan 18protein ectoenzymeGuanylin Fibroleukin mitochondrial

Insulin-likegrowthfactorbindingprotein3 Phosphatidylinositol-glycanspecific Transforminggrowthfactorbeta-2 Fibroblastgrowthfactor-bindingprotein1 Vasopressin-neurophysin2copeptin Protransforminggrowthfactoralpha Cysteine-richwithEGFlikedomain Inter-alphatrypsininhibitorheavychain Lamininsubunitalpha-2Lamininsubunitalpha-4 Lamininsubunitbeta-1 VitaminK-dependentproteinZSalivaryacidicproline-rich phosphoprotein1/2 Transferrinreceptorprotein1 Tumornecrosisfactorligandsuperfamily Tumornecrosisfactorreceptorsuperfamily Tumornecrosisfactorreceptorsuperfamily Cysteine-richsecretoryprotein2 Insulin-likegrowthfactorIA Iggamma-1chainCregion Iggamma-2chainCregionIggamma-3chainCregion Protein-lysine6oxidase PhospholipaseA2, Phospholipidtransferprotein Pregnancyzoneprotein Haptoglobin-relatedprotein UPF0378proteinKIAA0100 Kininogen-1 Multimerin-1 phospholipaseD Prostaticacidphosphatase Semaphorin-4Dproteinhomolog2Slit Trefoilfactor3 Acidicmammalianchitinase C-Xmotifchemokine16 Insulin-like3 Nidogen-1 Perforin-1 Fibrocystin ProrelaxinH2 Alpha-tectorin Tenascin-X member6 1Bmember member5 Thrombopoietin VIPpeptides C-motifchemokine26 Collectin-11 protein2 US 10 ,414 ,793 B2 43 44

Sclerostindomain-containingprotein1 Scavengerreceptorcysteine-richdomain WAPfour-disulfidecoredomainprotein3 Adipocyteplasmamembrane-associated Fibronectintype-IIIdomaincontaining Lysocardiolipinacyltransferase1 C3andPZP-likealpha2macroglobulin Post-GPIattachmenttoproteinsfactor3Germcell-specificgene1protein Adisintegrinandmetalloproteinasewith Progressiveankylosisproteinhomolog Angiopoietin-relatedprotein5 Coiled-coildomaincontainingprotein126 Stromalcell-derivedfactor2 Lysozyme-likeprotein6 domain-containingprotein8 Retinoicacidreceptorresponder Cartilageacidicprotein1 containingprotein4 containinggroupBprotein Tumornecrosisfactorreceptormember27 superfamily Thioredoxindomain-containing thrombospondinmotifs14 Chitinase-3likeprotein1 Proteincanopyhomolog4 PlasmaglutamatecarboxypeptidaseSlithomolog3protein Stanniocalcin-1 Interleukin-21receptor V-setandimmunoglobulindomain Semaphorin-4A Toll-likereceptor7 UPF0672proteinCXorf36 ArylsulfataseJ Plateletbasicprotein SerpinA9 protein2 16protein Alpha-2antiplasmin Peroxidasinhomolog CD177antigen proteinC4orf31 Interferonepsilon Beta-tectorin Prothyroliberin ProteinWFDC9 protein Cortistatin Ceruloplasmin ProteinFAM180A

Pregnancy-specificbeta1glycoprotein8 ComplementC1rsubcomponent-like Secretoglobinfamily1Cmember1 Secretoglobinfamily1Dmember1Secretoglobinfamily1Dmember2 Zymogengranulemembraneprotein16 Uncharacterizedaarfdomain-containing EGFfactorCandWillebrandvon Adisintegrinandmetalloproteinasewith Sodiumchannelsubunitbeta-2 Metalloproteinaseinhibitor4 Adisintegrinandmetalloproteinasewith matrixassociatedcartilage-Unique ComplementClqtumornecrosisfactor Proto-oncogeneproteinWnt3 Zonapellucida-bindingprotein1 Anteriorgradientprotein3homolog FMRFamide-relatedpeptides thrombospondinmotifs15 thrombospondinmotifs10 Transmembraneprotein130 relatedprotein9-like Growthinhibitionanddifferentiation CytokineSCM-1beta C5orf46proteinUncharacterized Fibroblastgrowthfactor18 Serineprotease33 Uncharacterizedglycosyltransferase Semaphorin-3G domain-containingprotein T-cellimmunomodulatoryprotein Thymicstromallymphopoietin Urocortin-3( relatedprotein88 ProteinWnt-10a ProteinWnt-3a ProteinWnt-6 ProteinWnt-9a proteinkinase1 C-Xmotifchemokine11 RibonucleaseK6 RibonucleaseT2 Urocortin-2 Retbindin Repetin protein AER61 SerpinA12 Serpin12 protein ProteinAMBP Amelotin Draxin TABLE1-continued

Leukocyteimmunoglobulin-likereceptor Leucine-richrepeatandfibronectintypeIII GPItransamidasecomponentPIG-S Transmembrane9superfamilymember3 Transmembranechannel-likeprotein2 Pregnancy-specificbeta1glycoprotein3 Thioredoxindomain-containingprotein5 Pregnancy-specificbeta1glycoprotein9 ProbableG-proteincoupledreceptor78 Tudordomain-containingprotein10 VonWillebrandfactorDandEGFdomain VascularendothelialgrowthfactorD Acidphosphatase-likeprotein2 proteinGPR177Integralmembrane HEPACAMfamilymember2Interleukin-27receptorsubunitalpha KTELmotif-containingprotein1 domain-containingprotein3 Protocadherinalpha-10 Protocadherinbeta-10 Osteopetrosis-associatedtransmembrane Beta-galactosidealpha2,6 Proline-richtransmembraneprotein3 Adisintegrinandmetalloproteinasewith thrombospondinmotifs16 SH2domain-containingprotein3A Disintegrinandmetalloproteinasedomain Transducinbeta-likeprotein2 Adisintegrinandmetalloproteinasewiththrombospondinmotifs17 ApolipoproteinO-like Adisintegrinandmetalloproteinasewith thrombospondinmotifs12 Beta-galactosidase1likeprotein Proenkephalin-A Integrinalpha-10 subfamilyAmember5 Protocadherin-12 sialyltransferase1 Sulfhydryloxidase2 SHCtransforming-protein4 containingprotein23 Pannexin-1 containingprotein Tetraspanin-6 Semaphorin-3F Uteroglobin Netrin-G1ligand protein1 Tenomodulin Beta-defensin119 ProteinFAM131A ProteinFAM3B

Lysophosphatidicacidphosphatasetype6 -II)AII(APOAAApoApolipoproteinAPOA-IV)AIV(ApolipoproteinAApo ApolipoproteinC-II(ApoCII)ApoC CellsurfaceglycoproteinCD200receptor1 Thrombospondintype-1domaincontaining WNT1inducible-signalingpathwayprotein2 Signalpeptide,CUBandEGF-likedomain ComplementcomponentC8alphachain Bromodomain-containingprotein9 acetylgalactosaminyltransferase14 Leucine-richrepeatcontainingprotein17 Leucine-richrepeatneuronalprotein2 vonWillebrandfactorAdomain-containing memberfamilyBdomain1typelectinC- Interleukin-1familymember5Interleukin-family 1member9 NucleotideexchangefactorSIL1 CD99antigen-likeprotein2 Carbohydratesulfotransferase12ProbableserinecarboxypeptidaseCPVL CUBandzonapellucida-likedomain BifunctionalheparansulfateN deacetylase/N-sulfotransferase3 Disintegrinandmetalloproteinasedomain Apoptosis-relatedprotein3 Histo-bloodgroupABOsystemtransferase Collagenalpha-1(VI)chain UncharacterizedproteinClorf159 containingprotein1 Brainmitochondrialcarrierprotein containingprotein3 Alpha-1acidglycoproteinAlpha-1acidglycoprotein2 containingprotein9 Calcium-activatedchloridechannel Beta-2glycoprotein1 Kallikrein-5 Matrilin-2 PolypeptideN Galectin-9 14-3proteinsigma Beta-secretase2 CathepsinL2 C-motifchemokine3 protein4 Mucin-3A Tuftelin protein1 Angiotensinogen regulator1Chymase US 10 ,414 ,793 B2 4545 91

cDNAFLJ77863,highlysimilartoHomo Putativekillercellimmunoglobulin-like Carcinoembryonicantigen-relatedcell Glycosyltransferase8domain-containing Notchhomolog2N-terminallikeprotein Alpha-1,3mannosylglycoprotein4beta sapienssecretedandtransmembrane1 Epididymal-specificlipocalin6 SecretoryphospholipaseA2receptor Bonemorphogeneticprotein3 Bonemarrowstromalantigen2 Bactericidal/permeabilityincreasing- GroupIIFsecretoryphospholipaseA2 GDNFfamilyreceptoralpha-like Probableglutathioneperoxidase8 Solutecarrierfamily22member12 Guanylatecyclaseactivator2B Probablecationtransporting-ATPase Glutathioneperoxidase3 Proteindpy-19homolog2 Platelet-activatingfactoracetylhydrolase Chorionicsomatomammotropinhormone Regulatorofmicrotubuledynamics Catechol-Omethyltransferasedomain Tripeptidyl-peptidase1 Trem-liketranscript1protein N-acetylglucosaminyltransferaseAMatrixextracellularphosphoglycoprotein KIR3DP1likeproteinreceptor adhesionmolecule20 CytochromeP45020A1 CarboxypeptidaseB Pappalysin-1 Retinoldehydrogenase14 Transcobalamin-2 containingprotein1 InducibleT-cellcostimulator Intelectin-2 (SECTM1),mRNA Claudin-18 like3protein- protein2 ProteinFAM19A1 Cystatin-D Cystatin-F protein3 Afamin 13A5 Haptoglobin like1 Galanin

Carcinoembryonicantigen-relatedcell Ly6/PLAURdomain-containingprotein6 Secretoglobinfamily1Dmember4 SisterchromatidcohesionproteinDCC1 Dyneinheavychaindomain-containing ExtracellularsulfataseSulf-2 Chymotrypsin-likeelastasefamily glycosylphosphatidylinositolanchor Matrixmetalloproteinase-27 Inactiveserineprotease35 UncharacterizedproteinC2orf82 Insulingrowthfactor-likefamily Bonemorphogeneticprotein15 Polymericimmunoglobulinreceptor Hyaluronanandproteoglycanlink domain-containingprotein30fusedhomolog ofSuppressor chainalphaXII)Collagen-1( Collagenalpha-1(XIV)chain ErythropoietinreceptorMAMdomain-containing Coiled-coildomaincontaining containingprotein2A Cadherin-likeprotein29 Plasmaserineproteaseinhibitor adhesionmolecule21 Galectin-3bindingprotein Fattyacyl-CoAreductase1Fin budinitiationfactorhomolog Prion-likeproteindoppel Disintegrinandmetalloproteinase Tumornecrosisfactorreceptor superfamilymember14 V-setandtransmembranedomain C-motifchemokine17 C-Xmotifchemokine6 C-Xmotifchemokine10 Folatereceptorbeta Interleukin-21 Interleukin-3 Interleukin-7 member1 protein2 134protein member1 Alpha-lactalbumin protein1 Beta-defensin1 protein2 Beta-defensin2 Suprabasin ADM Artemin TABLE1-continued

Spermacrosome-associatedprotein5 Multipleepidermalgrowthfactor-like Arylacetamidedeacetylase-like1 vonWillebrandfactorAdomain-containing familyDehydrogenasereductaseSDR/ CarboxypeptidaseNsubunit2 Collagentriplehelixrepeat-containing Inter-alphatrypsininhibitorheavychain likedomain-containingnogoreceptor Surfactant-associatedprotein2Adiponectinreceptorprotein1 WAP,kazalimmunoglobulinkunitzandNTRdomaincontaining-protein2 Pro-neuregulin3,membranebound Lamininsubunitgamma-1 HEATrepeat-containingproteinC7orf27 Collagenalpha-2(IX)chain Collagenalpha-3(IX)chain Collagenalpha-1(XXVII)chain ZincfingerRAD18domain-containing Growthdifferentiationfactor15 Lysozymeg-likeprotein1 Leucine-richrepeatandimmunoglobulin interactingprotein2 Neuroendocrineprotein7B2 Cathelicidinantimicrobialpeptide Progonadoliberin-1 Interferonalpha-17 Interferonalpha-21 Interferonalpha-8 Alpha-1Bglycoprotein Agouti-signalingprotein UPF0454proteinC12orf49 C-motifchemokine16 C-motifchemokine24 Leucine-richrepeattransmembrane protein4neuronal Endothelin-2 Fcreceptor-likeB H5-likeprotein domains6 Histatin-3 Claudin-8 protein5B1 Cadherin-6 member7B protein1 Clorfl24protein Glia-derivednexin GranzymeK isoform Colipase Fibromodulin

Receptortyrosine-proteinkinaseerbB3 Endoplasmicreticulumaminopeptidase1 ComplementfactorH-relatedprotein1 MajorhistocompatibilitycomplexclassI Insulin-likegrowthfactorbindingprotein6 Oxidizedlow-densitylipoproteinreceptor1 Pulmonarysurfactant-associatedproteinD Stimulatedbyretinoicacidgene6protein DnajhomologsubfamilymemberB11 pyrophosphatase/phosphodiesterasefamily Endoplasmicreticulumresidentprotein acetylgalactosaminyltransferase2 Lowdensity-lipoproteinreceptorrelated FibronectintypeIIIdomain-containing Prostatetumoroverexpressedgene1 Receptor-interactingserine/threonine Equilibrativenucleosidetransporter3 Tissuefactorpathwayinhibitor2 Intercellularadhesionmolecule4 ComplementcomponentC9 Glucose-fructoseoxidoreductasedomaincontainingprotein2 Hepatocytegrowthfactoractivator JunctionaladhesionmoleculeC UncharacterizedproteinKIAA0319Lamininsubunitalpha-5 Matrixmetalloproteinase-9 IgGFc-bindingprotein PolypeptideN relatedgeneprotein IgdeltachainCregion Interleukin-1beta Lipoproteinlipase proteinkinase2 SelenoproteinP Trefoilfactor1 Toll-likereceptor9 Interleukin-19 member7 protein10 protein4 Interstitialcollagenase Mucin-16 Mucin-2 Mucin-5B Isthmin-2 Ectonucleotide ERp44 Hemopexin Myocilin protein homolog Prothrombin US 10 ,414 ,793 B2 47 48

Regeneratingislet-derivedprotein4 ComplementClqtumornecrosisfactor Basicsalivaryproline-richprotein4 Dehydrogenase/reductaseSDRfamily oligosaccharide1,2-alphamannosidase Lamininsubunitbeta-2 EGF-containingfibulinlikeextracellular Receptor-typetyrosineprotein Matrixmetalloproteinase-23 Pentraxin-relatedproteinPTX3 Majorfacilitatorsuperfamilydomain Butyrophilin-likeprotein3Butyrophilin-likeprotein9 Lamininsubunitgamma-2 Endoplasmicreticulummannosyl Pancreaticsecretorygranulemembrane Guanylate-bindingprotein5 Carboxylesterase8Thioredoxin-relatedtransmembrane containingprotein2 Tumornecrosisfactorreceptor superfamilymember18 Beta-1,4galactosyltransferase diphosphohydrolase6 Neuropilin-2 matrixprotein1 phosphatasekappa Tachykinin-4 relatedprotein5 Pre-small/secretedglycoprotein Brevicancoreprotein C-motifchemokine23 majorglycoproteinGP24BSemaphorin- Semaphorin-5B Ectonucleosidetriphosphate Kallikrein-12 Testican-3 BrotherofCDO Tenascin-R Epsilon-sarcoglycan Opticin protein4 Porimin Torsin-1A member9 Eppin Otoancorin Growthfactor TSPEARProtein Hephaestin ProteinLMBRIL Mucin-21

Fibroblastgrowthfactorreceptor-like1 ComplementfactorH-relatedprotein5 Dehydrogenase/reductaseSDRfamily Pregnancy-specificbeta1glycoprotein6 Dehydrogenase/reductaseSDRfamily Regeneratingislet-derivedprotein3 Transmembraneandcoiled-coildomain Hydroxysteroid11-betadehydrogenase GDNFfamilyreceptoralpha-3 Magnesiumtransporterprotein1 Transmembraneandubiquitin-like Lamininsubunitalpha-3 FXYDdomain-containingiontransport Serinebeta-lactamaselikeprotein protein3relatedDickkopf- VEGFco-regulatedchemokine1 Amiloride-sensitiveamineoxidase domain-containingprotein1 memberonchromosomeX Serineincorporator2 phosphoprotein1Secreted RINGfingerprotein43 Semenogelin-2 Growth-regulatedalphaprotein containingprotein3 Progonadoliberin-2 DNAdamage-regulatedautophagymodulatorprotein2 TransmembraneproteinC17orf87 FK506-bindingprotein7 Serineincorporator1 Inhibinalphachain Stromelysin-3 Bonesialoprotein2 Delta-likeprotein1 PlateletreceptorGi24 Kallikrein-7 ApolipoproteinF VIP36-likeprotein containing]copper-[ regulator6 LACTB,mitochondrial Galectin-3 Pancreaticprohormone member11 Mucin-15 R-spondin2 Ephrin-A1 gamma Lymphotactin ADM2 1-likeprotein ProteinCASC4 TABLE1-continued

PeptidoglycanrecognitionproteinI-beta Choriogonadotropinsubunitbetavariant2 Transforminggrowthfactorbeta-3 Proproteinconvertasesubtilisin/kexin Carcinoembryonicantigen-relatedcell Earlyplacentainsulin-likepeptide Fibronectintype3andankyrinrepeat Immunoglobulinsuperfamilycontaining TranslocationproteinSEC63homolog Insulin-likegrowthfactorbindingprotein WAPfour-disulfidecoredomainprotein6 Adisintegrinandmetalloproteinasewith Cocaine-andamphetamineregulated Interferonomega-1 EGFlatrophilin,andseventransmembranedomain-containingprotein1 Lysyloxidasehomolog4 Leucine-richrepeattransmembraneprotein leucine-richrepeatprotein2 containingprotein2 Retinol-bindingprotein3 complexacidlabilechain Amelogenin,Yisoform thrombospondinmotifs1mitochondrial ProteinCOQ10A,proteinC19orf41 Uncharacterized UncharacterizedproteinC21orf63 domainsprotein1 Nucleobindin-2 PhospholipaseA2 Proenkephalin-B V-setandimmunoglobulindomain ProteinWnt-10b CarboxypeptidaseA4 Olfactomedin-4 ArylsulfataseF adhesionmolecule19 PeptideYY Beta-defensin104 Beta-defensin105 Beta-defensin107 Claudin-6 Proteindeltahd transcriptprotein Adropin type4 ProteinWFDC11 EpigenFAM19A5 Protein Lumican FLRT1 Atherin Renalase A

Pairedimmunoglobulin-liketype2receptor Regeneratingislet-derivedprotein3alpha Secretedfrizzled-relatedprotein1,isoform Scavengerreceptorcysteine-richtype1 Lactosylceramidealpha-2,3 Latent-transforminggrowthfactorbeta Plasminogen-relatedproteinB Angiopoietin-relatedprotein1 Beta-galactosidase1likeprotein2 WAPfour-disulfidecoredomainprotein5 Disintegrinandmetalloproteinasedomain DiacylglycerolO-acyltransferase2 Junctionaladhesionmolecule-like Plexindomain-containingprotein2 E3ubiquitin-proteinligaseRNF5 ProbablepalmitoyltransferaseZDHHC16 ERdegradation-enhancingalpha Interleukin-17receptorE PDZdomaincontaining-protein11 Retinoid-inducibleserinecarboxypeptidase Shortpalate,lungandnasalepithelium carcinoma-associatedprotein2 Platelet-derivedgrowthfactorC BSDdomain-containingprotein1 Interleukin-17receptorC Interleukin-17receptorD Integratorcomplexsubunit1 ligaseLNXproteinubiquitin-E3 Methionineadenosyltransferase2 protein2Podocalyxinlike- KinofIRRE-likeprotein1 bindingprotein4 Protachykinin-1 UPF0510proteinC19orf63 mannosidase-like2 containingprotein33 Celladhesionmolecule3 CDC45-relatedprotein Leucine-richrepeattransmembraneneuronalprotein3 Roundabouthomolog4 Kallikrein-10 Interleukin-20 Interleukin-25 Relaxin-3 Prominin-2 CRA_a M160protein 3-ketosteroidreductase alpha Chondrolectin subunitbeta sialyltransferase US 10 ,414 ,793 B2 49 50

ScavengerreceptorclassAmember5 Latent-transforminggrowthfactorbeta Secretedandtransmembrane1precusor kexinsubtilisinconvertase/Proprotein ProteinRMD5homologB protein108TransmembraneSushidomain-containingprotein3 Collagenalpha-1(XX)chain ATP-dependentmetalloproteaseYME1L1 Semaphorin-6B bindingprotein2Putativeuncharacterizedprotein NetrinreceptorUNC5D SerpinB3 UNQ6190/PRO20217 Mucin-13 variant type5

ComplementClqtumornecrosisfactor Secretedandtransmembrane1precusor Cysteine-richsecretoryprotein3 Putativeuncharacterizedprotein ComplementC4-APutativeuncharacterizedprotein Calcium-activatedchloridechannel Neuroblastomasuppressorof Toll-likereceptor10 relatedprotein1 UNQ6494/PRO21346 variant PRO2829 regulator2 TABLE1-continued lectin11binding-likeIgSialicacidLeucine-richrepeatcontainingprotein3 Cateyesyndromecriticalregionprotein1C-typelectindomainfamily18memberA LipomaHMGICfusionpartner-like1proteinProteinERGIC53 Leucine-richrepeatcontainingproteinToll25likereceptor8 Sortingnexin-24Ly6/PLAURdomaincontainingprotein4 SelenoproteinTLeucine-richrepeatcontainingprotein3B receptor-likeproteinKIR3DX1(Leukocytetumorigenicity1 Putativekillercellimmunoglobulin-like Leucine-richrepeatcontainingprotein18 Adisintegrinandmetalloproteinasewith receptorclustermember12) VitaminKepoxidereductasecomplex thrombospondinmotifs20Putativeuncharacterizedprotein Testis-expressedprotein101 Xylosyltransferase2 immunoglobulinTransmembraneanddomain-containingprotein1 subunit1 ENSP00000381830 ProteinFAM20A

factorsite-1

SID1transmembranefamilymember2 Transmembraneprotease,serine2 galactosamide-alpha2,3sialyltransferase containing)3(HakataantigenNL3 containing)3(Hakataantigen,isoform Sushidomain-containingprotein1Serine/threonine-proteinkinaseTAO2 UDP-glucuronicaciddecarboxylase1 Transmembraneprotein119 Transmembraneprotein98 PutativeuncharacterizedproteinC14orf144 Ficolin(Collagen/fibrinogendomain UncharacterizedproteinC10orf58 Thioredoxin-relatedtransmembrane CMP-Nacetylneuraminatebeta Putativeuncharacterizedprotein Pre-Blymphocyteprotein3 Membrane-boundtr (FicolinCollagen/fibrinogendomain protein2 ENSP00000380674 protease CRA_b) US 10 ,414 ,793 B2 51 52 The therapeutic proteins provided herein should not be for the substrate ( FIX and FX ) into closer proximity and by considered to be exclusive . Rather , as is apparent from the inducing a conformational change , which enhances the disclosure provided herein , the methods of the invention are enzymatic activity of FVIIa . applicable to any protein wherein attachment of a water The activation of FX is the common point of the two soluble polymer is desired according to the invention . For 5 pathways . Along with phospholipid and calcium , factors Va example , therapeutic proteins are described in US 2007 / (FVa ) and Xa convert prothrombin to thrombin (prothrom 0026485 , incorporated herein by reference in its entirety . binase complex ) , which then cleaves fibrinogen to form Blood Coagulation Proteins fibrin monomers . The monomers polymerize to form fibrin In one aspect , the starting material of the present inven strands . Factor XIIIa ( FXIIIa ) covalently bonds these tion is a blood coagulation protein , which can be derived 10 strands to one another to form a rigid mesh . from human plasma, or produced by recombinant engineer - Conversion of FVII to FVIIa is also catalyzed by a ing techniques, as described in U . S . Pat. Nos . 4 ,757 ,006 ; number of proteases , including thrombin , FIXa , FXa, factor 5 ,733 ,873 ; 5 , 198 , 349; 5 ,250 ,421 ; 5 ,919 , 766 ; and EP 306 Xla (FXla ), and factor XIIa (FXIIa ) . For inhibition of the 968 . early phase of the cascade, tissue factor pathway inhibitor Therapeutic polypeptides such as blood coagulation pro - 15 targets FVIIa / tissue factor /FXa product complex . teins including Factor IX (FIX ) , Factor VIII (FVIII ) , Factor Factor VIIa VIIa (FVIIa ) , Von Willebrand Factor (VWF ), Factor FV FVII ( also known as stable factor or proconvertin ) is a ( FV ) , Factor X (FX ), Factor XI (FXI ) , Factor XII ( FXII ), vitamin K - dependent serine protease glycoprotein with a thrombin (FII ), protein C , protein S , PA , PAI- 1 , tissue factor pivotal role in hemostasis and coagulation (Eigenbrot , Curr ( TF ) and ADAMTS 13 protease are rapidly degraded by 20 Protein Pept Sci. 2002 ; 3 : 287 - 99 ) . proteolytic enzymes and neutralized by antibodies . This FVII is synthesized in the liver and secreted as a single reduces their half- life and circulation time, thereby limiting chain glycoprotein of 48 kD . FVII shares with all vitamin their therapeutic effectiveness . Relatively high doses and K -dependent serine protease glycoproteins a similar protein frequent administration are necessary to reach and sustain domain structure consisting of an amino - terminal gamma the desired therapeutic or prophylactic effect of these coagu - 25 carboxyglutamic acid (Gla ) domain with 9 - 12 residues lation proteins. As a consequence , adequate dose regulation responsible for the interaction of the protein with lipid is difficult to obtain and the need of frequent intravenous membranes, a carboxy - terminal serine protease domain administrations imposes restrictions on the patient' s way of ( catalytic domain ) , and two epidermal growth factor - like living. domains containing a calcium ion binding site that mediates As described herein , blood coagulation proteins includ - 30 interaction with tissue factor. Gamma- glutamyl carboxylase ing , but not limited to , Factor IX (FIX ) , Factor VIII (FVIII ) , catalyzes carboxylation of Gla residues in the amino - termi Factor VIIa (FVIIa ), Von Willebrand Factor (VWF ) , Factor nal portion of the molecule . The carboxylase is dependent on FV ( FV ) , Factor X (FX ) , Factor XI, Factor XII ( FXII ) , a reduced form of vitamin K for its action , which is oxidized thrombin (FII ) , protein C , protein S , IPA , PAI- 1 , tissue factor to the epoxide form . Vitamin K epoxide reductase is ( TF ) and ADAMTS 13 protease are contemplated by the 35 required to convert the epoxide form of vitamin K back to invention . As used herein , the term " blood coagulation the reduced form . protein ” refers to any Factor IX ( FIX ), Factor VIII (FVIII ), The major proportion of FVII circulates in plasma in Factor VIIa (FVIIa ), Von Willebrand Factor (VWF ), Factor zymogen form , and activation of this form results in cleav FV ( FV ) , Factor X ( FX ) , Factor XII (FXII ) , thrombin ( FII ) , age of the peptide bond between arginine 152 and isoleucine protein C , protein S , DPA , PAI- 1 , tissue factor ( TF ) and 40 153 . The resulting activated FVIIa consists of a NH2 ADAMTS 13 protease which exhibits biological activity derived light chain (20 kD ) and a COOH terminal- derived that is associated with that particular native blood coagula - heavy chain (30 kD ) linked via a single disulfide bond (Cys tion protein . 135 to Cys 262) . The light chain contains the membrane The blood coagulation cascade is divided into three binding Gla domain , while the heavy chain contains the distinct segments : the intrinsic, extrinsic , and common path - 45 catalytic domain . ways (Schenone et al ., Curr Opin Hematol. 2004 ; 11 : 272 -7 ) . The plasma concentration of FVII determined by genetic The cascade involves a series of serine protease enzymes and environmental factors is about 0 . 5 mg/ mL (Pinotti et al. , ( zymogens) and protein cofactors . When required , an inac - Blood . 2000 ; 95 : 3423 - 8 ) . Different FVII genotypes can tive zymogen precursor is converted into the active form , result in several- fold differences in mean FVII levels . which consequently converts the next enzyme in the cas - 50 Plasma FVII levels are elevated during pregnancy in healthy cade . females and also increase with age and are higher in females The intrinsic pathway requires the clotting factors VIII, and in persons with hypertriglyceridemia . FVII has the IX , X , XI, and XII . Initiation of the intrinsic pathway occurs shortest half -life of all procoagulant factors (3 - 6 h ). The when prekallikrein , high -molecular - weight kininogen , fac - mean plasma concentration of FVIIa is 3 .6 ng /mL in healthy tor XI ( FXI) and factor XII ( FXII ) are exposed to a nega - 55 individuals and the circulating half - life of FVIIa is relatively tively charged surface. Also required are calcium ions and long ( 2 . 5 h ) compared with other coagulation factors . phospholipids secreted from platelets . Hereditary FVII deficiency is a rare autosomal recessive The extrinsic pathway is initiated when the vascular bleeding disorder with a prevalence estimated to be 1 case lumen of blood vessels is damaged . The membrane glyco - per 500, 000 persons in the general population (Acharya et protein tissue factor is exposed and then binds to circulating 60 al. , J Thromb Haemost . 2004 ; 2248 - 56 ). Acquired FVII factor VII (FVII ) and to small preexisting amounts of its deficiency from inhibitors is also very rare . Cases have also activated form FVIIa . This binding facilitates full conver - been reported with the deficiency occurring in association sion of FVII to FVIIa and subsequently , in the presence of with drugs such as cephalosporins , penicillins , and oral calcium and phospholipids , the conversion of factor IX anticoagulants . Furthermore , acquired FVII deficiency has ( FIX ) to factor IXa ( FIXa) and factor X ( FX ) to factor Xa 65 been reported to occur spontaneously or with other condi (FXa ) . The association of FVIIa with tissue factor enhances tions , such as myeloma , sepsis , aplastic anemia , with inter the proteolytic activity by bringing the binding sites of FVII leukin - 2 and antithymocyte globulin therapy . US 10 ,414 ,793 B2 53 54 Reference polynucleotide and polypeptide sequences and the light chains, which alter the availability of different include , e . g . , GenBank Accession Nos . J02933 for the binding epitopes in FVIII , e . g . allowing FVIII to dissociate genomic sequence , M13232 for the cDNA (Hagen et al. from VWF and bind to a phospholipid surface or altering the PNAS 1986 ; 83 : 2412 -6 ) , and P08709 for the polypeptide binding ability to certain monoclonal antibodies. sequence (references incorporated herein in their entireties ). 5 The lack or dysfunction of FVIII is associated with the A variety of polymorphisms of FVII have been described , most frequent bleeding disorder , hemophilia A . The treat for example see Sabater- Lleal et al . (Hum Genet. 2006 ; ment of choice for the management of hemophilia A is 118 :741 -51 ) ( reference incorporated herein in its entirety ) . Factor IX replacement therapy with plasma derived or rFVIII concen FIX is a vitamin K -dependent plasma protein that par - 10 trates . Patients with severe hemophilia A with FVIII levels ticipates in the intrinsic pathway of blood coagulation by below 1 % , are generally on prophylactic therapy with the converting FX to its active form in the presence of calcium aim of keeping FVIII above 1 % between doses . Taking into ions, phospholipids and FVIIIa . The predominant catalytic account the average half - lives of the various FVIII products capability of FIX is as a serine protease with specificity for in the circulation , this result can usually be achieved by a particular arginine - isoleucine bond within FX . Activation 15 giving FVIII two to three times a week . of FIX occurs by FXla which causes excision of the acti Reference polynucleotide and polypeptide sequences vation peptide from FIX to produce an activated FIX mol - include , e . g . , UniProtKB /Swiss - Prot P00451 ecule comprising two chains held by one or more disulphide (FA8 _ HUMAN ) ; Gitschier J et al. , Characterization of the bonds. Defects in FIX are the cause of recessive X - linked human Factor VIII gene , Nature , 312 ( 5992 ) : 326 - 30 ( 1984 ) ; hemophilia B . 20 Vehar G H et al. , Structure of human Factor VIII, Nature , Hemophilia A and B are inherited diseases characterized 312 ( 5992 ) : 337 -42 ( 1984 ) ; Thompson A R . Structure and by deficiencies in FVIII and FIX polypeptides , respectively . Function of the Factor VIII gene and protein , Semin Thromb The underlying cause of the deficiencies is frequently the Hemost , 2003 :29 ; 11 -29 (2002 ) . result of mutations in FVIII and FIX genes, both of which Von Willebrand Factor are located on the X chromosome. Traditional therapy for 25 Von Willebrand factor (VWF ) is a glycoprotein circulat hemophilias often involves intravenous administration of ing in plasma as a series of multimers ranging in size from pooled plasma or semi- purified coagulation proteins from about 500 to 20 ,000 kD . Multimeric forms of VWF are normal individuals . These preparations can be contaminated by pathogenic agents or viruses, such as infectious prions, composed of 250 kD polypeptide subunits linked together HIV , parvovirus , hepatitis A , and hepatitis C . Hence , there is 30 bytot disulfide bonds. VWF mediates initial platelet adhesion an urgent need for therapeutic agents that do not require the to the sub - endothelium of the damaged vessel wall. Only the use of human serum . larger multimers exhibit hemostatic activity . It is assumed The level of the decrease in FIX activity is directly that endothelial cells secrete large polymeric forms of VWF proportional to the severity of hemophilia B . The current and those forms of VWF which have a low molecular weight treatment of hemophilia B consists of the replacement of the 3525 (( low10 molecular weight VWF ) arise from proteolytic cleav missing protein by plasma- derived or recombinant FIX age . The multimers having large molecular masses are ( so -called FIX substitution or replacement treatment or stored in the Weibel- Pallade bodies of endothelial cells and therapy ). liberated upon stimulation . Polynucleotide and polypeptide sequences of FIX can be VWF is synthesized by endothelial cells and megakaryo found for example in the UniProtKB /Swiss - Prot Accession 40 cytes as prepro -VWF that consists to a large extent of No . P00740 , U . S . Pat . No. 6 , 531, 298 and in FIG . 1 (SEQ ID repeated domains . Upon cleavage of the signal peptide , NO : 1 ). pro - VWF dimerizes through disulfide linkages at its C - ter Factor VIII minal region . The dimers serve as protomers for multim Coagulation factor VIII (FVIII ) circulates in plasma at a erization , which is governed by disulfide linkages between very low concentration and is bound non - covalently to Von 45 the free end termini. The assembly to multimers is followed Willebrand factor (VWF ) . During hemostasis , FVIII is sepa - by the proteolytic removal of the propeptide sequence rated from VWF and acts as a cofactor for activated factor ( Leyte et al. , Biochem . J. 274 ( 1991 ) , 257 - 261) . IX (FIXa )- mediated FX activation by enhancing the rate of The primary translation product predicted from the cloned activation in the presence of calcium and phospholipids or cDNA of VWF is a 2813 - residue precursor polypeptide cellular membranes . 50 (prepro - VWF ) . The prepro -VWF consists of a 22 amino acid FVIII is synthesized as a single -chain precursor of signal peptide and a 741 amino acid propeptide , with the approximately 270 - 330 kD with the domain structure mature VWF comprising 2050 amino acids (Ruggeri Z . A . , A1 - A2- B - A3- C1 - C2 . When purified from plasma ( e . g ., and Ware , J. , FASEB J . , 308 -316 ( 1993 ) . " plasma - derived ” or “ plasmatic ” ) , FVIII is composed of a Defects in VWF are causal to Von Willebrand disease heavy chain (A1 - A2 - B ) and a light chain ( A3 -C1 - C2 ) . The 55 (VWD ) , which is characterized by a more or less pro molecular mass of the light chain is 80 kD whereas, due to nounced bleeding phenotype . VWD type 3 is the most proteolysis within the B domain , the heavy chain is in the severe form , in which VWF is completely missing , and range of 90 - 220 kD . VWD type 1 relates to a quantitative loss of VWF and its FVIII is also synthesized as a recombinant protein for phenotype can be very mild . VWD type 2 relates to quali therapeutic use in bleeding disorders . Various in vitro assays 60 tative defects of VWF and can be as severe as VWD type 3 . have been devised to determine the potential efficacy of VWD type 2 has many sub forms, some being associated recombinant FVIII (rFVIII ) as a therapeutic medicine. These with the loss or the decrease of high molecular weight assays mimic the in vivo effects of endogenous FVIII. In multimers . Von Willebrand disease type 2a (VWD - 2A ) is vitro thrombin treatment of FVIII results in a rapid increase characterized by a loss of both intermediate and large and subsequent decrease in its procoagulant activity , as 65 multimers . VWD - 2B is characterized by a loss of highest measured by in vitro assays . This activation and inactivation molecular -weight multimers. Other diseases and disorders coincides with specific limited proteolysis both in the heavy related to VWF are known in the art . US 10 , 414 , 793 B2 55 56 The polynucleotide and amino acid sequences of prepro from which the analog is derived, based on one or more VWF are available atGenBank Accession Nos. NM _ 000552 mutations involving (i ) deletion of one or more amino acid and NP _ 000543, respectively . residues at one or more termini of the polypeptide and /or one Other blood coagulation proteins according to the present or more internal regions of the naturally -occurring polypep invention are described in the art, e . g . Mann KG , Thromb 5 tide sequence ( e . g ., fragments ) , ( ii) insertion or addition of Haemost , 1999 ; 82 : 165 -74 . one or more amino acids at one or more termini ( typically an “ addition ” or “ fusion ” ) of the polypeptide and /or one or A . Polypeptides more internal regions ( typically an “ insertion ” ) of the natu rally -occurring polypeptide sequence or ( iii ) substitution of In one aspect , the starting material of the present inven - 10 one or more amino acids for other amino acids in the tion is a protein or polypeptide. As described herein , the term naturally -occurring polypeptide sequence. By way of therapeutic protein refers to any therapeutic protein mol example , a “ derivative” is a type of analog and refers to a ecule which exhibits biological activity that is associated polypeptide sharing the same or substantially similar struc withinvention the ,therapeutic the therapeutic protein protein . In moleculeone embodiment is a full - lengthof the 15 ture as a reference polypeptide that has been modified , e . g . , protein . chemically . Therapeutic protein molecules contemplated include full A variant polypeptide is a type of analog polypeptide and length proteins, precursors of full length proteins, biologi - includes insertion variants , wherein one or more amino acid cally active subunits or fragments of full length proteins, as residues are added to a therapeutic protein amino acid well as biologically active derivatives and variants of any of 20 sequence of the invention . Insertions may be located at these forms of therapeutic proteins. Thus , therapeutic pro - either or both termini of the protein , and/ or may be posi tein include those that ( 1 ) have an amino acid sequence that tioned within internal regions of the therapeutic protein has greater than about 60 % , about 65 % , about 70 % , about amino acid sequence . Insertion variants , with additional 75 % , about 80 % , about 85 % , about 90 % , about 91 % , about residues at either or both termini, include for example , 92 % , a , about 94 % 93 % , abbot 94 % , about 95 % , about 96 % , 25 fusion proteins and proteins including amino acid tags or about 97 % , about 98 % or about 99 % or greater amino acid other amino acid labels. In one aspect , the blood coagulation sequence identity , over a region of at least about 25 , about protein molecule optionally contains an N - terminal Met , 50 , about 100 , about 200 , about 300 , about 400 , or more especially when the molecule is expressed recombinantly in amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein ; 30 a bacterial cell such as E . coli. and / or ( 2 ) specifically bind to antibodies, e . g . , polyclonal or In deletion variants , one or more amino acid residues in monoclonal antibodies, generated against an immunogen a therapeutic protein polypeptide as described herein are comprising a referenced amino acid sequence as described removed . Deletions can be effected at one or both termini of herein , an immunogenic fragment thereof, and/ or a conser the therapeutic protein polypeptide, and /or with removal of vatively modified variant thereof. one or more residues within the therapeutic protein amino According to the present invention , the term “ recombi - acid sequence . Deletion variants , therefore , include frag nant therapeutic protein ” includes any therapeutic protein ments of a therapeutic protein polypeptide sequence . obtained via recombinant DNA technology . In certain In substitution variants, one or more amino acid residues embodiments , the term encompasses proteins as described of a therapeutic protein polypeptide are removed and herein . 40 replaced with alternative residues . In one aspect , the sub As used herein , “ endogenous therapeutic protein " stitutions are conservative in nature and conservative sub includes a therapeutic protein which originates from the stitutions of this type are well known in the art . Alterna mammal intended to receive treatment. The term also tively , the invention embraces substitutions that are also includes therapeutic protein transcribed from a transgene or non - conservative . Exemplary conservative substitutions are any other foreign DNA present in said mammal . As used 45 described in Lehninger, ( Biochemistry , 2nd Edition ; Worth herein , " exogenous therapeutic protein ” includes a blood Publishers , Inc. , New York ( 1975 ), pp . 71- 77 ] and are set out coagulation protein which does not originate from the mam immediately below . mal intended to receive treatment. As used herein , " plasma - derived blood coagulation pro CONSERVATIVE SUBSTITUTIONS tein ” or “ plasmatic ” includes all forms of the protein found 50 in blood obtained from a mammal having the property participating in the coagulation pathway . SIDE CHAIN As used herein “ biologically active derivative ” or “ bio CHARACTERISTIC AMINO ACID logically active variant” includes any derivative or variant of a molecule having substantially the same functional and /or 55 Non - polar (hydrophobic ) : biological properties of said molecule , such as binding A . Aliphatic ALIVP properties , and/ or the same structural basis , such as a B . Aromatic FW peptidic backbone or a basic polymeric unit . C . Sulfur - containing An " analog , ” such as a “ variant” or a “ derivative , ” is a D . Borderline compound substantially similar in structure and having the 60 Uncharged -polar : same biological activity , albeit in certain instances to a A . Hydroxyl STY differing degree, to a naturally occurring molecule . For B . Amides NO example , a polypeptide variant refers to a polypeptide shar C . Sulfhydryl D . Borderline ing substantially similar structure and having the same Positively charged (basic ) KRH biological activity as a reference polypeptide . Variants or 65 Negatively charged ( acidic ) DE analogs differ in the composition of their amino acid sequences compared to the naturally - occurring polypeptide US 10 ,414 ,793 B2 57 58 Alternatively , exemplary conservative substitutions are by genetic engineering , ( ii ) introducing recombinant DNA set out immediately below . into prokaryotic or eukaryotic cells by , for example and without limitation , transfection , electroporation or microin CONSERVATIVE SUBSTITUTIONS II jection , ( iii ) cultivating said transformed cells , ( iv ) express 5 ing therapeutic protein , e . g . constitutively or upon induction , and ( v ) isolating said blood coagulation protein , e . g . from the culture medium or by harvesting the transformed cells , EXEMPLARY in order to obtain purified therapeutic protein . ORIGINAL RESIDUE SUBSTITUTION In other aspects , the therapeutic protein is produced by Ala ( A ) Val , Leu , Ile 10 expression in a suitable prokaryotic or eukaryotic host Arg ( R ) Lys , Gin , Asn system characterized by producing a pharmacologically Asn ( N ) Gln , His , Lys , Arg Asp ( D ) Glu acceptable blood coagulation protein molecule . Examples of Cys ( C ) Ser eukaryotic cells are mammalian cells , such as CHO , COS , Gln ( Q ) Asn HEK 293 , BHK , SK -Hep , and HepG2 . Glu ( E ) Asp His (H ) Asn , Gln , Lys , Arg A wide variety of vectors are used for the preparation of Ile ( I ) Leu , Val, Met, Ala , Phe , the therapeutic protein and are selected from eukaryotic and Leu ( L ) Ile , Val, Met, Ala , Phe prokaryotic expression vectors . Examples of vectors for Lys ( K ) Arg , Gln , Asn prokaryotic expression include plasmids such as, and with Met ( M ) Leu , Phe , Ile Phe ( F ) Leu , Val, Ile , Ala out limitation , PRSET , PET, and PBAD , wherein the pro Pro (P ) Gly 20 moters used in prokaryotic expression vectors include one or Ser ( S ) Thr more of, and without limitation , lac , trc , trp , recA , or Thr ( T ) Ser Trp ( W ) Tyr araBAD . Examples of vectors for eukaryotic expression Tyr ( Y ) Trp , Phe , Thr , Ser include: ( i ) for expression in yeast, vectors such as , and Ile , Leu , Met , Phe , Ala without limitation , PAO , PPIC , PYES , or PMET, using Val ( V ) 25 promoters such as , and without limitation , AOX1, GAP , GALT, or AUG1; ( ii ) for expression in insect cells , vectors such as and without limitation , pMT, pAc5 , PIB , PMIB , or B . Polynucleotides PBAC , using promoters such as and without limitation PH , Nucleic acids encoding a therapeutic protein of the inven p10 , MT, Ac5 , OPIE2, gp64 , or polh , and ( iii ) for expression tion include , for example and without limitation , genes , 30 in mammalian cells , vectors such as and without limitation pre -mRNAs , mRNAs, cDNAs, polymorphic variants , PSVL , PCMV, pRc /RSV , pcDNA3 , or pBPV , and vectors alleles , synthetic and naturally occurring mutants . derived from , in one aspect , viral systems such as and Polynucleotides encoding a therapeutic protein of the without limitation vaccinia virus, adeno -associated viruses , invention also include , without limitation , those that ( 1 ) herpes viruses, or retroviruses, using promoters such as and specifically hybridize under stringent hybridization condi- 35 without limitation CMV, SV40 , EF - 1 , UbC , RSV, ADV , tions to a nucleic acid encoding a referenced amino acid BPV , and ß -actin . sequence as described herein , and conservatively modified variants thereof ; ( 2 ) have a nucleic acid sequence that has D . Administration greater than about 95 % , about 96 % , about 97 % , about 98 % , about 99 % , or higher nucleotide sequence identity , over a 40 In one embodiment a conjugated therapeutic protein of region of at least about 25 , about 50 , about 100 , about 150 , the present invention may be administered by injection , such about 200 , about 250 , about 500 , about 1000 , or more as intravenous, intramuscular, or intraperitoneal injection . nucleotides ( up to the full length sequence of 1218 nucleo To administer compositions comprising a conjugated tides of the mature protein ), to a reference nucleic acid therapeutic protein of the present invention to human or test sequence as described herein . Exemplary “ stringent hybrid - 15 animals in one aspect the compositions comprise one or ization " conditions include hybridization at 42° C . in 50 % more pharmaceutically acceptable carriers . The terms" phar formamide , 5xSSC , 20 mM Na. PO4, pH 6 . 8 ; and washing maceutically " or " pharmacologically acceptable " refer to in 1xSSC at 55° C . for 30 minutes . It is understood that variation in these exemplary conditions can be made based molecular entities and compositions that are stable , inhibit on the length and GC nucleotide content of the sequences to protein degradation such as aggregation and cleavage prod be hybridized . Formulas standard in the art are appropriate 50 ucts, and in addition do not produce allergic , or other for determining appropriate hybridization conditions . See adverse reactions when administered using routes well Sambrook et al. , Molecular Cloning : A Laboratory Manual known in the art , as described below . “ Pharmaceutically (Second ed ., Cold Spring Harbor Laboratory Press , 1989 ) SS acceptable carriers ” include any and all clinically useful 9 .47 - 9 .51 . solvents , dispersion media , coatings , antibacterial and anti A “ naturally - occurring ” polynucleotide or polypeptide 55 fungal agents, isotonic and absorption delaying agents and sequence is typically derived from a mammal including , but the like, including those agents disclosed above . not limited to , primate, e . g. , human ; rodent, e . g. , rat, mouse , As used herein , “ effective amount includes a dose suit hamster ; cow , pig , horse , sheep , or any mammal . The able for treating a disease or disorder or ameliorating a nucleic acids and proteins of the invention can be recombi- symptom of a disease or disorder . In one embodiment, nant molecules ( e . g . , heterologous and encoding the wild 60 “ effective amount" includes a dose suitable for treating a type sequence or a variant thereof , or non -naturally occur mammal having a bleeding disorder as described herein . ring ) . The compositions may be administered orally , topically , transdermally , parenterally, by inhalation spray, vaginally , C . Production of Therapeutic Proteins rectally , or by intracranial injection . The term parenteral as 65 used herein includes subcutaneous injections, intravenous , Production of a therapeutic protein includes any method intramuscular , intracisternal injection , or infusion tech known in the art for (i ) the production of recombinant DNA niques . Administration by intravenous, intradermal , intra US 10 , 414 , 793 B2 59 60 muscular, intramammary , intraperitoneal, intrathecal, ret- chemical linker is MBPH ( 4 - [ 4 - N -Maleimidophenyl ]butyric robulbar, intrapulmonary injection and or surgical acid hydrazide ) containing a carbohydrate -selective hydraz implantation at a particular site is contemplated as well. ide and a sulfhydryl- reactive maleimide group (Chamow et Generally , compositions are essentially free of pyrogens, as al. , J Biol Chem 1992 ; 267: 15916 - 22 ). Other exemplary and well as other impurities that could be harmful to the recipi- 5 preferred linkers are described below . ent . In one embodiment, the derivative retains the full func Single or multiple administrations of the compositions tional activity of native therapeutic protein products , and can be carried out with the dose levels and pattern being selected by the treating physician . For the prevention or provides an extended half - life in vivo , as compared to native treatment of disease , the appropriate dosage will depend on 10 therapeutic protein products . In another embodiment, the the type of disease to be treated , as described above , the derivative retains at least 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27, 28 , severity and course of the disease , whether drug is admin 29 , 30 , 31 , 32 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 . 45 , istered for preventive or therapeutic purposes , previous 46 , 47 , 48 , 49 , 50 , 51, 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61, therapy , the patient' s clinical history and response to the 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , drug , and the discretion of the attending physician . 15 78 , 79 , 80 , 81, 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , The present invention also relates to a pharmaceutical 94 , 95 , 96 , 97 , 98 , 99 , 100 , 110 , 120 , 130 , 140 , or 150 composition comprising an effective amount of a conjugated percent ( % ) biological activity relative to native blood therapeutic protein as defined herein . The pharmaceutical coagulation protein . In a related aspect , the biological activi composition may further comprise a pharmaceutically ties of the derivative and native blood coagulation protein acceptable carrier , diluent, salt , buffer , or excipient. The 20 are determined by the ratios of chromogenic activity to pharmaceutical composition can be used for treating the blood coagulation factor antigen value ( blood coagulation above - defined bleeding disorders . The pharmaceutical com factor :Chr : blood coagulation factor :Ag ) . In still another position of the invention may be a solution or a lyophilized embodiment of the invention , the half - life of the construct is product . Solutions of the pharmaceutical composition may decreased or increased 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 , 1 . 0 , 1 . 1 , 1 . 2 , be subjected to any suitable lyophilization process . 1 . 3 , 1 . 4 , 1 .5 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 - fold relative to the As an additional aspect, the invention includes kits which in vivo half - life of native therapeutic protein . comprise a composition of the invention packaged in a manner which facilitates its use for administration to sub A . Sialic Acid and PSA jects . In one embodiment, such a kit includes a compound or composition described herein ( e .g ., a composition compris - 30 N PSAs consist of polymers (generally homopolymers ) of ing a conjugated therapeutic protein ), packaged in a conS - 30 N - acetylneuraminic acid . The secondary amino group nor tainer such as a sealed bottle or vessel, with a label affixed mally bears an acetyl group , but it may instead bear a to the container or included in the package that describes use glycolyl group . Possible substituents on the hydroxyl groups of the compound or composition in practicing the method . In include acetyl, lactyl, ethyl, sulfate , and phosphate groups . one embodiment, the kit contains a first container having a 35 composition comprising a conjugated therapeutic protein and a second container having a physiologically acceptable HO OH reconstitution solution for the composition in the first con COO tainer. In one aspect, the compound or composition is packaged in a unit dosage form . The kit may further include 40 AcHNE / OH a device suitable for administering the composition accord ?? ?? ing to a specific route of administration . Preferably , the kit contains a label that describes use of the therapeutic protein N - Acetylneuraminic acid or peptide composition . Neu5Ac Water Soluble Polymers 45 In one aspect, a therapeutic protein derivative ( i. e . , a Structure of Sialic Acid ( N - Acetylneuraminic Acid ) conjugated therapeutic protein )molecule provided is bound PSAs and mPSAs generally comprise linear polymers to a water - soluble polymer including , but not limited to , consisting essentially of N - acetylneuraminic acid moieties polyethylene glycol (PEG ) , branched PEG , polysialic acid linked by 2 , 8 - or 2 , 9 - glycosidic linkages or combinations of ( PSA ) , hydroxyalkyl starch (HAS ) , hydroxylethyl starch 50 these ( e .g . alternating 2 ,8 - and 2 ,9 - linkages ). In particularly (HES ) , carbohydrate , polysaccharides , pullulane , chitosan , preferred PSAs and mPSAs, the glycosidic linkages are hyaluronic acid , chondroitin sulfate , dermatan sulfate , - 2 , 8 . Such PSAs and mPSAs are conveniently derived starch , dextran , carboxymethyl- dextran , polyalkylene oxide from colominic acids, and are referred to herein as “ CAs” (PAO ), polyalkylene glycol (PAG ), polypropylene glycol and “mCAs ” . Typical PSAs and mPSAs comprise at least 2 , (PPG ) polyoxazoline , poly acryloylmorpholine, polyvinyl 55 preferably at least 5 , more preferably at least 10 and most alcohol (PVA ), polycarboxylate , polyvinylpyrrolidone, preferably at least 20 N - acetylneuraminic acid moieties. polyphosphazene , polyoxazoline , polyethylene - co -maleic Thus, they may comprise from 2 to 300 N - acetylneuraminic acid anhydride , polystyrene - co -maleic acid anhydride , poly acid moieties, preferably from 5 to 200 N - acetylneuraminic ( 1 - hydroxymethylethylene hydroxymethylformal) ( PHF ) , acid moieties , or most preferably from 10 to 100 N -acetyl 2 -methacryloyloxy - 2 ' -ethyltrimethylammoniumphosphate 60 neuraminic acid moieties . PSAs and CAs preferably are (MPC ) . In one embodiment of the invention , the water essentially free of sugar moieties other than N - acetyl soluble polymer is consisting of sialic acid molecule having neuraminic acid . Thus PSAs and CAs preferably comprise at a molecular weight range of 350 to 120 ,000 , 500 to 100 , 000 , least 90 % ,more preferably at least 95 % and most preferably 1000 to 80 , 000, 1500 to 60 , 000 , 2 ,000 to 45, 000 Da , 3 ,000 at least 98 % N - acetylneuraminic acid moieties . to 35 ,000 Da, and 5 , 000 to 25 ,000 Da . The coupling of the 65 Where PSAs and CAs comprise moieties other than water soluble polymer can be carried out by direct coupling N -acetylneuraminic acid ( as, for example in mPSAS and to the protein or via linker molecules. One example of a mCAs ) these are preferably located at one or both of the US 10 , 414 , 793 B2 61 62 ends of the polymer chain . Such “ other” moieties may, for The naturally occurring polymer PSA is available as a example , be moieties derivedd fromfrom terminal N -acetyl acetyl- polydisperse preparation showing a broad size distribution neuraminic acid moieties by oxidation or reduction . For example , WO - A -0187922 describes such mPSAs and ( e . g . Sigma C -5762 ) and high polydispersity (PD ) . Because mCAs in which the non -reducing terminal N -acetyl - 5 the polysaccharides are usually produced in bacteria carry neuraminic acid unit is converted to an aldehyde group by ing the inherent risk of copurifying endotoxins, the purifi reaction with sodium periodate . Additionally , WO 2005 / cation of long sialic acid polymer chains may raise the 016974 describes such mPSAs and mCAs in which the probability of increased endotoxin content . Short PSA mol reducing terminal N - acetylneuraminic acid unit is subjected ecules with 1 - 4 sialic acid units can also be synthetically to reduction to reductively open the ring at the reducing 10 prepared (Kang S H et al ., Chem Commun . 2000 ; 227 -8 ; terminal N -acetylneuraminic acid unit , whereby a vicinal Ress D K and Linhardt RJ, Current Organic Synthesis . diol group is formed , followed by oxidation to convert the 2004 ; 1: 31 -46 ) , thus minimizing the risk of high endotoxin vicinal diol group to an aldehyde group . levels . However PSA preparations with a narrow size dis Sialic acid rich glycoproteins bind selectin in humans and other organisms. They play an important role in human 15 tribution and low polydispersity , which are also endotoxin influenza infections . E . g . , sialic acid can hide mannose free , can now be manufactured . Polysaccharide compounds antigens on the surface of host cells or bacteria from of particular use for the invention are , in one aspect, those mannose - binding lectin . This prevents activation of comple - produced by bacteria . Some of these naturally - occurring ment. Sialic acids also hide the penultimate galactose resi - polysaccharides are known as glycolipids. In one embodi due thus preventing rapid clearance of the glycoprotein by 20 ment, the polysaccharide compounds are substantially free the galactose receptor on the hepatic parenchymal cells . of terminal galactose units .

HO COONa HO . HOITO COONa AcHN HO HO . COONa AcHN HO . AcHN HO

Structure of Colominic Acid (Homopolymer of N - Acetyl - 35 B . Polyethylene Glycol (PEG ) and Pegylation neuraminic Acid ) Colominic acids ( a sub - class of PSAs ) are homopolymers In certain aspects , therapeutic proteins are conjugated to of N -acetylneuraminic acid (NANA ) with a (208 ) ketosidic a water soluble polymer by any of a variety of chemical linkage, and are produced , inter alia , by particular strains of methods (Roberts J M et al. , Advan Drug Delivery Rev Escherichia coli possessing K1 antigen . Colominic acids * 2002 ; 54 : 459 - 76 ) . For example , in one embodiment a thera have many physiological functions. They are important as a peutic protein is modified by the conjugation of PEG to free raw material for drugs and cosmetics . amino groups of the protein using N -hydroxysuccinimide Comparative studies in vivo with polysialylated and (NHS ) esters . In another embodiment the water soluble unmodified asparaginase revealed that polysialylation 45 polymer, for example PEG , is coupled to free SH groups increased the half - life of the enzyme (Fernandes and Gre - using maleimide chemistry or the coupling of PEG hydraz goriadis , Biochimica Biophysica Acta 1341 : 26 - 34 , 1997 ) . ides or PEG amines to carbohydrate moieties of the thera As used herein , “ sialic acid moieties” includes sialic acid peutic protein after prior oxidation . monomers or polymers (“ polysaccharides” ) which are The conjugation is in one aspect performed by direct soluble in an aqueous solution or suspension and have little 50 coupling (or coupling via linker systems) of the water or no negative impact, such as side effects , to mammals upon soluble polymer to a therapeutic protein under formation of administration of the PSA -blood coagulation protein conju - stable bonds. In addition degradable , releasable or hydroly gate in a pharmaceutically effective amount . The polymers sable linker systems are used in certain aspects the present are characterized , in one aspect , as having 1 , 2 , 3 , 4 , 5 , 10 , invention ( Tsubery et al. J Biol Chem 2004 ; 279 : 38118 - 24 / 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 200 , 300 , 400 , or 500 55 Greenwald et al. , J Med Chem 1999 ; 42 : 3657 -67 / Zhao et al. , sialic acid units . In certain aspects , different sialic acid units Bioconj Chem 2006 ; 17 :341 - 51 /WO2006 / 138572A2 / U . S . are combined in a chain . Pat . No . 7 ,259 ,224B2 / U .S . Pat. No . 7, 060 , 259B2) . In one embodiment of the invention , the sialic acid In one embodiment of the invention , a therapeutic protein portion of the polysaccharide compound is highly hydro - is modified via lysine residues by use of polyethylene glycol philic , and in another embodiment the entire compound is 60 derivatives containing an active N -hydroxysuccinimide highly hydrophilic . Hydrophilicity is conferred primarily by ester (NHS ) such as succinimidyl succinate , succinimidyl the pendant carboxyl groups of the sialic acid units , as well glutarate or succinimidyl propionate . These derivatives react as the hydroxyl groups . The saccharide unit may contain with the lysine residues of the therapeutic protein under mild other functional groups, such as, amine, hydroxyl or sul- conditions by forming a stable amide bond . In one embodi phate groups, or combinations thereof. These groups may be 65 ment of the invention , the chain length of the PEG derivative present on naturally - occurring saccharide compounds , or is 5 , 000 Da . Other PEG derivatives with chain lengths of introduced into derivative polysaccharide compounds . 500 to 2, 000 Da, 2 ,000 to 5 ,000 Da , greater than 5 ,000 up US 10 ,414 ,793 B2 63 64 to 10 ,000 Da or greater than 10 ,000 up to 20 ,000 Da , or Structure of a Branched PEG -Derivative (Nektar greater than 20 ,000 up to 150 ,000 Da are used in various Therapeutics) embodiments , including linear and branched structures . Branched PEG N -Hydroxysuccinimide Alternative methods for the PEGylation of amino groups 5 (mPEG2 -NHS ) are , without limitation , the chemical conjugation with PEG carbonates by forming urethane bonds , or the reaction with aldehydes or ketones by reductive amination forming sec ondary amide bonds. In one embodiment of the present invention a therapeutic 10 MPEG protein molecule is chemically modified using PEG deriva - C - 0 - N tives that are commercially available . These PEG deriva tives in alternative aspects have linear or branched struc mPEG tures. Examples of PEG - derivatives containing NHS groups are listed below . 15 This reagent with branched structure is described in more The following PEG derivatives are non - limiting examples detail by Kozlowski et al. (BioDrugs 2001 ; 5 :419 - 29 ). of those commercially available from Nektar Therapeutics Other non - limiting examples of PEG derivatives are com (Huntsville , Ala . ; see www .nektar . com /PEG reagent catalog ; mercially available from NOF Corporation ( Tokyo , Japan ; Nektar Advanced PEGylation , price list 2005 - 2006 ) : 20 see www .nof . co . jp / english : Catalogue 2005 ) General Structure of Linear PEG - Derivatives (NOF mPEG -Succinimidyl Propionate (mPEG - SPA ) Corp . )

MPEG – CH2CH2 — - O - N CH30 (CH2CH2O )n - X - N 30

35as X = carboxymethyl

MPEG -Succinimidyl a -Methylbutanoate (MPEG - SMB ) 40 CH3O (CH2CH20 )n - CH2 - - 0 - N

45 X =carboxypentyl MPEG – CH2CH2CH - 0 - 0 - N CH3

50 CH30 (CH2CH2O )n - (CH2 ) 5 — C — 0 - N

MPEG -CM -HBA -NHS (CM + Carboxymethyl; 55 HBA = Hydroxy Butyric Acid ) x = succinate

60 O CH30 (CH2CH20 )n - ö - CH2CH2 - 0 - 0 - N MPEG - CH20 - 0 - CHCH20 - 0 - N CH3 65 MPEG Succinimidyl succinate US 10 ,414 ,793 B2 65 66 x = glutarate 65 up to 95 % by weight. HES exhibits advantageous biological ** elutarane properties and is used as a blood volume replacement agent and in hemodilution therapy in the clinics ( Sommermeyer et al. , 1987 , Krankenhauspharmazie , 8 (8 ), 271 -278 ; and Wei 5 dler et al ., 1991 , Arzneim .- Forschung/ Drug Res. g 419 494 - 498 ). CH3O (CH2CH20 )n - ö — (CH2 ) 3 — — 0 - N Amylopectin consists of glucose moieties, wherein in the main chain alpha - 1 , 4 - glycosidic bonds are present and at the branching sites alpha - 1 , 6 - glycosidic bonds are found . The MPEG Succinimidyl glutarate 10 physical - chemical properties of this molecule are mainly determined by the type of glycosidic bonds . Due to the nicked alpha - 1 , 4 - glycosidic bond , helical structures with Structures of Branched PEG -Derivatives (NOF about six glucose -monomers per turn are produced . The Corp . ) : 2 , 3 - Bis (methylpolyoxyethylene - oxy ) - 1 - ( 1 , 5 16 physico - chemical as well as the biochemical properties of dioxo - 5 - succinimidyloxy, pentyloxy ) propane the polymer can be modified via substitution . The introduc tion of a hydroxyethyl group can be achieved via alkaline hydroxyethylation . By adapting the reaction conditions it is possible to exploit the different reactivity of the respective H3C - (OCH2 - CH2) n - O - CH2 hydroxy group in the unsubstituted glucose monomer with O

H3C - (OCH2 - CH2) n - O - CH = respect to a hydroxyethylation . Owing to this fact, the CH2 - 0 - C - CH2CH2CH2 - 0 - 0 - N skilled person is able to influence the substitution pattern to a limited extent. HAS refers to a starch derivative which has been substi 25 tuted by at least one hydroxyalkyl group . Therefore, the term hydroxyalkyl starch is not limited to compounds where the terminal carbohydrate moiety comprises hydroxyalkyl groups R1, R2, and /or R3, but also refers to compounds in 2 , 3 -Bis (methylpolyoxyethylene -oxy ) - 1 - ( succinimidyl which at least one hydroxy group present anywhere , either carboxypentyloxy ) propane in the terminal carbohydrate moiety and /or in the remaining H3C — (OCH2 - CH2n – O – CH2 H3C — (OCH2 - CH2) n - O - CH CH2 - O - CH2CH2CH2CH2CH2 - C - 0 - N

40 These propane derivatives show a glycerol backbone with part of the starch molecule , HAS ', is substituted by a a 1, 2 substitution pattern . In the present invention branched hydroxyalkyl group R1, R2 , or R3 . PEG derivatives based on glycerol structures with 1 , 3 sub stitution or other branched structures described in US2003/ 0143596A1 are also contemplated . 220031 as PEG derivatives with degradable ( for example , hydroly ORI sable ) linkers as described by Tsubery et al . ( J Biol Chem H . 2004 ; 279 : 38118 -24 ) and Shechter et al. HAS' (W0004089280A3 ) are also contemplated . . 50 R20 OH Surprisingly , the PEGylated therapeutic protein of this | H OR3 invention exhibits functional activity , combined with an H extended half -life in vivo . In addition the PEGylated rFVIII, FVIIa , FIX , or other blood coagulation factor seems to be The alkyl group may be a linear or branched alkyl group more resistant against thrombin inactivation . 33 which may be suitably substituted . Preferably , the hydroxy alkyl group contains 1 to 10 carbon atoms, more preferably C . Hydroxyalkyl Starch (HAS ) and Hydroxylethyl from 1 to 6 carbon atoms, more preferably from 1 to 4 Starch (HES ) carbon atoms, and even more preferably 2 - 4 carbon atoms. In various embodiments of the present invention , a thera - 60 “ Hydroxyalkyl starch ” therefore preferably comprises peutic protein molecule is chemically modified using hydroxyethyl starch , hydroxypropyl starch and hydroxybu hydroxyalkyl starch (HAS ) or hydroxylethyl starch (HES ) or tyl starch , wherein hydroxyethyl starch and hydroxypropyl derivatives thereof. starch are particularly preferred . HES is a derivative of naturally occurring amylopectin Hydroxyalkyl starch comprising two or more different and is degraded by alpha - amylase in the body . HES is a 65 hydroxyalkyl groups is also comprised in the present inven substituted derivative of the carbo - hydrate polymer amylo - tion . The at least one hydroxyalkyl group comprised in HAS pectin , which is present in corn starch at a concentration of may contain two or more hydroxy groups. According to one US 10 ,414 ,793 B2 67 68 embodiment, the at least one hydroxyalkyl group comprised deacetylated 5 -amino group , or an aldehyde group of a HAS contains one hydroxy group . molecule of a therapeutic protein forms a Schiff base with The term HAS also includes derivatives wherein the alkyl the N -deacetylated 5 - amino group of a sialic acid residue . group is mono - or polysubstituted . In one embodiment, the Alternatively , the polysaccharide compound is associated alkyl group is substituted with a halogen , especially fluorine , 5 in a non -covalent manner with a therapeutic protein . For or with an aryl group , provided that the HAS remains example , the polysaccharide compound and the pharmaceu soluble in water. Furthermore , the terminal hydroxy group a tically active compound are in one aspect linked via hydro of hydroxyalkyl group may be esterified or etherified . HAS phobic interactions . Other non - covalent associations include derivatives are described in WO /2004 /024776 , which is electrostatic interactions, with oppositely charged ions incorporated by reference in its entirety . 10 attracting each other. In various embodiments , the therapeutic protein is linked D . Methods of Attachment to or associated with the polysaccharide compound in stoi chiometric amounts (e . g. , 1 : 1, 1 : 2, 1: 3 , 1: 4 , 1 :5 , 1 :6 , 1 :7 , 1 : 7 , A therapeutic protein may be covalently linked to the 1 : 8 , 1 : 9 , or 1: 10 , etc . ) . In various embodiments , 1 - 6 , 7 - 12 or polysaccharide compounds by any of various techniques 15 13 - 20 polysaccharides are linked to the blood coagulation known to those of skill in the art . In various aspects of the protein . In still other embodiments , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , invention , sialic acid moieties are bound to a therapeutic 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 or more polysac protein , e . g ., FIX , FVIII , FVIIa or VWF, for example by the charides are linked to the blood coagulation protein . method described in U . S . Pat. No. 4 , 356 , 170 , which is in various embodiments , the therapeutic protein is modi herein incorporated by reference . 20 fied to introduce glycosylation sites ( i. e ., sites other than the Other techniques for coupling PSA to polypeptides are native glycosylation sites ) . Such modification may be also known and contemplated by the invention . For accomplished using standard molecular biological tech example , US Publication No . 2007 /0282096 describes con - niques known in the art. Moreover , the therapeutic protein , jugating an amine or hydrazide derivative of, e. g ., PSA , to prior to conjugation to a water soluble polymer via one or proteins . In addition , US Publication No . 2007/ 0191597 25 more carbohydrate moieties , may be glycosylated in vivo or describes PSA derivatives containing an aldehyde group for in vitro . These glycosylated sites can serve as targets for reaction with substrates ( e . g . , proteins) at the reducing end . conjugation of the proteins with water soluble polymers (US These references are incorporated by reference in their Patent Application No. 20090028822 , US Patent Applica entireties . tion No. 2009/ 0093399 , US Patent Application No . 2009 / Various methods are disclosed at column 7 , line 15 , 30 0081188 , US Patent Application No . 2007 /0254836 , US through column 8 , line 5 of U .S . Pat. No. 5 ,846 ,951 ( incor - Patent Application No. 2006 /0111279 , and DeFrees S . et al. , porated by reference in its entirety ) . Exemplary techniques Glycobiology, 2006 , 16 , 9 , 833 -43 ). For example , a protein include linkage through a peptide bond between a carboxyl that is not naturally glycoslyated in vivo ( e . g ., a protein that group on one of either the blood coagulation protein or is not a glycoprotein ) may be modified as described above . polysaccharide and an amine group of the blood coagulation 35 protein or polysaccharide, or an ester linkage between a E . Aminooxy Linkage carboxyl group of the blood coagulation protein or polysac charide and a hydroxyl group of the therapeutic protein or In one embodiment of the invention , the reaction of polysaccharide . Another linkage by which the therapeutic hydroxylamine or hydroxylamine derivatives with alde protein is covalently bonded to the polysaccharide com - 40 hydes ( e . g . , on a carbohydrate moiety following oxidation pound is via a Schiff base , between a free amino group on by sodium periodate ) to form an oxime group is applied to the blood coagulation protein being reacted with an alde - the preparation of conjugates of blood coagulation protein . hyde group formed at the non -reducing end of the polysac - For example , a glycoprotein ( e . g . , a therapeutic protein charide by periodate oxidation ( Jennings H J and Lugowski according to the present invention ) is first oxidized with a C , J Immunol. 1981 ; 127 : 1011 - 8 ; Fernandes A l and Gre - 45 oxidizing agent such as sodium periodate (NalO4 ) (Rothfus goriadis G , Biochim Biophys Acta . 1997 ; 1341 ; 26 -34 ) . The JA et Smith E L ., J Biol Chem 1963 , 238 , 1402 - 10 ; and Van generated Schiff base is in one aspect stabilized by specific Lenten L and Ashwell G ., J Biol Chem 1971 , 246 , 1889 - 94 ) . reduction with NaCNBH3 to form a secondary amine . An The periodate oxidation of glycoproteins is based on the alternative approach is the generation of terminal free amino classical Malaprade reaction described in 1928 , the oxida groups in the PSA by reductive amination with NH4C1 after 50 tion of vicinal diols with periodate to form an active alde prior oxidation . Bifunctional reagents can be used for link - hyde group (Malaprade L . , Analytical application , Bull Soc ing two amino or two hydroxyl groups. For example , PSA Chim France, 1928 , 43, 683 - 96 ). Additional examples for containing an amino group is coupled to amino groups of the such an oxidizing agent are lead tetraacetate ( Pb (OAC ) 4 ) , protein with reagents like BS3 ( Bis ( sulfosuccinimidyl) sub - manganese acetate (MnO ( Ac ) 3 ), cobalt acetate (Co (OAc ) 2 ) , erate /Pierce , Rockford , ill . ) . In addition heterobifunctional 55 thallium acetate ( TLOAC) , cerium sulfate (Ce ( SO4 ) 2 ) ( U . S . cross linking reagents like Sulfo -EMCS ( N - E -Maleimido Pat . No. 4 ,367 ,309 ) or potassium perruthenate (KRu04 ) caproyloxy ) sulfosuccinimide ester / Pierce ) is used for (Marko et al. , J Am Chem Soc 1997 , 119, 12661 - 2 ). By instance to link amine and thiol groups. " oxidizing agent ” a mild oxidizing compound which is In another approach , a PSA hydrazide is prepared and capable of oxidizing vicinal diols in carbohydrates, thereby coupled to the carbohydrate moiety of the protein after prior 60 generating active aldehyde groups under physiological reac oxidation and generation of aldehyde functions. tion conditions is meant. As described above, a free amine group of the therapeutic The second step is the coupling of the polymer containing protein reacts with the 1 -carboxyl group of the sialic acid an aminooxy group to the oxidized carbohydrate moiety to residue to form a peptidyl bond or an ester linkage is formed form an oxime linkage . In one embodiment of the invention , between the 1 -carboxylic acid group and a hydroxyl or other 65 this step can be carried out in the presence of catalytic suitable active group on a blood coagulation protein . Alter - amounts of the nucleophilic catalyst aniline or aniline natively , a carboxyl group forms a peptide linkage with derivatives (Dirksen A et Dawson P E , Bioconjugate Chem . US 10 , 414 , 793 B2 70 2008 ; Zeng Y et al. , Nature Methods 2009; 6 :207 - 9 ). The -continued aniline catalysis dramatically accelerates the oxime ligation allowing the use of very low concentrations of the reagents . In another embodiment of the invention the oxime linkage is stabilized by reduction with NaCNBH3 to form an 5 alkoxyamine linkage ( FIG . 2 ) . Additional catalysts are NH , OH described below . HR HR Additional information on aminooxy technology can be found in the following references , each of which is incor- 10 -HO + H + porated in their entireties: EP 1681303A1 (HASylated eryth ropoietin ); WO 2005 /014024 ( conjugates of a polymer and a protein linked by an oxime linking group ) ; W096 / 40662 (aminooxy - containing linker compounds and their applica protonated aniline + H tion in conjugates ); WO 2008 /025856 (Modified proteins ) ; 15 Schiff base 2 Peri F et al. , Tetrahedron 1998 , 54 , 12269 -78 ; Kubler- Kielb = J et . Pozsgay V . , J Org Chem 2005 , 70 , 6887 - 90 ; Lees A et al. , Vaccine 2006 , 24 ( 6 ) , 716 - 29 ; and Heredia K L et al. , Macromoecules 2007, 40 ( 14 ), 4772 - 9. In various embodiments of the invention , the water 20 - NH2 soluble polymer which is linked according to the aminooxy technology described herein to an oxidized carbohydrate moiety of a therapeutic protein ( e . g . , FVIII , FVIIa , or FIX ) N include , but are not limited to polyethylene glycol ( PEG ) , 25 ?? ?? HR branched PEG , polysialic acid ( PSA ) , carbohydrate , poly - aniline saccharides , pullulane, chitosan , hyaluronic acid , chondroi oxime ligation HR tin sulfate, dermatan sulfate, starch , dextran , carboxym product ethyl- dextran , polyalkylene oxide (PAO ) , polyalkylene glycol ( PAG ) , polypropylene glycol (PPG ) polyoxazoline , 30 However, considering the numerous health risks associ poly acryloylmorpholine, polyvinyl alcohol (PVA ) , polycar - ated with aniline , alternative catalysts are desirable . The boxylate, polyvinylpyrrolidone , polyphosphazene , polyox - present invention provides aniline derivatives as alternative azoline, polyethylene- co -maleic acid anhydride, polysty oxime ligation catalysts . Such aniline derivatives include , rene - co -maleic acid anhydride , poly ( 1 - but are not limited to , o -amino benzoic acid , m -amino hydroxymethylethylene hydroxymethylformal) (PHF ) , 35 benzoic acid , p -amino benzoic acid , sulfanilic acid , o - amin 2 -methacryloyloxy - 2 ' - ethyltrimethylammoniumphosphate obenzamide, o - toluidine , m - toluidine , p - toluidine , o -ani (MPC ). sidine, m - anisidine , and p -anisidine . Nucleophilic Catalysts In one embodiment of the invention , m - toluidine (aka As described herein , the conjugation of water soluble 40in meta - toluidine, m -methylaniline , 3 -methylaniline , or polymers to therapeutic proteins can be catalyzed by aniline . 3 - amino - 1 -methylbenzene ) is used to catalyze the conjuga tion reactions described herein . M - toluidine and aniline have Aniline strongly catalyzes aqueous reactions of aldehydes similar physical properties and essentially the same pKa and ketones with amines to form stable imines such as value (m - toluidine : pKa 4 .73 , aniline: pKa 4 .63 ) . hydrazones and oximes . The following diagram compareslidation 45 The nucleophilic catalysts of the invention are useful for an uncatalyzed versus the aniline -catalyzed oxime ligation 45 oxime ligation ( e . g , using aminooxy linkage ) or hydrazone reaction (Kohler J J , ChemBioChem 2009 ; 10 :2147 - 50 ) : formation ( e . g . , using hydrazide chemistry ) . In various embodiments of the invention , the nucleophilic catalyst is provided in the conjugation reaction at a concentration of 50 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 , HR 10 . 0 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 25 , 30 , 35 , 40 , 45 , or 50 mM . In one embodiment, the nucleophilic catalyst uncatalized aniline - catalized reaction is provided between 1 to 10 mM . In various embodiments of reaction 55 the invention , the pH range of conjugation reaction is 4 . 5 , 5 . 0 , 5 . 5 , 6 . 0 , 6 . 5 , 7 . 0 and 7 . 5 . In one embodiment, the pH is excess between 5 . 5 to 6 . 5 . Purification of Conjugated Proteins to- H HN In various embodiments , purification of a protein that has 60 been incubated with an oxidizing agent and /or a therapeutic HR " NH OH protein that has been conjugated with a water soluble HR polymer according to the present disclosure , is desired . Numerous purification techniques are known in the art and include , without limitation , chromatographic methods such \\ -H20 65 as ion - exchange chromatography , hydrophobic interaction chromatography, size exclusion chromatography and affinity chromatography or combinations thereof, filtration methods, US 10 , 414 , 793 B2 71 72 and precipitation methods (Guide to Protein Purification , in a two step organic reaction employing a modified Gabriel Meth . Enzymology Vol 463 (edited by Burgess R R and Synthesis of primary amines . In the first step one molecule Deutscher MP) , 2nd edition , Academic Press 2009 ). of hexaethylene glycol dichloride was reacted with two The following examples are not intended to be limiting molecules of Endo - N -hydroxy - 5 -norbornene - 2 , 3 -dicarboxi but only exemplary of specific embodiments of the inven - 5 mide in DMF. The desired homobifunctional product was tion . prepared from the resulting intermediate by hydrazinolysis in ethanol. EXAMPLES Example 4 Example 1 10 Preparation of the Homobifunctional Linker NH , Detailed Synthesis of the Aminooxy - PSA Reagent [OCH2CH2 ) 2ONH2 3 - oxa -pentane - 1 , 5 dioxyamine was synthesized according The homobifunctional linker NH2[ OCH2CH2) 2ONH2 15 to Botyryn et al ( Tetrahedron 1997 ; 53 :5485 - 92 ) in a two step organic synthesis as outlined in Example 1 . H2N - NH2 Step 1 : " O To a solution of Endo - N - hydroxy - 5 -norbonene - 2 , 3 - dicar boxiimide (59 . 0 g ; 1 .00 eq ) in 700 ml anhydrous N , N ( 3 -oxa - pentane - 1 , 5 - dioxyamine ) containing two active dimetylformamide anhydrous K2CO3 (45 .51 g ; 1 .00 eq ) and aminooxy groups was synthesized according to Boturyn et 2 ,2 -dichlorodiethylether (15 .84 ml; 0 .41 eq ) were added . al . ( Tetrahedron 1997 ; 53 : 5485 - 92 ) in a two step organic The reaction mixture was stirred for 22 h at 50° C . The reaction employing a modified Gabriel- Synthesis of primary mixture was evaporated to dryness under reduced pressure . amines (FIG . 3 ) . In the first step , one molecule of 2 , 2 - 25 The residue was suspended in 2 L dichloromethane and chlorodiethylether was reacted with twomolecules of Endo extracted two times with saturated aqueous NaCl- solution N -hydroxy - 5 - norbornene- 2 , 3 -dicarboximide in dimethyl ( each 1 L ). The Dichloromethane layer was dried over formamide (DMF ) . The desired homobifunctional product Na2SO4 and then evaporated to dryness under reduced was prepared from the resulting intermediate by pressure and dried in high vacuum to give 64 . 5 g of hydrazinolysis in ethanol. 3 - oxapentane - 1 , 5 - dioxy - endo - 2 ' , 3 ' - dicarboxydiimidenor 30 bornene as a white -yellow solid ( intermediate 1 ) . Example 2 Step 2 : To a solution of intermediate 1 (64 .25 g ; 1 .00 eq ) in 800 Preparation of the Homobifunctional Linker NH , ml anhydrous Ethanol , 31. 0 ml Hydrazine hydrate ( 4 . 26 eq ) [OCH2CH2 ]4ONH2 35 were added . The reaction mixture was then refluxed for 2 hrs . The mixture was concentrated to the half of the starting The homobifunctional linker NH [OCH , CH2] 4ONH , volume by evaporating the solvent under reduced pressure . The occurring precipitate was filtered off . The remaining ethanol layer was evaporated to dryness under reduced HNHNNA NH2 4040 Ppressure . The residue containing the crude product 3 - oxa pentane- 1 , 5 - dioxyamine was dried in vacuum to yield 46 . 3 ( 3 , 6 , 9 -trioxa - undecane - 1 , 11 - dioxyamine ) containing two g . The crude product was further purified by column chro active aminooxy groups was synthesized according to Botu matography ( Silicagel 60 ; isocratic elution with Dichlo ryn et al . ( Tetrahedron 1997 ; 53 : 5485 - 92 ) in a two step us romethane /Methanol mixture , 9 / 1 ) to yield 11 . 7 g of the pure organic reaction employing a modified Gabriel- Synthesis of 45 final product 3 -oxa -pentane - 1 , 5 - dioxyamine . primary amines (FIG . 3 ) . In the first step one molecule of Bis -( 2 -( 2 -chlorethoxy ) -ethyl ) - ether was reacted with two Example 5 molecules of Endo - N -hydroxy - 5 - norbornene - 2 , 3 - dicarboxi mide in DMF. The desired homobifunctional product was prepared from the resulting intermediate by hydrazinolysis 50 Preparation of Aminooxy -PSA in ethanol. 1000 mg of oxidized PSA (MW = 20 kD ) obtained from Example 3 the Serum Institute of India (Pune , India ) was dissolved in 16 ml 50 mM phosphate buffer pH 6 . 0 . Then 170 mg Preparation of the Homobifunctional Linker NH , 55 3 -oxa - pentane - 1 , 5 - dioxyamine was given to the reaction [OCH2CH2 ] ONH2 mixture . After shaking for 2 hrs at RT 78 . 5 mg sodium cyanoborohydride was added and the reaction was per The homobifunctional linker NH [OCH ,CH , ]6ONH , formed for 18 hours over night. The reaction mixture was

HN IL

( 3 ,6 , 9 ,12 , 15 - penatoxa -heptadecane - 1 , 17 -dioxyamine ) 65 then subjected to a ultrafiltration / diafiltration procedure containing two active aminooxy groups was synthesized (UF /DF ) using a membrane with a 5 kD cutoff made of according to Boturyn et al. ( Tetrahedron 1997 ; 53 : 5485 - 92 ) regenerated cellulose (50 cm ? , Millipore ) . US 10 ,414 ,793 B2 73 74 Example 6 mM phosphate buffer pH 6 .0 ( Bufffer A ). Then 94 mg 3 - oxa - pentane - 1 , 5 - dioxyamine is given to this solution . Preparation of Aminooxy -PSA Employing a Subsequently 2 . 3 ml of a 50 mM m -toluidine stock solution Chromatographic Purification Step are added to this reaction mixture. After shaking for 2 h at 1290 mg of oxidized PSA (MW = 20 kD ) obtained from 5 RT the mixture is then subjected to a weak anion exchange the Serum Institute of India ( Pune, India ) was dissolved in chromatography step employing a Fractogel EMD DEAE 25 ml 50 mM phosphate buffer pH 6 . 0 (Bufffer A ) . Then 209 650 - M chromatography gel (column dimension : XK16 / mg 3 -oxa - pentane - 1 , 5 - dioxyamine was given to the reaction 105) . The reaction mixture is diluted with 50 ml Buffer A mixture . After shaking for 1 h at RT 101 mg sodium and loaded onto the DEAE column pre - equilibrated with cyanoborohydride was added and the reaction was per 10 Buffer A at a flow rate of 1 cm /min . Then the column is formed for 3 hours . Then the mixture was then subjected to washed with 20 CV Buffer B ( 20 mM Hepes , pH 6 . 0 ) to a weak anion exchange chromatography step employing a remove free 3 - oxa -pentane - 1, 5 - dioxyamine and cyanide at a Fractogel EMD DEAE 650 - M chromatography ge ] ( column flow rate of 2 cm /min . The aminooxy - PSA reagent is the dimension : XK26 / 135 ). The reaction mixture was diluted eluted with a step gradient consisting of 67 % Buffer B and with 110 ml Buffer A and loaded onto the DEAE column 15 43 % Buffer C ( 20 mM Hepes, 1 M NaCl, pH 7 . 5 ) . The eluate pre - equilibrated with Buffer A at a flow rate of 1 cm /min . is concentrated by UF /DF using a 5 kD membrane made of Then the column was washed with 20 CV Buffer B ( 20 mM polyether sulfone (50 cm ? , Millipore ) . The final diafiltration Hepes, pH 6 . 0 ) to remove free 3 -oxa - pentane - 1, 5 -di - step is performed against Buffer D (20 mM Hepes, 90 mm oxyamine and cyanide at a flow rate of 2 cm /min . The NaCl, pH 7 . 4 ) . The preparation is analytically characterized aminooxy - PSA reagent was then eluted with a step gradient 20 by measuring total PSA (Resorcinol assay ) and total ami consisting of 67 % Buffer B and 43 % Buffer C (20 mM nooxy groups ( TNBS assay ) to determine the degree of Hepes, 1M NaC1, pH 7 . 5 ) . The eluate was concentrated by UF /DF using a 5 kD membrane made of polyether sulfone modification . Furthermore the polydispersity as well as free (50 cm , Millipore ). The final diafiltration step was per 3 -oxa - pentane - 1 , 5 - dioxyamine is determined . formed against Buffer D (20 mM Hepes , 90 mM NaCl, pH 25 7 . 4 ) . The preparation was analytically characterized by Example 9 measuring total PSA (Resorcinol assay ) and total aminooxy groups ( TNBS assay ) to determine the degree of modifica Preparation of Aminooxy - PSA Reagent tion . Furthermore the polydispersity as well as free 3 -oxa An Aminooxy — PSA reagent was prepared according to pentane - 1 , 5 -dioxyamine and cyanide was determined . 30 the Examples 4 - 8 . After diafiltration , the product was frozen Example 7 at - 80° C . and lyophilized . After lyophilization the reagent was dissolved in the appropriate volume of water and used Preparation of Aminooxy -PSA without a Reduction for preparation of PSA -protein conjugates via carbohydrate Step modification . 35 573 mg of oxidized PSA (MW = 20 kD ) obtained from the Example 10 Serum Institute of India (Pune , India ) was dissolved in 11 . 3 ml 50 mM phosphate buffer pH 6 .0 (Bufffer A ) . Then 94 mg Evaluation of the Efficacy of Different Alternative 3 -oxa - pentane - 1 , 5 -dioxyamine was given to the reaction Nucleophilic Catalysts mixture . After shaking for 5 h at RT the mixture was then 40 subjected to a weak anion exchange chromatography step rFIX was incubated with sodium periodate , aminooxy employing a Fractogel EMD DEAE 650 - M chromatography PSA reagent under standardized conditions ( 1 mg/ ml rFIX in gel ( column dimension : XK16 / 105 ). The reaction mixture 20 mM L - histidine, 150 mM NaC1, 5 mM CaCl ,, pH 6 .0 , was diluted with 50 ml Buffer A and loaded onto the DEAE column pre - equilibrated with Buffer A at a flow rate of 1 5 - fold molar aminooxy -PSA reagent excess, 100 um NaO2) cm /min . Then the column was washed with 20 CV Buffer B 45 using different nucleophilic catalysts (aniline , m - toluidine , ( 20 mM Hepes, pH 6 . 0 ) to remove free 3 - oxa -pentane - 1 , 5 0 - anisidine, m - anisidine, o - aminobenzoic acid , m - amin dioxyamine and cyanide at a flow rate of 2 cm /min . The obenzoic acid , p -aminobenzoic acid , p -aminobenzamide , aminooxy -PSA reagent was the eluted with a step gradient sulfanilic acid / standard concentration : 10 mM ) The reaction consisting of 67 % Buffer B and 43 % Buffer C ( 20 mM was carried out for 2 hrs in the dark at room temperature Henes . 1 M NaCl. pH 7 . 5 ) . The eluate was concentrated by 50 under gentle stirring and quenched for 15 min at room UF /DF using a 5 kd membrane made of polyether sulfone temperature by the addition of aqueous cysteine solution (50 cm , Millipore ). The final diafiltration step was per with a final concentration of 1 mM . formed against Buffer D (20 mM Hepes , 90 mM NaCl, pH The coupling efficiency was determined by SDS -PAGE 7 . 4 ) . The preparation was analytically characterized by using an Invitrogen X -cell mini system . Samples were measuring total PSA (Resorcinol assay ) and total aminooxy 55 spiked with lithium dodecyl sulfate (LDS ) buffer and dena groups ( TNBS assay ) to determine the degree of modifica - tured for 10 min at 70° C . Then the samples were applied on tion . Furthermore the polydispersity as well as free 3 - oxa 3 - 8 % TRIS -acetate gels and ran at 150 V for 60 min . pentane - 1 , 5 -dioxyamine was determined . Subsequently the gels were stained with Coomassie . In addition the samples were characterized by use of a Example 8 60 SEC -HPLC system using a Agilent 1200 HPLC system equipped with a Shodex KW 803 column under conditions Preparation of Aminooxy -PSA without a Reduction as previously described (Kolarich et al, Transfusion 2006 ; Step in the Presence of the Nucleophilic Catalyst 46 : 1959 -77 ). m - Toluidine 50 ul of samples were injected undiluted and eluted 65 isocratically with a 0 .22 m filtered solution of 20 mM 573 mg of oxidized PSA (MW = 20 kD ) obtained from the NaH2PO4 , 50 mM Na2SO4, pH 6 . 1 at a flow rate of 0 . 5 Serum Institute of India (Pune , India ) is dissolved in 9 ml50 ml/min . The elution pattern was recorded at 280 nm . US 10 ,414 , 793 B2 75 76 The results are summarized in FIGS. 5A - C and 6 (SDS Method 2 : PAGE) and Table 2 (SEC - HPLC results ) . The catalytic effect 12 . 3 mgrFIX is dissolved in L -histidine buffer, pH 6 . 0 (20 of the different preparations is demonstrated . It is shown that mM L -histidine , 150 mM NaC1, 5 mM CaCl2 ) to get a final the use of m -toluidine leads to equivalent results as obtained protein concentration of 1 mg rFIX /ml . A 5 mM aqueous with aniline. sodium periodate solution is added to get a final concentra tion of 100 uM and the reaction mixture is incubated for 1 TABLE 2 hour in the dark at 4° C . under gentle stirring at pH 6 . 0 and quenched for 15 min at room temperature by the addition of di- PSAylated mono free an 1 M aqueous L - cysteine solution ( or other quenching nucleophilic catalysts rFIX PSAylated rFIX rFIX 10 reagents ) to get a final concentration of 10 mM . Themixture no catalyst 4 . 5 % 24 . 9 % 70 . 6 % is subsequently subjected to UF /DF employing Vivaspin 10 mM aniline 47 . 7 % 33 .6 % 18 . 7 % 15R 10 kD centrifugal filtrators to remove excess periodate , 10 mM m -toluidine 31. 4 % 40 . 8 % 27 . 8 % quencher and the byproducts thereof . 10 mM o - aminobenzioc acid 30 . 9 % 38 . 5 % 30 . 6 % 10 mM m - aminobenzioc acid 27 . 6 % 38 . 0 % 34 . 4 % The obtained retentate ( 8 . 8 ml) , containing oxidized rFIX , 10 mM p -aminobenzioc acid 18 . 1 % 39 . 3 % 42 .6 % 15 is mixed with an aqueous m -toluidine solution (50 mM ) to 10 mM o -aminobenzamide 15 . 9 % 38 . 4 % 45 . 7 % give a final concentration of 10 mM and incubated for 30 10 mM sulfanilic acid 11 . 8 % 35 . 8 % 52 . 4 % min at room temperature . Then aminooxy - PSA reagent with a MW of 20 kD ( described above ) is added to give a 5 - fold molar reagent excess . This mixture was incubated at pH 6 . 0 Example 11 20 for 2 . 5 hours at room temperature ; 0 . 5 hours to 18 hours at + 4° C . ) in the dark under gentle stirring . Polysialylation of rFIX Using Aminooxy -PSA and The free rFIX is removed by means of anion exchange m - Toluidine as a Nucleophilic Catalyst chromatography (AEC ). The reaction mixture is diluted with appropriate amounts of Buffer A (50 mM Hepes, 5 mM Method 1 : 25 CaCl2 , pH 7 . 5 ) to correct the solutions conductivity and pH 12 . 3 mg rFIX was dissolved in 6 . 1 ml histidine buffer, pH prior to load onto a 20 ml HiPrep QFF 16 / 10 column (GE 6 . 0 ( 20 mM L -histidine , 150 mM NaC1, 5 mM CaC12 ) . 254 Healthcare , Fairfield , Conn . ) pre - equilibrated with buffer A . ulof an aqueous sodium periodate solution (5 mM ) was then Then the column is eluted with Buffer B (50 mM Hepes , 1 added and the reaction mixture is incubated for 1 h in the M NaC1, 5 mM CaCl2 , pH 7 . 5 ) . Free rFIX is eluted by a step dark at 4° C . under gentle stirring and quenched for 15 min 30 gradient using 25 % of Buffer B , which results in a conduc at room temperature by the addition of 6 .5 ul of a 1 M tivity between 12 - 25 mS /cm in the obtained fraction and the aqueous cysteine solution . The mixture was subsequently conjugate using a step gradient of 50 % Buffer B , which subjected to UF /DF employing Vivaspin 15R 10 kD cen results in a conductivity between 27 -45 mS/ cm in the trifugal filtrators to remove excess periodate , quencher and conjugate fraction . The conductivity of the conjugate con the byproducts thereof. 35 taining fraction is subsequently raised to 190 mS / cm with The retentate ( 8 . 8 ml) , containing oxidized rFIX was Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaCl2 , pH 6 . 9 mixed with 2 .46 ml of an aqueous m - toluidine solution (50 or by use of anti - chaotropic salts e . g . ammonium sulphate , mM ) and incubated for 30 min at room temperature . Then ammonium acetate etc . ) and loaded onto a 20 ml HiPrep aminooxy -PSA reagent with a MW of 20 kD ( described Butyl FF 16 / 10 column (GE Healthcare , Fairfield , Conn . or above ) was added to give a 5 - fold molar reagent excess . This 40 comparable HIC media ) pre - equilibrated with Buffer D ( 50 mixture was incubated for 2 . 5 h at RT in the dark under mM Hepes, 3 M NaCl, 5 mM CaCl2 , pH 6 . 9 ) . Free ami gentle stirring . nooxy -PSA reagent is washed out within 5 CV Buffer D . The free rFIX was removed by means of anion exchange Subsequently, the conjugate is eluted with 100 % Buffer E chromatography (AEC ) . The reaction mixture was diluted (50 mM Hepes, 5 mM CaCl2 , pH 7 . 4 ) . The conjugate with 15 ml Buffer A (50 mM Hepes , 5 mM CaC12 , pH 7 . 5 ) 45 containing fractions are concentrated by UF /DF using a 10 and loaded onto a 20 ml HiPrep QFF 16 / 10 column (GE KD membrane made of regenerated cellulose (88 cm2, Healthcare, Fairfield , Conn . ) pre -equilibrated with Buffer A . cutoff 10 kD , Millipore ). The final diafiltration step is The column was then eluted with Buffer B (50 mM Hepes, performed against L - histidine buffer, pH 7 . 2 containing 150 1 M NaCl, 5 mM CaCl2 , pH 7 . 5 ) . Free rFIX elutes at a mM NaCl and 5 mM CaC12 . The preparation is analytically conductivity between 12 - 25 mS /cm and the conjugate 50 characterized by measuring total protein ( Bradford and BCA between 27 - 45 mS/ cm . The conductivity of the conjugate procedure ) and FIX chromogenic - and clotting activity . For containing fractions was subsequently raised to 190 mS/ cm the PSA -rFIX conjugate a specific activity of > 50 % in with Buffer C (50 mM Hepes, 5M NaCl, 5 mM CaCl2 , pH comparison to native rFIX is determined . 6 . 9 ) and loaded onto a 20 ml HiPrep Butyl FF 16 / 10 column Method 3 : (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with Buf- 55 25 . 4 mg rFIX was dissolved in 18 . 7 ml histidine buffer, fer D ( 50 mM Hepes , 3 M NaC1, 5 mM CaC12 , pH 6 . 9 ) . Free pH 6 . 0 (20 mM L -histidine , 150 mM NaCl, 5 mM CaCl2 ) . aminooxy -PSA reagent was washed out within 5 CV Buffer 531 ul of an aqueous sodium periodate solution ( 5 mM ) and D . Subsequently the conjugate is eluted with 100 % Buffer E 5 .07 ml of an aqueous m - toluidine solution (50 mM ) were (50 mM Hepes , 5 mM CaC12 , pH 7 . 4 ) . The conjugate then added . Subsequently, the aminooxy -PSA reagent with a containing fractions were concentrated by UF /DF using 60 MW of 20 kD ( described above ) was added to give a 5 - fold Vivaspin 15R 10 kD centrifugal filtrator . The final diafiltra - molar reagent excess . The mixture was incubated for 2 h in tion step was performed against histidine buffer , pH 7 . 2 the dark at room temperature under gentle stirring and containing 150 mM NaCl and 5 mM CaC12 . The preparation quenched for 15 min at room temperature by the addition of was analytically characterized by measuring total protein 25 ul of 1 M aqueous cysteine solution . ( Bradford ) and FIX chromogenic activity. The PSA -rFIX 65 The free rFIX was removed by means of anion exchange conjugate showed a specific activity of > 50 % in comparison chromatography (AEC ) . The reaction mixture was diluted to native rFIX is determined . with 20 ml Buffer A (50 mM Hepes, 5 mM CaC12 , pH 7 . 5 ) US 10 , 414 , 793 B2 77 78 and loaded onto a 20 ml HiPrep QFF 16 / 10 column (GE mM Hepes , 5 mM CaC12 , pH 7 . 4 ) . The conjugate containing Healthcare, Fairfield , Conn . ) pre -equilibrated with Buffer A . fractions were concentrated by UF /DF using a 10 kD Then the column was eluted with Buffer B (50 mM Hepes , membrane made of regenerated cellulose (88 cm2, cutoff 10 1 M NaC1, 5 mM CaC12 , pH 7 . 5 ) . Free rFIX eluted at a kD , Millipore ) . The final diafiltration step was performed conductivity between 12 - 25 mS/ cm and the conjugate 5 against L -histidine buffer , pH 7 . 2 containing 150 mM NaCl between 27 - 45 mS/ cm . The conductivity of the conjugate and 5 mM CaC12 . The preparation was analytically charac containing fractions was subsequently raised to 190 mS/ cm terized by measuring total protein (Bradford and BCA with Buffer C (50 mM Hepes , 5 M NaCl, 5 mM CaC12 , pH procedure ) and FIX chromogenic - and clotting activity . For 6 . 9 ) and loaded onto a 20 ml HiPrep Butyl FF 16 / 10 column the PSA - rFIX conjugate a specific activity of > 50 % in (GE Healthcare, Fairfield , Conn .) pre -equilibrated with Buf- 10 comparison to native rFIX was determined . The conjugate fer D (50 mM Hepes , 3 M NaC1, 5 mM CaCl2 , pH 6 . 9 ) . Free was additionally analytically characterized by Size Exclu aminooxy -PSA reagent was washed out within 5 CV Buffer sion HPLC using a Agilent 1200 HPLC system equipped D . Subsequently , the conjugate was eluted with 100 % Buffer with a Shodex KW 803 column under conditions as previ E (50 mM Hepes , 5 mM CaC12 , pH 7 . 4 ) . The conjugate ously described (Kolarich et al , Transfusion 2006 ; 46 : 1959 containing fractions were concentrated by UF /DF using a 10 15 77 ) . It was shown that the preparation contains no free FIX . kD membrane made of regenerated cellulose (88 cm2, The conjugate consisted of 57 % mono -polysialylated and cutoff 10 kD , Millipore ) . The final diafiltration step was 31 % di -polysialylated and 12 % tri - polysialyated product. performed against histidine buffer , pH 7 . 2 containing 150 mM NaCl and 5 mM CaCl2 . The preparation was analyti Example 12 cally characterized by measuring total protein (Bradford ) 20 and FIX chromogenic activity . For the PSA - rFIX conjugate Polysialylation of rFVIII Using Aminooxy -PSA and a specific activity of > 50 % in comparison to native rFIX was m - Toluidine as a Nucleophilic Catalyst determined . The conjugate was additionally analytically characterized by Size Exclusion HPLC using a Agilent 1200 Method 1 : HPLC system equipped with a Shodex KW 803 column 25 50 mg rFVIII was transferred into reaction buffer ( 50 mM under conditions as previously described (Kolarich et al, Hepes, 350 mM sodium chloride , 5 mM calcium chloride, Transfusion 2006 ; 46 : 1959 -77 ) . It was shown that the prepa - pH 6 . 0 ) and diluted to obtain a protein concentration of 1 ration contains no free FIX . The conjugate consisted of 57 % mg/ ml . To this solution , Nalo , was added to give a final mono -polysialylated and 31 % di- polysialylated and 12 % concentration of 200 uM . The oxidation was carried at RT tri - polysialyated product . 30 for 30 min in the dark under gentle shaking . Then the Method 4 : reaction was quenched with cysteine ( final concentration : 10 25 . 4 mg rFIX was dissolved in L - histidine buffer, pH 6 . 0 mM ) for 60 min at RT. The solution was subjected to an IEX (20 mM L -histidine , 150 mM NaCl, 5 mM CaC12 ) to get a column with a volume of 20 ml (Merck EMD TMAE ( M ) ) final protein concentration of 2 mg rFIX /ml . Subsequently which was equilibrated with Buffer A ( 20 mM Hepes , 5 mM an 5 mM aqueous sodium periodate solution was added 35 CaCl2, pH 7 .0 ). The column was equilibrated with 5 CV within 15 minutes to give a final concentration of 100 uM , Buffer A . Then the oxidized rFVIII was eluted with Buffer followed by addition of an 50 mM aqueous m - toluidine B (20 mM Hepes, 5 mM CaC1, , IM NaCl, pH 7 . 0 ) . The solution to get a final concentration of 10 mM within a time r FVIII containing fractions were collected . The protein period of 30 minutes. Then the aminooxy - PSA reagent with content was determined ( Coomassie , Bradford ) and adjusted a MW of 20 kD ( described above ) was added to give a 5 - fold 40 to 1 mg/ ml with reaction buffer and adjusted to pH 6 . 0 by molar reagent excess . After correction of the pH to 6 . 0 the dropwise addition of 0 . 5 M HCl. Then a 50 - fold molar mixture was incubated for 2 h in the dark at room tempera - excess of a aminooxy - PSA reagent with a MW of 20 kD ture under gentle stirring and quenched for 15 min at room (described above ) was added followed by m - toluidine as a temperature by the addition of a 1 M aqueous L - cysteine nucleophilic catalyst ( final concentration : 10 mM ) . The solution to give a final concentration of 10 mM . 45 coupling reaction was performed for 2 hours in the dark The free rFIX was removed by means of ion exchange under gentle shaking at room temperature . The excess of chromatography (IEC ) . The reaction mixture was diluted aminooxy - PSA reagent was removed by means of HIC . The with appropriate amounts of Buffer A (50 mM Hepes , 5 mM conductivity of the reaction mixture was raised to 130 CaC12 , pH 7 . 5 ) to correct the solutions conductivity and pH mS /cm by adding a buffer containing ammonium acetate ( 50 value prior to load onto a 20 ml HiPrep QFF 16 / 10 column 50 mM Hepes , 350 mM sodium chloride, 5 mM calcium (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with Buf - chloride , 8 M ammonium acetate , pH 6 . 9 ) and loaded onto fer A . Then the column was eluted with Buffer B (50 mM a column filled with 80 ml Phenyl Sepharose FF (GE Hepes , 1 M NaCl, 5 mM CaC12 , pH 7 . 5 ) . Free rFIX was Healthcare , Fairfield , Conn .) pre - equilibrated with 50 mM eluted by a step gradient using 25 % of Buffer B , which Hepes , 2 . 5 M ammonium acetate , 350 mM sodium chloride , results in a conductivity between 12 - 25 mS/ cm in the 55 5 mM calcium chloride, pH 6 . 9 . Subsequently , the conjugate obtained fraction and the conjugate using a step gradient of was eluted with 50 mM Hepes buffer pH 7 . 5 containing 5 50 % Buffer B , which results in a conductivity between mM CaCl, . Finally, the PSA - rFVIII containing fractions 27 -45 mS/ cm in the conjugate fraction . The conductivity of were collected and subjected to UF /DF by use of a 30 kD the conjugate containing fraction was subsequently raised to membrane made of regenerated cellulose (88 cm ? , Milli 190 mS/ cm with Buffer C (50 mM Hepes , 5 M NaC1, 5 mM 60 pore ) . The preparation was analytically characterized by CaCl2 , pH 6 . 9 ; by use of anti -chaotropic salts e . g . ammo measuring total protein (Coomassie , Bradford ) and FVIII nium acetate ) and loaded onto a 20 ml HiPrep Butyl FF chromogenic activity . The PSA - rFVIII conjugate showed a 16 / 10 column (GE Healthcare , Fairfield , Conn . ; or compa - specific activity of > 70 % in comparison to native rFVIII was rable HIC media ) pre - equilibrated with Buffer D (50 mM determined . Hepes , 3 M NaC1, 5 mM CaC12 , pH 6 . 9 ) . Free aminooxy - 65 Method 2 : PSA reagent was washed out within 5 CV Buffer D . Sub - 58 mg of recombinant factor VIII (rFVIII ) derived from sequently the conjugate was eluted with 100 % Buffer E (50 the ADVATE process in Hepes buffer (50 mM HEPES , ~ 350 US 10 , 414 , 793 B2 79 80 mM sodium chloride , 5 mM calcium chloride , 0 . 1 % Poly - Finally the purified conjugate is concentrated by ultra -/ sorbate 80 , pH 7 .4 ) is dissolved in reaction buffer (50 mM diafiltration (UF /DF ) using a membrane made of regener Hepes, 350 mM sodium chloride , 5 mM calcium chloride , ated cellulose with a molecular weight cut off 30 kD (88 pH 6 . 0 ) to get a final protein concentration of 1 . 0 + / - 0 . 25 cm ? , Millipore ). mg/ ml . Then the pH of the solution is corrected to 6 . 0 by 5 The conjugate prepared by use of this procedure are drop wise addition of a 0 . 5 N aqueous HCl solution . Sub analytically characterized by measuring total protein , FVIII sequently , a 40 mM aqueous sodium periodate solution is chromogenic activity and determination of the polysialya added within 10 minutes to give a concentration of 200 uM . tion degree by measuring the PSA content ( resorcinol The oxidation reaction is carried out for 30 + / – 5 min at a assay ). For the conjugate obtained a specific activity > 50 % temperature ( T ) of T = + 22 + / - 2° C . Then the reaction is " and a PSA degree > 5 .0 is calculated . stopped by addition of an aqueous L -cysteine solution ( 1 M ) Method 3 : within 15 minutes at T = + 22 + / - 2° C . to give a final concen - 50 mg rFVIII was transferred into reaction buffer ( 50 mM tration of 10 mM in the reaction mixture and incubation for Hepes, 350 mM sodium chloride, 5 mM calcium chloride , 60 + / - 5 min . 16 pH 6 . 0 ) and diluted to obtain a protein concentration of 1 The oxidized rFVIII is further purified by anion exchange mg/ ml . A 50 - fold molar excess of aminooxy -PSA reagent chromatography on EMD TMAE (M ) (Merck ). The mixture with a MW of 20 kD (described above ) was added followed is diluted with Buffer A (20 mM Hepes, 5 mM CaCl2 , pH by m - toluidine as a nucleophilic catalyst (final concentra 6 . 5 ) to give a conductivity of 5 ms/ cm . This solution is tion : 10 mM ) and NaI04 ( final concentration : 400 UM ) . The loaded onto the IEX column ( bed height: 5 . 4 cm ) with a 20 coupling reaction was performed for 2 hours in the dark column volume of 10 ml using a flow rate of 1. 5 cm /min . under gentle shaking at room temperature . Subsequently , the This column is subsequently washed ( flow rate : 1 . 5 cm /min ) reaction was quenched with cysteine for 60 min at RT ( final with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and Buffer concentration : 10 mM ) . Then the conductivity of the reac B (20 mM Hepes , 5 mM CaCl ,, 1 . 0 M NaCl, pH 7 . 0 ) . Then tion mixture was raised to 130 mS / cm by adding a buffer the oxidized rFVIII is eluted with a 50 :50 ( w /w ) mixture of 25 containing ammonium acetate (50 mM Hepes , 350 mm Buffer A and Buffer B followed by a postelution step with 5 sodium chloride , 5 mM calcium chloride, 8 M ammonium CV of Buffer B . The elution steps are carried out by use of acetate , pH 6 . 9 ) and loaded onto a column filled with 80 ml a flow rate of 1 . 0 cm /min . Phenyl Sepharose FF (GE Healthcare , Fairfield , Conn .) Subsequently , the aminooxy -polysialic acid (PSA -ONH2 ) pre -equilibrated with 50 mM Hepes, 2 .5 M ammonium reagent is added in a 50 - fold molar excess to the eluate 30 containing the purified oxidized rFVIII within a maximum acetate , 350 mM sodium chloride , 5 mM calcium chloride , time period ( t ) of 15 minutes under gentle stirring . Then an 0 .01 % Tween 80 , pH 6 . 9 . Subsequently , the conjugate was aqueous m - toluidine solution ( 50 mM ) is added within 15 eluted with 50 mM Hepes, 5 mM calcium chloride , pH 7 . 5 . minutes to get a final concentration of 10 mM . The reaction Finally , the PSA -rFVIII containing fractions were collected mixture is incubated for 120 + / – 10 min . in the dark at a 3525 and subjected to UF /DF by use of a 30 kD membrane made temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . of regenerated cellulose ( 88 cm ? , Millipore ). The prepara The obtained PSA - rFVIII conjugate is purified by Hydro tion was analytically characterized by measuring total pro phobic Interaction Chromatography (HIC ) using a Phenyl tein ( Bradford ) and FVIII chromogenic activity . For the Sepharose FF low sub resin (GE Healthcare ) packed into a PSA - rFVIII conjugate a specific activity of > 70 % in com column manufactured by GE Healthcare with a bed height 40 parison to native rFVIII was determined . ( h ) of 15 cm and a resulting column volume (CV ) of 81 ml. Method 4 : The reaction mixture is spiked with ammonium acetate by 50 mg recombinant factor VIII ( rFVIII ) derived from the addition of 50 mM Hepes buffer , containing 350 mM sodium ADVATE process in 50 mM Hepes buffer (50 mM HEPES , chloride, 8 M ammonium acetate , 5 mM calcium chloride , ~ 350 mM sodium chloride, 5 mM calcium chloride, 0 . 1 % pH 6 . 9 . Two volumes of the reaction mixture are mixed with 45 Polysorbate 80 , pH 7 . 4 ) was dissolved in reaction buffer (50 1 volume of the ammonium acetate containing buffer system mM Hepes , 350 mM sodium chloride, 5 mM calcium and the pH value is corrected to pH 6 . 9 by drop wise chloride , pH 6 . 0 ) to get a final protein concentration of addition of a 0 . 5 N aqueous NaOH solution . This mixture is 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution was cor loaded onto the HIC column at flow rate of 1 cm /min rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 followed by a washing step using > 3 CV equilibration buffer 50 solution . (50 mM Hepes , 350 mM sodium chloride , 2 . 5 M ammonium Subsequently , the aminooxy - polysialic acid ( PSA -ONH2 ) acetate , 5 mM calcium chloride , pH 6 . 9 ). reagent was added in a 50 - fold molar excess to this rFVIII For removal of reaction by - products and anti- chaotropic solution within a maximum time period ( t) of 15 minutes salt a second washing step is performed with > 5 CV washing under gentle stirring . Then an aqueous m - toluidine solution buffer 1 ( 50 mM Hepes , 3 M sodium chloride , 5 mM 55 (50 mM ) was added within 15 minutes to get a final calcium chloride , pH 6 . 9 ) in upflow mode at a flow rate of concentration of 10 mM . Finally , a 40 mM aqueous sodium 2 cm /min . Then elution of purified PSA -rFVIII conjugate is periodate solution was added to give a concentration of 400 performed in down flow mode using a step gradient of 40 % UM . washing buffer 2 (50 mM Hepes , 1 . 5 M sodium chloride , 5 The reaction mixture was incubated for 120 + / – 10 min . in mM calcium chloride , pH 6 . 9 ) and 60 % elution buffer (20 60 the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle mM Hepes , 5 mM calcium chloride , pH 7 . 5 ) at a flow rate shaking. Then the reaction was stopped by the addition of an of 1 cm /min . The elution of the PSA -rFVIII conjugate is aqueous L -cysteine solution ( 1 M ) to give a final concen monitored at UV 280 nm and the eluate containing the tration of 10 mM in the reaction mixture and incubation for conjugate is collected within < 4 CV . The post elution step is 60 + / - 5 min . performed with > 3 CV elution buffer under the same con - 65 The obtained PSA - rFVIII conjugate was purified by ditions to separate minor and /or non modified rFVIII from Hydrophobic Interaction Chromatography (HIC ) using a the main product . Phenyl Sepharose FF low sub resin (GE Healthcare ) packed US 10 ,414 ,793 B2 81 82 into a column manufactured by GE Healthcare with a bed The retentate (10 . 9 ml) , containing oxidized rFVIII , is height ( h ) of 15 cm and a resulting column volume (CV ) of mixed with 2 . 94 ml of an aqueous m - toluidine solution ( 50 81 ml. mM ) and incubated for 30 min at room temperature . Then The reaction mixture was spiked with ammonium acetate aminooxy -PEG reagent with a MW of 20 kD is added to by addition of 50 mM Hepes buffer , containing 350 mM 5 give a 5 -fold molar reagent excess. This mixture was incu sodium chloride, 8 M ammonium acetate , 5 mM calcium bated for 2. 5 h at room temperature in the dark under gentle chloride , pH 6 .9 . Two volumes of the reaction mixture was stirringFinally : , the PEG -rFVIII conjugate is purified by ion mixed with 1 volume of the ammonium acetate containing exchange chromatography on Sepharose FF. 1 . 5 mg buffer system and the pH value was corrected to pH 6 . 9 by 10 protein /ml gel is loaded on the column equilibrated with 50 drop wise addition of an 0 . 5 N aqueous NaOH solution . This mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The mixture was loaded onto the HIC column using a flow rate conjugate is eluted with 50 mM Hepes buffer containing 5 of 1 cm /min followed by a washing step using > 3 CV mM CaCl2 and 500 mM sodium chloride, pH 7 .4 and is then equilibration buffer (50 mM Hepes , 350 mM sodium chlo subjected to UF /DF using a 30 kD membrane (50 cm2, ride , 2 . 5 M ammonium acetate , 5 mM calcium chloride , pH 15 Millipore ). The preparation is analytically characterized by 6 . 9 ) . measuring total protein (Coomassie , Bradford ) and FVIII For removal of reaction by -products and anti -chaotropic chromogenic activity . It is expected that the PEG - rFVIII salt a second washing step was performed with > 5 CV conjugate will demonstrate a specific activity of > 70 % in washing buffer 1 (50 mM Hepes , 3 M sodium chloride, 5 comparison to native rFVIII was determined . mM calcium chloride, pH 6 . 9 ) in upflow mode at a flow rate 20 Method 2 : of 2 cm /min . Then elution of purified rFVIII conjugate was rFVIII is PEGylated by use of a linear 20 kD PEGylation performed in down flow mode using a step gradient of 40 % reagent containing an aminooxy group . An example of this washing buffer 2 (50 mM Hepes, 1 . 5 M sodium chloride , 5 type of reagent is the Sunbright® CA series from NOF (NOF mM calcium chloride , pH 6 . 9 ) and 60 % elution buffer (20 Corp ., Tokyo , Japan ) . A starting weight or concentration of mM Hepes , 5 mM calcium chloride, pH 7 . 5 ) at a flow rate 25 rFVIII is dissolved in or transferred to a reaction buffer (50 of 1 cm /min . The elution of the PSA - rFVIII conjugate was mM Hepes , 350 mM sodium chloride , 5 mM calcium monitored at UV 280 nm and the eluate containing the chloride , pH 6 . 0 ) to get a final protein concentration of conjugate was collected within < 4 CV . The post elution step 1. 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected was performed with > 3 CV elution buffer under the same to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 conditions to separate minor and /or non modified rFVIII 30 solution . Subsequently a 40 mM aqueous sodium periodate from the main product . solution is added within 10 minutes to give a concentration Finally, the purified conjugate was concentrated by ultra - / of 200 UM . The oxidation reaction is carried out for 30 + / - 5 diafiltration ( UF/ DF ) using a membrane made of regener- min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction ated cellulose with a molecular weight cut off 30 kD (88 is stopped by addition of an aqueous L -cysteine solution ( 1 cm2, Millipore ) . 35 . M ) within 15 minutes at T = + 22 + / - 2° C . to give a final The conjugates prepared by use of this procedure were concentration of 10 mM in the reaction mixture and incu analytically characterized by measuring total protein , FVIII bation for 60 + / - 5 min . chromogenic activity and determination of the polysialya - The oxidized rFVIII is further purified by anion exchange tion degree by measuring the PSA content ( resorcinol chromatography on EMD TMAE ( M ) (Merck ) . The mixture assay ). 40 is diluted with Buffer A ( 20 mM Hepes , 5 mM CaC12 , pH Analytical data (mean of 6 consecutive batches ): 6 . 5 ) to give a conductivity of 5 ms/ cm . This solution is Process yield (Bradford ) : 58 . 9 % loaded onto the IEX column (bed height: 5 . 4 cm ) with a Process yield ( FVIII chrom . ) : 46 . 4 % column volume of 10 ml using a flow rate of 1 . 5 cm /min . Specific activity : ( FVIII chrom ./ mg protein ) : 4148 IU /mg This column is subsequently washed ( flow rate : 1 . 5 cm /min ) Specific activity ( % of starting material) : 79 .9 % 45 with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and Buffer PSA degree (mol / mol ) : 8 . 1 B (20 mM Hepes , 5 mM CaC12 , 1 . 0 M NaCl, pH 7 . 0 ) . Then the oxidized rFVIII is eluted with a 50 :50 ( w / w ) mixture of Example 13 Buffer A and Buffer B followed by a postelution step with 5 CV of Buffer B . The elution steps are carried out by use of PEGylation of r FVIII Using an Aminooxy - PEG 50 a flow rate of 1 . 0 cm /min . Reagent and m - Toluidine as a Nucleophilic Subsequently, the aminooxy - PEG reagent with a MW of Catalyst 20 kD reagent is added in a 50 -fold molar excess to the eluate containing the purified oxidized rFVIII within a Method 1 : maximum time period (t ) of 15 minutes under gentle stirring . rFVIII is PEGylated by use of a linear 20 kD PEGylation 55 Then an aqueous m - toluidine solution (50 mM ) is added reagent containing an aminooxy group . An example of this within 15 minutes to get a final concentration of 10 mM . The type of reagent is the Sunbright® CA series from NOF (NOF reaction mixture is incubated for 120 + / - 10 min . in the dark Corp ., Tokyo , Japan ). 14 . 7 mg rFVIII is dissolved in 7 . 0 ml at a temperature ( T ) of T = + 22 + /- 2° C . under gentle shaking . histidine buffer , pH 6 . 0 ( 20 mM L - histidine , 150 mM NaCl, The obtained PEG - rFVIII conjugate is purified by Hydro 5 mM CaC12 ) . Then 296 ul of an aqueous sodium periodate 60 phobic Interaction Chromatography (HIC ) using a Phenyl solution (5 mM ) is added and the reaction mixture is Sepharose FF low sub resin (GE Healthcare ) packed into a incubated for 1 h in the dark at 4° C . under gentle stirring column manufactured by GE Healthcare with a bed height and quenched for 15 min at room temperature by the ( h ) of 15 cm and a resulting column volume (CV ) of 81 ml. addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The T he reaction mixture is spiked with ammonium acetate by mixture was subsequently subjected to UF/ DF employing 65 addition of 50 mM Hepes buffer , containing 350 mM sodium Vivaspin 15R 10 kD centrifugal filtrators to remove excess chloride , 8 M ammonium acetate , 5 mM calcium chloride , periodate , quencher and the byproducts thereof. pH 6 . 9 . Two volumes of the reaction mixture are mixed with US 10 ,414 , 793 B2 83 84 1 volume of the ammonium acetate containing buffer system aminooxy -PEG reagent with a MW of 20 kD (described and the pH value is corrected to pH 6 . 9 by drop wise above ) is added to give a 20 - fold molar excess . After addition of a 0 . 5 N aqueous NaOH solution . This mixture is correction of the pH to 6 . 0 the mixture is incubated for 2 h loaded onto the HIC column using a flow rate of 1 cm /min in the dark at room temperature under gentle stirring and followed by a washing step using > 3 CV equilibration buffer 5 quenched for 15 min at room temperature by the addition of ( 50 mM Hepes , 350 mM sodium chloride , 2 . 5 M ammonium a 1 M aqueous L - cysteine solution to give a final concen acetate , 5 mM calcium chloride , pH 6 . 9 ) . tration of 10 mM . For removal of reaction by - products and anti -chaotropic The free rFVIII is removed by means of ion exchange salt a second washing step is performed with > 5 CV washing chromatography ( IEC ) . The reaction mixture was diluted buffer 1 (50 mM Hepes , 3 M sodium chloride , 5 mM 10 with appropriate amounts of Buffer A (50 mM Hepes, 5 mM calcium chloride , pH 6 .9 ) in upflow mode at a flow rate of CaC12 , pH 7 . 5 ) to correct the solutions conductivity and pH 2 cm /min . Then elution of purified rFVIII conjugate is value prior to load onto a 20 ml HiPrep QFF 16 / 10 column performed in down flow mode using a step gradient of 40 % GE( Healthcare , Fairfield , Conn . ) pre - equilibrated with Buf washing buffer 2 (50 mM Hepes, 1 . 5 M sodium chloride , 5 fer A . Then the column was eluted with Buffer B (50 mM mM calcium chloride , pH 6 . 9 ) and 60 % elution buffer ( 20 15 Hepes , 1 M NaC1, 5 mM CaC12 , pH 7 . 5 ) . Free rFVIII was mM Hepes , 5 mM calcium chloride , pH 7 . 5 ) at a flow rate eluted by a step gradient using 25 % of Buffer B , which of 1 cm /min . The elution of the PEG -rFVIII conjugate is results in a conductivity between 12 -25 mS/ cm in the monitored at UV 280 nm and the eluate containing the obtained fraction and the conjugate using a step gradient of conjugate is collected within < 4 CV . The post elution step is 50 % Buffer B , which results in a conductivity between performed with > 3 CV elution buffer under the same con - 20 27 - 45 mS / cm in the conjugate fraction . The conductivity of ditions to separate minor and /or non modified rFVIII from the conjugate containing fraction is subsequently raised with the main product . Buffer C (50 mM Hepes , 5 M NaC1, 5 mM CaC12 , pH 6 . 9 ; Finally , the purified conjugate is concentrated by ultra - / by use of anti- chaotropic salts e . g . ammonium acetate , diafiltration (UF /DF ) using a membrane made of regener - ammonium sulphate etc . ) and loaded onto a 20 ml HiPrep ated cellulose with a molecular weight cut off 30 kD 25 Butyl FF 16 / 10 column (GE Healthcare , Fairfield , Conn .; or (Millipore ). comparable HIC media ) pre - equilibrated with Buffer D (50 The conjugate prepared by use of this procedure are mM Hepes , 3 M NaC1, 5 mM CaCl2 , pH 6 . 9 ) . Free PEG analytically characterized by measuring total protein and reagent was washed out within 5 CV Buffer D . Subse biological activity according to methods known in the art . quently , the conjugate was eluted with 100 % Buffer E ( 50 Method 3 : 30 mM Hepes, 5 mM CaC12 , pH 7 . 4 ) . The conjugate containing rFVIII is PEGylated by use of a linear 20 kD PEGylation fractions are concentrated by UF /DF using a 10 kD mem reagent containing an aminooxy group . An example of this brane made of regenerated cellulose (88 cm2, cutoff 10 kD , type of reagent is the Sunbright @ CA series from NOF Millipore ). The final diafiltration step is performed against (NOF Corp ., Tokyo , Japan ) . 7 .84 mg rFVIII , dissolved in 6 Hepes buffer (50 mM Hepes , 5 mM CaCl2 , pH 7 .5 ) . ml Hepes buffer (50 mM Hepes , 150 mM sodium chloride , 35 The preparation is analytically characterized by measur 5 mM calcium chloride , pH 6 . 0 ) are mixed with 314 ul of an ing total protein ( Bradford and BCA procedure ) and bio aqueous sodium periodate solution ( 10 mM ), and 1 .57 ml of logical activity according to known methods. an aqueous m - toluidine solution ( 50 mm ). Subsequently the aminooxy reagent is added to give a 20 - fold molar reagent Example 14 excess . The mixture is incubated for 2 h in the dark at room 40 temperature under gentle stirring and quenched for 15 min Polysialylation of rFVIIa Using Aminooxy - PSA at room temperature by the addition of 8 ul of aqueous and m - Toluidine as a Nucleophilic Catalyst cysteine solution ( 1 M ) . Finally the PEG -rFVIII conjugate is purified by ion Method 1 : exchange chromatography on Q - Sepharose FF. 1. 5 mg pro - 45 A starting concentration or weight of recombinant factor tein /ml gel is loaded on the column pre equilibrated with 50 VIIa (rFVIIa ) is transferred or dissolved in reaction buffer mM Hepes buffer , pH 7 .4 containing 5 mM CaCl2 . The (50 mM Hepes , 350 mM sodium chloride, 5 mM calcium conjugate is eluted with 50 mM Hepes buffer containing 5 chloride , pH 6 . 0 ) to get a final protein concentration of mM CaCl, and 500 mM sodium chloride , pH 7 . 4 and is then 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is corrected subjected to UF /DF using a 30 kD membrane (88 cm " , 50 to 6 . 0 by drop wise addition of a 0 . 5 N aqueous NaOH Millipore ) . The analytical characterization of the conjugate solution . Subsequently, a 40 mM aqueous sodium periodate by FVIII chromogenic assay and determination of total solution is added within 10 minutes to give a concentration protein (Bradford ) shows a specific activity of >60 % com of 50 ?M . The oxidation reaction is carried out for 30 + / - 5 pared to the rFVIII starting material . min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction Method 4 : 55 is stopped by addition of an aqueous L -cysteine solution ( 1 rFVIII is PEGylated by use of a linear 20 kD PEGylation M ) within 15 minutes at T = + 22 + / - 2° C . to give a final reagent containing an aminooxy group . An example of this concentration of 10 mM in the reaction mixture and incu type of reagent is the Sunbright® CA series from NOF (NOF bation for 60 + / – 5 min . Corp . , Tokyo , Japan ) . An initial concentration or weight of The oxidized rFVIIa is further purified by anion exchange rFVIII is transferred or dissolved in Hepes buffer (50 mM 60 chromatography on EMD TMAE ( M ) (Merck ) . The mixture Hepes , 150 mM sodium chloride, 5 mM calcium chloride , is diluted with Buffer A (20 mM Hepes, 5 mM CaCl2 , pH pH 6 . 0 ) to get a final protein concentration of 2 mg rFVIII/ 6 . 5 ) to give a conductivity of 5 ms/ cm . This solution is ml. Subsequently , an 5 mM aqueous sodium periodate loaded onto the IEX column (bed height: 5 .4 cm ) with a solution is added within 15 minutes to give a final concen - column volume of 10 ml using a flow rate of 1 . 5 cm /min . tration of 100 UM , followed by addition of an 50 mM 65 This column is subsequently washed ( flow rate : 1 . 5 cm /min ) aqueous m - toluidine solution to get a final concentration of with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and Buffer 10 mM within a time period of 30 minutes . Then the B (20 mM Hepes , 5 mM CaCl2 , 1 . 0 M NaCl, pH 7 . 0 ) . Then US 10 , 414 , 793 B2 85 86 the oxidized rFVIIa is eluted with a 50 : 50 ( w / w ) mixture of shaking. Then the reaction is stopped by the addition of an Buffer A and Buffer B followed by a postelution step with 5 aqueous L -cysteine solution ( 1 M ) to give a final concen CV of Buffer B . The elution steps are carried out by use of tration of 10 mM in the reaction mixture and incubation for a flow rate of 1. 0 cm /min . 60 + / - 5 min . Subsequently , the aminooxy -polysialic acid (PSA -ONH2 ) 5 The obtained PSA -rFVIIa conjugate is purified by Hydro reagent is added in a 50 - fold molar excess to the eluate phobic Interaction Chromatography (HIC ) using a Phenyl containing the purified oxidized rFVIIa within a maximum Sepharose FF low sub resin (GE Healthcare ) packed into a time period ( t ) of 15 minutes under gentle stirring . Then an aqueous m - toluidine solution (50 mM ) is added within 15 column manufactured by GE Healthcare with a bed height minutes to get a final concentration of 10 mM . The reaction 10 ( h ) of 15 cm and a resulting column volume (CV ) of 81 ml. mixture is incubated for 120 + / – 10 min . in the dark at a The reaction mixture is spiked with ammonium acetate by temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . addition of 50 mM Hepes buffer, containing 350 mM sodium The obtained PSA - rFVIIa conjugate is purified by Hydro chloride , 8 M ammonium acetate , 5 mM calcium chloride , phobic Interaction Chromatography (HIC ) using a Phenyl pH 6 . 9 . Two volumes of the reaction mixture is mixed with Sepharose FF low sub resin (GE Healthcare ) packed into a 1515 lT volume of the ammonium acetate containing buffer system column manufactured by GE Healthcare with a bed height and the pH value is corrected to pH 6 . 9 by drop wise ( h ) of 15 cm and a resulting column volume (CV ) of 81 ml. addition of an 0 .5 N aqueous NaOH solution . This mixture The reaction mixture is spiked with ammonium acetate by is loaded onto the HIC column using a flow rate of 1 cm /min addition of 50 mM Hepes buffer, containing 350 mM sodium followed by a washing step using > 3 CV equilibration buffer chloride , 8 M ammonium acetate , 5 mM calcium chloride , 20 (50 mM Hepes , 350 mM sodium chloride, 2 .5 M ammonium pH 6 . 9 . Two volumes of the reaction mixture are mixed with acetate , 5 mM calcium chloride, pH 6 . 9 ) . 1 volume of the ammonium acetate containing buffer system For removal of reaction by -products and anti -chaotropic and the pH value is corrected to pH 6 . 9 by drop wise salt a second washing step is performed with > 5 CV washing addition of a 0 . 5 N aqueous NaOH solution . This mixture is buffer 1 (50 mM Hepes, 3 M sodium chloride , 5 mM loaded onto the HIC column using a flow rate of 1 cm /min 25 calcium chloride , pH 6 . 9 ) in upflow mode at a flow rate of followed by a washing step using > 3 CV equilibration buffer 2 cm /min . Then elution of purified rFVIIa conjugate is ( 50 mM Hepes, 350 mM sodium chloride, 2 . 5 M ammonium performed in down flow mode using a step gradient of 40 % acetate , 5 mM calcium chloride, pH 6 .9 ) . washing buffer 2 (50 mM Hepes, 1. 5 M sodium chloride, 5 For removal of reaction by -products and anti- chaotropic mM calcium chloride, pH 6 . 9 ) and 60 % elution buffer ( 20 salt a second washing step is performed with > 5 CV washing 30 mM Hepes, 5 mM calcium chloride , pH 7 . 5 ) at a flow rate buffer 1 (50 mM Hepes, 3 M sodium chloride , 5 mM calcium chloride , pH 6 . 9 ) in upflow mode at a flow rate of of 1 cm /min . The elution of the PSA - rFVIIa conjugate is 2 cm /min . Then elution of purified rFVIIa conjugate is monitored at UV 280 nm and the eluate containing the performed in down flow mode using a step gradient of 40 % conjugate was collected within < 4 CV . The post elution step washing buffer 2 ( 50 mM Hepes , 1 . 5 M sodium chloridees, 5 35 15is performedI with > 3 CV elution buffer under the same mM calcium chloride, pH 6 .9 ) and 60 % elution buffer (20 conditions to separate minor and /or non modified rFVIII mM Hepes , 5 mM calcium chloride , pH 7 . 5 ) at a flow rate from the main product . of 1 cm /min . The elution of the PSA -rFVIIa conjugate is Finally , the purified conjugate is concentrated by ultra - / monitored at UV 280 nm and the eluate containing the diafiltration (UF /DF ) using a membrane made of regener conjugate is collected within < 4 CV. The post elution step is 40 ated cellulose (Millipore ) . performed with > 3 CV elution buffer under the same con The conjugates prepared by use of this procedure are ditions to separate minor and / or non modified rFVIIa from analytically characterized by measuring total protein , bio the main product. logical activity according to methods known in the art, and Finally , the purified conjugate is concentrated by ultra - / determination of the polysialyation degree by measuring the diafiltration (UF / DF ) using a membrane made of regener - 45 PSA content (resorcinol assay ) . ated cellulose with an appropriate molecular weight cut off ( e. g . 10 kD MWCO , 88 cm², Millipore ). Example 15 The conjugate prepared by use of this procedure is analytically characterized by measuring total protein , bio PEGylation of rFIX Using an Aminooxy - PEG logical activity , and determination of the polysialyation 50 Reagent and m - Toluidine as a Nucleophilic degree by measuring the PSA content ( resorcinol assay ) . Catalyst Method 2 : A starting weight or concentration of rFVIIa is dissolved Method 1 : in or transferred to a reaction buffer (50 mM Hepes, 350 mM r FIX is PEGylated by use of a linear 20 kD PEGylation sodium chloride, 5 mM calcium chloride, pH 6 . 0 ) to get a 55 reagent containing an aminooxy group . An example of this final protein concentration of 1 . 0 + / - 0 .25 mg/ ml. Then the type of reagent is the Sunbright® CA series from NOF (NOF pH of the solution is corrected to 6 . 0 by drop wise addition Corp ., Tokyo , Japan ). A starting weight or concentration of of a 0 .5 N aqueous NaOH solution . rFIX is dissolved in or transferred to a reaction buffer (50 Subsequently , the aminooxy -polysialic acid (PSA -ONH , ) mM Hepes , 350 mM sodium chloride , 5 mM calcium reagent is added in a 50 - fold molar excess to this rFVIIa 60 chloride , pH 6 . 0 ) to get a final protein concentration of solution within a maximum time period (t ) of 15 minutes 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is corrected under gentle stirring. Then an aqueous m -toluidine solution to 6 .0 by drop wise addition of a 0 .5 N aqueous HCI (50 mM ) is added within 15 minutes to get a final concen - solution . Subsequently , a 40 mM aqueous sodium periodate tration of 10 mM . Finally a 40 mM aqueous sodium perio - solution is added within 10 minutes to give a concentration date solution is added to give a concentration of 150 uM . 65 of 200 uM . The oxidation reaction is carried out for 30 + / - 5 The reaction mixture is incubated for 120 + / - 10 min . in min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle is stopped by addition of an aqueous L - cysteine solution ( 1 US 10 ,414 ,793 B2 88 M ) within 15 minutes at T = + 22 +/ - 2° C . to give a final rFIX is transferred or dissolved in Hepes buffer (50 mM concentration of 10 mM in the reaction mixture and incu Hepes , 150 mM sodium chloride , 5 mM calcium chloride , bation for 60 + / - 5 min . pH 6 .0 ) to get a final protein concentration of 2 mg rFIX /ml . The oxidized rFVIII is further purified by anion exchange Subsequently , an 5 mM aqueous sodium periodate solution chromatography on EMD TMAE (M ) (Merck ) . The mixture 5 is added within 15 minutes to give a final concentration of is diluted with Buffer A ( 20 mM Hepes, 5 mM CaCl2 , pH 100 uM , followed by addition of an 50 mM aqueous 6 . 5 ) to give a conductivity of 5 mS / cm . This solution is m - toluidine solution to get a final concentration of 10 mM loaded onto the IEX column (bed height: 5 . 4 cm ) with a column volume of 10 ml using a flow rate of 1 . 5 cm /min . within a time period of 30 minutes. Then the aminooxy -PEG This column is subsequently washed ( flow rate : 1 . 5 cm /min ) 10 reagent with a MW of 20 kD (described above ) is added to with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and Buffer give a 20 -fold molar reagent excess. After correction of the B (20 mM Hepes, 5 mM CaC12 , 1 . 0 M NaCl, pH 7 . 0 ) . Then pH to 6 . 0 the mixture is incubated for 2 h in the dark at room the oxidized rFIX is eluted with a 50 :50 ( w /w ) mixture of temperature under gentle stirring and quenched for 15 min Buffer A and Buffer B followed by a postelution step withth 5 at room temperature by the addition of a 1 M aqueous CV of Buffer B . The elution steps are carried out by use of 15 L - cysteine solution to give a final concentration of 10 mM . a flow rate of 1 . 0 cm /min . The free rFIX is removed by means of ion exchange Subsequently , the aminooxy - PEG reagent with a MW of chromatography ( IEC ) . The reaction mixture was diluted 20 kD reagent is added in a 50 - fold molar excess to the with appropriate amounts of Buffer A (50 mM Hepes, 5 mM eluate containing the purified oxidized rFIX within a maxi- CaCl2 , pH 7 . 5 ) to correct the solutions conductivity and pH mum time period ( t ) of 15 minutes under gentle stirring . 20 value prior to load onto a 20 ml HiPrep QFF 16 / 10 column Then an aqueous m - toluidine solution (50 mM ) is added (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with Buf within 15 minutes to get a final concentration of 10 mM . The fer A . Then the column was eluted with Buffer B ( 50 mM reaction mixture is incubated for 120 + / – 10 min . in the dark Hepes, 1 M NaCl, 5 mM CaCl2 , pH 7 . 5 ) . Free FIX was at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . eluted by a step gradient using 25 % of Buffer B , which The obtained PEG - FIX conjugate is purified by Hydro - 25 results in a conductivity between 12 - 25 mS/ cm in the phobic Interaction Chromatography (HIC ) using a Phenyl obtained fraction and the conjugate using a step gradient of Sepharose FF low sub resin (GE Healthcare ) packed into a 50 % Buffer B , which results in a conductivity between column manufactured by GE Healthcare with a bed height 27 -45 mS/ cm in the conjugate fraction . The conductivity of ( h ) of 15 cm and a resulting column volume (CV ) of 81 ml. the conjugate containing fraction is subsequently raised with The reaction mixture is spiked with ammonium acetate by 30 Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaCl2 , pH 6 . 9 ; addition of 50 mM Hepes buffer , containing 350 mM sodium by use of anti - chaotropic salts e . g . ammonium acetate , etc ) chloride , 8 M ammonium acetate , 5 mM calcium chloride , and loaded onto a 20 ml HiPrep Butyl FF 16 / 10 column (GE pH 6 . 9 . Two volumes of the reaction mixture are mixed with Healthcare, Fairfield , Conn . ; or comparable HIC media ) 1 volume of the ammonium acetate containing buffer system pre -equilibrated with Buffer D (50 mM Hepes , 3 M NaCl, 5 and the pH value is corrected to pH 6 . 9 by drop wise 35 mM CaC12 , pH 6 . 9 ) . Free aminooxy -PEG reagent was addition of a 0 . 5 N aqueous NaOH solution . This mixture is washed out within 5 CV Buffer D . Subsequently, the con loaded onto the HIC column using a flow rate of 1 cm /min jugate was eluted with 100 % Buffer E ( 50 mM Hepes , 5 mM followed by a washing step using > 3 CV equilibration buffer CaC12 , pH 7 . 4 ) . The conjugate containing fractions are (50 mM Hepes , 350 mM sodium chloride , 2 . 5 M ammonium concentrated by UF /DF using a 10 kD membrane made of acetate , 5 mM calcium chloride , pH 6 . 9 ) . 40 regenerated cellulose (88 cm2, cutoff 10 kD , Millipore ) . For removal of reaction by -products and anti- chaotropic The final diafiltration step is performed against Hepes buffer salt a second washing step is performed with > 5 CV washing (50 mM Hepes , 5 mM CaC12 , pH 7 . 5 ) . buffer 1 (50 mM Hepes , 3 M sodium chloride, 5 mM The preparation is analytically characterized by measur calcium chloride , pH 6 . 9 ) in upflow mode at a flow rate of ing total protein ( Bradford and BCA procedure ) and bio 2 cm /min . Then elution of purified rFIX conjugate is per - 45 logical activity according to known methods . formed in down flow mode using a step gradient of 40 % washing buffer 2 ( 50 mM Hepes , 1 . 5 M sodium chloride, 5 Example 16 mM calcium chloride , pH 6 .9 ) and 60 % elution buffer (20 mM Hepes , 5 mM calcium chloride , pH 7 . 5 ) at a flow rate PEGylation of rFVIIa Using an Aminooxy - PEG of 1 cm /min . The elution of the PEG - rFIX conjugate is 50 Reagent and m - Toluidine as a Nucleophilic monitored at UV 280 nm and the eluate containing the Catalyst conjugate is collected within < 4 CV. The post elution step is performed with > 3 CV elution buffer under the same con Method 1 : ditions to separate minor and/ or non modified rFIX from the rFVIIa is PEGylated by use of a linear 20 kD PEGylation main product. 55 reagent containing an aminooxy group . An example of this Finally , the purified conjugate is concentrated by ultra- / type of reagent is the Sunbright® CA series from NOF (NOF diafiltration (UF /DF ) using a membrane made of regener Corp ., Tokyo , Japan ) . A starting weight or concentration of ated cellulose with a molecular weight cut off 10 kD (88 rFVIIa is dissolved in or transferred to a reaction buffer (50 cm2 , Millipore ). mM Hepes, 350 mM sodium chloride , 5 mM calcium The conjugate prepared by use of this procedure are 60 chloride , pH 6 . 0 ) to get a final protein concentration of analytically characterized by measuring total protein and 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is corrected biological activity according to methods known in the art . to 6 .0 by drop wise addition of a 0 .5 N aqueous NaOH Method 2 : solution . Subsequently , a 40 mM aqueous sodium periodate rFIX is PEGylated by use of a linear 20 kD PEGylation solution is added within 10 minutes to give a concentration reagent containing an aminooxy group . An example of this 65 of 50 uM . The oxidation reaction is carried out for 30 + / – 5 type of reagent is the Sunbright® CA series from NOF (NOF min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction Corp ., Tokyo , Japan ). An initial concentration or weight of is stopped by addition of an aqueous L -cysteine solution ( 1 US 10 ,414 ,793 B2 89 90 M ) within 15 minutes at T = + 22 + / - 2° C . to give a final rFVIIa is transferred or dissolved in Hepes buffer ( 50 mM concentration of 10 mM in the reaction mixture and incu Hepes, 150 mM sodium chloride , 5 mM calcium chloride , bation for 60 + / - 5 min . pH 6 . 0 ) to get a final protein concentration of 2 mg rFVIIa / The oxidized rFVIIa is further purified by anion exchange ml. Subsequently an 5 mM aqueous sodium periodate solu chromatography on EMD TMAE ( M ) (Merck ) . The mixture 5 tion is added within 15 minutes to give a final concentration is diluted with Buffer A ( 20 mM Hepes , 5 mM CaC12 , pH of 100 UM , followed by addition of an 50 mM aqueous 6 . 5 ) to give a conductivity of 5 mS/ cm . This solution is m - toluidine solution to get a final concentration of 10 mM loaded onto the IEX column (bed height: 5 . 4 cm ) with a within a time period of 30 minutes . Then the aminooxy - PEG column volume of 10 ml using a flow rate of 1 . 5 cm /min . reagent with a MW of 20 kD ( described above ) is added to This column is subsequently washed ( flow rate : 1 . 5 cm /min ) 10 give a 20 - fold molar reagent excess . After correction of the with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and Buffer pH to 6 . 0 the mixture is incubated for 2 h in the dark at room B ( 20 mM Hepes, 5 mM CaC12 , 1 . 0 M NaCl, pH 7 . 0 ) . Then temperature under gentle stirring and quenched for 15 min the oxidized rFVIIa is eluted with a 50 :50 ( w / w ) mixture of at room temperature by the addition of a 1 M aqueous Buffer A and Buffer B followed by a postelution step with 5 L -cysteine solution to give a final concentration of 10 mM . CV of Buffer B . The elution steps are carried out by use of 15 The free rFVIIa is removed by means of ion exchange a flow rate of 1 . 0 cm /min . chromatography ( IEC ) . The reaction mixture was diluted Subsequently , the aminooxy - PEG reagent with a MW of with appropriate amounts of Buffer A ( 50 mM Hepes , 5 mM 20 kD reagent is added in a 50 - fold molar excess to the CaC12 , pH 7 . 5 ) to correct the solutions conductivity and pH eluate containing the purified oxidized rFVIIa within a value prior to load onto a 20 ml HiPrep QFF 16 / 10 column maximum time period (t ) of 15 minutes under gentle stirring . 20 (GE Healthcare, Fairfield , Conn .) pre - equilibrated with Buf Then an aqueous m -toluidine solution (50 mM ) is added fer A . Then the column was eluted with Buffer B (50 mM within 15 minutes to get a final concentration of 10 mM . The Hepes, 1 M NaCl, 5 mM CaC12 , PH 7 . 5 ). Free rFVIla was reaction mixture is incubated for 120 + / - 10 min . in the dark eluted by a step gradient using 25 % of Buffer B , which at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking results in a conductivity between 12 -25 mS /cm in the The obtained PEG -rFVIIa conjugate is purified by Hydro - 25 obtained fraction and the conjugate using a step gradient of phobic Interaction Chromatography (HIC ) using a Phenyl 50 % Buffer B , which results in a conductivity between Sepharose FF low sub resin (GE Healthcare ) packed into a 27 -45 mS / cm in the conjugate fraction . The conductivity of column manufactured by GE Healthcare with a bed height the conjugate containing fraction is subsequently raised with ( h ) of 15 cm and a resulting column volume (CV ) of 81 ml. Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaC12 , pH 6 . 9 ; The reaction mixture is spiked with ammonium acetate by 30 by use of anti -chaotropic salts e. g . ammonium acetate ) and addition of 50 mM Hepes buffer , containing 350 mM sodium loaded onto a 20 ml HiPrep Butyl FF 16 / 10 column (GE chloride, 8 M ammonium acetate , 5 mM calcium chloride , Healthcare , Fairfield , Conn . ; or comparable HIC media ) pH 6 . 9 . Two volumes of the reaction mixture are mixed with pre -equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 1 volume of the ammonium acetate containing buffer system mM CaC12 , pH 6 . 9 ). Free PEG -reagent was washed out and the pH value is corrected to pH 6 . 9 by drop wise 35 within 5 CV Buffer D . Subsequently the conjugate was addition of a 0 . 5 N aqueous NaOH solution . This mixture is eluted with 100 % Buffer E (50 mM Hepes, 5 mM CaC12 , pH loaded onto the HIC column using a flow rate of 1 cm /min 7 . 4 ) . The conjugate containing fractions are concentrated by followed by a washing step using > 3 CV equilibration buffer UF /DF using a 10 kD membrane made of regenerated (50 mM Hepes , 350 mM sodium chloride , 2 . 5 M ammonium cellulose ( 88 cm2 , cutoff 10 kD , Millipore ) . The final acetate , 5 mM calcium chloride , pH 6 . 9 ) . 40 diafiltration step is performed against Hepes buffer ( 50 mM For removal of reaction by - products and anti - chaotropic Hepes, 5 mM CaCl2 , pH 7 . 5 ) . salt a second washing step is performed with > 5 CV washing The preparation is analytically characterized by measur buffer 1 (50 mM Hepes, 3 M sodium chloride , 5 mM ing total protein (Bradford and BCA procedure ) and bio calcium chloride , pH 6 . 9 ) in upflow mode at a flow rate of logical activity according to known methods . 2 cm /min . Then elution of purified rFVIIa conjugate is 45 performed in down flow mode using a step gradient of 40 % Example 17 washing buffer 2 (50 mM Hepes, 1 .5 M sodium chloride , 5 mM calcium chloride, pH 6 . 9 ) and 60 % elution buffer (20 Polysialylation of rFIX in the Presence of o - Amino mM Hepes, 5 mM calcium chloride , pH 7 .5 ) at a flow rate Benzoic Acid of 1 cm /min . The elution of the PEG - rFVIIa conjugate is 50 monitored at UV 280 nm and the eluate containing the Method 1 : conjugate is collected within < 4 CV. The post elution step is 8 . 2 mg rFIX is dissolved in 4 .0 ml histidine buffer , pH 6 .0 performed with > 3 CV elution buffer under the same con - (20 mM L -histidine , 150 mM NaCl, 5 mM CaCl2 ) . Then 82 ditions to separate minor and / or non modified rFVIIa from ul of an aqueous sodium periodate solution ( 5 mM ) is added the main product. 55 and the reaction mixture is incubated for 1 h in the dark at Finally , the purified conjugate is concentrated by ultra - / 4° C . under gentle stirring and quenched for 15 min at room diafiltration (UF / DF ) using a membrane made of regener temperature by the addition of 4 ul of a 1 Maqueous cysteine ated cellulose with a molecular weight cut off 10 kD solution . The mixture is subsequently subjected to UF /DF (Millipore ) . employing Vivaspin 6 10 kD centrifugal filtrators to remove The conjugate prepared by use of this procedure are 60 excess periodate , quencher and the byproducts thereof. analytically characterized by measuring total protein and The retentate ( 6 . 5 ml) , containing oxidized rFIX , is mixed biological activity according to methods known in the art with 1 .64 ml of an aqueous o - amino benzoic acid ( 50 mm ) Method 2 : and incubated for 30 min at room temperature . Then ami rFVIIa is PEGylated by use of a linear 20 kD PEGylation nooxy - PSA reagent with a MW of 20 kD (described above ) reagent containing an aminooxy group . An example of this 65 is added to give a 5 - fold molar reagent excess . This mixture type of reagent is the Sunbright® CA series from NOF (NOF was incubated for 2 . 5 h at room temperature in the dark Corp . , Tokyo , Japan ) . An initial concentration or weight of under gentle stirring . US 10 ,414 ,793 B2 91 92 The further purification of the conjugate is carried out as 10 mg EPO is dissolved in 5 ml histidine buffer, pH 6 . 0 described herein . ( 20 mM L - histidine , 150 mM NaCl) . 100 ul of an aqueous Method 2 : sodium periodate solution ( 5 mm ) is then added and the A solution of 1 mg rFIX in 0 .65 ml sodium phosphate reaction mixture is incubated for 1 h in the dark at 4° C . buffer, pH 6 . 0 containing a 5 - fold molar excess of ami- 5 under gentle stirring and quenched for 15 min at room nooxy -PSA reagent with a MW of 20 kD (described above ) temperature by the addition of 50 ul of a 1 M aqueous wasW : prepared . Then 333 ul of an aqueous O - amino benzoic cysteine solution . The mixture is subsequently subjected to acid solution ( 30 mM ) was added as nucleophilic catalyst to UF /DF employing Vivaspin 15R 10 kD centrifugal filtrators give a final concentration of 10 mM . Subsequently 20 ul of to remove excess periodate , quencher and the byproducts an aqueous solution of NaI04 ( 5 mM ) was added yielding in The retentate ( approx . 7 ml) , containing oxidized EPO , is a final concentration of 100 ?M . The coupling process was mixed with 2 ml of an aqueous m - toluidine solution ( 50 performed for 2 hours in the dark under gentle shaking at mM ) and incubated for 30 min at room temperature . Then room temperature and quenched for 15 min at room tem aminooxy -PSA reagent with a MW of 20 kD ( described perature by the addition of 1 ul of aqueous cysteine solution 15 ( 1 M ). The further purification of the conjugate is carried outon 15 mixtureabove ) isis added incubated to give for a2 .55 - hfold at RTmolar in thereagent dark excessunder gentle. This as described herein . stirring. The free EPO is removed by means of anion exchange Example 18 chromatography (AEC ) . The reaction mixture is diluted with 20 20 ml Buffer A ( 50 mM Hepes, pH 7 . 5 ) and loaded onto a Polysialylation of EPO Using Aminooxy - PSA and 20 ml HiPrep QFF 16 / 10 column (GE Healthcare , Fairfield , m - Toluidine as a Nucleophilic Catalyst Conn . ) pre - equilibrated with Buffer A . Then the column is eluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 7 .5 ). Method 1 : Free EPO is eluted by washing the column with 25 % Buffer A starting concentration of erythropoietin ( EPO ) is trans - 25 B and the conjugate at 50 % Buffer B . The conductivity of ferred into a reaction buffer ( e . g . 50 mM Hepes, 350 mm the conjugate containing fractions is subsequently raised to sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and - 190 mS/ cm with Buffer C ( 50 mM Hepes , 5 M NaC1, pH diluted to obtain a protein concentration of 1 mg/ ml . To this 6 . 9 ) and loaded onto a 20 ml HiPrep Butyl FF 16 / 10 column solution , NalO is added to give a final concentration of 200 (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with Buf uM . The oxidation is carried at RT for 30 min in the dark 30 fer D ( 50 mM Hepes, 3 M NaCl, pH 6 . 9 ) . Free PSA -reagent under gentle shaking . The reaction is then quenched with is washed out within 5 CV Buffer D . Subsequently , the cysteine ( final concentration : 10 mM ) for 60 min at RT. conjugate is eluted with 100 % Buffer E (50 mM Hepes , pH The solution is next subjected to UF /DF employing 7 . 4 ). The conjugate containing fractions are concentrated by Vivaspin centrifugal filtrators to remove excess periodate , UF /DF using a 10 kD membrane made of regenerated quencher and the byproducts thereof or, in the alternative , to 35 cellulose ( 88 cm2, cut - off 10 kD /Millipore ) . The final dia an IEX column with a volume of 20 ml (Merck EMD TMAE filtration step is performed against histidine buffer , pH 7 . 2 ( M )) which is equilibrated with Buffer A ( 20 mM Hepes , 5 containing 150 mM NaCl. The preparation is analytically mM CaCl2, pH 7 . 0 ) . The column is equilibrated with 5 CV characterized by measuring total protein (Bradford ) and Buffer A . The oxidized EPO is eluted with Buffer B ( 20 mM biological activity according to methods known in the art . Hepes, 5 mM CaCl, , 1M NaCl, pH 7 . 0 ) . The EPO contain - 40 For the PSA - EPO conjugate a specific activity of > 50 % in ing fractions are collected . The protein content is determined comparison to native EPO is determined . The conjugate is ( Coomassie , Bradford ) and adjusted to 1 mg/ml with reac - additionally analytically characterized by Size Exclusion tion buffer and adjusted to pH 6 . 0 by dropwise addition of HPLC using a Agilent 1200 HPLC system equipped with a 0 .5M HC1. Shodex KW 803 column under conditions as previously A 50 - fold molar excess of a aminooxy - PSA reagent with 45 described (Kolarich et al, Transfusion 2006 ; 46 : 1959 -77 ) . It a MW of 20 kD ( described above) is added followed by is shown that the preparation contains no free EPO . m - toluidine as a nucleophilic catalyst ( final concentration : Method 2 : 10 mM ) . The coupling reaction is performed for 2 hours in EPO is transferred or dissolved in reaction buffer ( e . g . 50 the dark under gentle shaking at room temperature . The mM Hepes, 350 mM sodium chloride, 5 mM calcium excess of aminooxy - PSA reagent is removed by means of 50 chloride , pH 6 . 0 ) to get a final protein concentration of HIC . The conductivity of the reaction mixture is adjusted by 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is corrected adding a buffer containing ammonium acetate (50 mM to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 Hepes, 350 mM sodium chloride , 5 mM calcium chloride , 8 solution . Subsequently , a 40 mM aqueous sodium periodate M ammonium acetate , pH 6 . 9 ) and loaded onto a column solution is added within 10 minutes to give a concentration filled with 80 ml Phenyl Sepharose FF (GE Healthcare, 55 of 200 UM . The oxidation reaction is carried out for 30 + / - 5 Fairfield , Conn .) pre -equilibrated with 50 mM Hepes, 2 . 5 M min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction ammonium acetate , 350 mM sodium chloride , 5 mM cal - is stopped by addition of an aqueous L - cysteine solution ( 1 cium chloride, pH 6 .9 . Subsequently , the conjugate is eluted M ) within 15 minutes at T = + 22 +/ - 2° C . to give a final with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC1, . concentration of 10 mM in the reaction mixture and incu Finally the PSA - EPO containing fractions are collected and 60 bation for 60 + / - 5 min . subjected to UF /DF by use of a membrane made of regen - The oxidized EPO is further purified by ion exchange erated cellulose (MWCO 10 kD , 50 cm2, Millipore ) . The chromatography . The oxidized EPO containing fractions of preparation is next analytically characterized by measuring the eluate are collected and used for the conjugation reac total protein ( Coomassie , Bradford ) and biological activity tion . according to methods known in the art. 65 The aminooxy - polysialic acid (PSA - ONH2) reagent is In an alternative embodiment, Method 1 is carried out as added in a 50 - fold molar excess to the eluate containing the follows. purified oxidized EPO within a maximum time period ( t ) of US 10 ,414 ,793 B2 93 94 15 minutes under gentle stirring . Then an aqueous m - tolui - is washed out within 5 CV Buffer D . Subsequently , the dine solution (50 mM ) is added within 15 minutes to get a conjugate is eluted with 100 % Buffer E ( 50 mM Hepes, pH final concentration of 10 mM . The reaction mixture is 7 . 4 ) . The conjugate containing fractions are concentrated by incubated for 120 + / – 10 min . at pH 6 .0 in the dark at a UF /DF using a 10 kD membrane made of regenerated temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking 5 cellulose ( 88 cm2 , cutoff 10 kD , Millipore ) . The final (protein concentration : 1 mg/ ml ) . diafiltration step is performed against histidine buffer, pH The obtained PSA - EPO conjugate is further purified by 7 .2 containing 150 mM NaCl. The preparation is analyti ion exchange chromatography. The PSA - EPO conjugate c ally characterized by measuring total protein (Bradford ) containing fractions are collected and concentrated by ultra - and biological activity according to methods known in the diafiltration (UF / DF ) using a membrane made of regener - 10 art . For the PSA - EPO conjugate a specific activity of > 50 % ated cellulose with an appropriate molecular weight cut off in comparison to native EPO is determined . The conjugate (Millipore ). is additionally analytically characterized by Size Exclusion The conjugate prepared by use of this procedure is HPLC using a Agilent 1200 HPLC system equipped with a analytically characterized by measuring total protein , bio - Shodex KW 803 column under conditions as previously logical activity, and determination of the polysialyation 15 described (Kolarich et al , Transfusion 2006 ; 46 : 1959 - 77 ) . It degree by measuring the PSA content (resorcinol assay ) . is shown that the preparation contains no free EPO . Method 3 : Method 4 : Erythropoietin (EPO ) is transferred into reaction buffer EPO is dissolved in or transferred to a reaction buffer (e . g . (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium 50 mM Hepes , 350 mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to obtain a protein concentra - 20 chloride , pH 6 . 0 ) to get a final protein concentration of tion of 1 mg/ ml . A 50 fold molar excess of a aminooxy - PSA 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected reagent with a MW of 20 kD (described above ) is added to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 followed by m - toluidine as a nucleophilic catalyst (10 mM solution . final concentration ) and NaI04 ( final concentration : 400 Subsequently , the aminooxy -polysialic acid (PSA -ONH2 ) uM ) . The coupling reaction is performed for 2 hours in the 25 reagent is added in a 50 - fold molar excess to this EPO dark under gentle shaking at room temperature . Subse - solution within a maximum time period ( t ) of 15 minutes quently , the reaction is quenched with cysteine for 60 min at under gentle stirring. Then an aqueous m - toluidine solution RT ( cysteine concentration : 10 mM ). Then the conductivity (50 mM ) is added within 15 minutes to get a final concen of the reaction mixture is adjusted by adding a buffer tration of 10 mM . Finally a 40 mM aqueous sodium perio containing ammonium acetate ( 50 mM Hepes, 350 mM 30 date solution is added to give a concentration of 400 UM . sodium chloride , 5 mM calcium chloride , 8 M ammonium The reaction mixture is incubated for 120 + / – 10 min . in acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle Sepharose FF (GE Healthcare , Fairfield , Conn . ) pre - equili - shaking . Then the reaction is stopped by the addition of an brated with 50 mM Hepes , 2 . 5 M ammonium acetate , 350 aqueous L -cysteine solution ( 1 M ) to give a final concen mM sodium chloride , 5 mM calcium chloride , 0 . 01 % Tween 35 tration of 10 mM in the reaction mixture and incubation for 80 , pH 6 . 9 . Subsequently , the conjugate is eluted with 50 60 + / - 5 min . mM Hepes , 5 mM calcium chloride , pH 7 .5 . Finally , the The obtained PSA -EPO conjugate is purified by ion PSA - EPO containing fractions are collected and subjected to exchange chromatography. The PSA - EPO containing frac UF /DF by use of a membrane made of regenerated cellulose tions of the eluate are collected and concentrated by ultra - / (MWCO 10 kD , 88 cm2, Millipore ) . The preparation is 40 diafiltration (UF / DF ) using a membrane made of analytically characterized by measuring total protein (Brad - regenerated cellulose (MWCO 10 kD , 88 cm2, Millipore ) . ford ) and biological activity according to methods known in The conjugates prepared by use of this procedure are the art . analytically characterized by measuring total protein , bio In an alternative embodiment, Method 3 is carried out as logical activity according to methods known in the art, and follows. 10 mg EPO is dissolved in 8 ml histidine buffer, pH 45 determination of the polysialyation degree by measuring the 6 . 0 (20 mM L -histidine , 150 mM NaCl) . 200 ul of an PSA content (resorcinol assay ) . aqueous sodium periodate solution ( 5 mM ) and 2 ml of an aqueous m - toluidine solution (50 mm ) are then added . Example 19 Subsequently , the aminooxy - PSA reagent with a MW of 20 kD (described above ) is added to give a 5 - fold molar reagent 50 Polysialylation of Ang - 2 Using Aminooxy - PSA and excess . The mixture is incubated for 2 h in the dark at room m - Toluidine as a Nucleophilic Catalyst temperature under gentle stirring and quenched for 15 min at room temperature by the addition of 100 ul of 1 M Method 1 : aqueous cysteine solution . A starting concentration of angiopoietin - 2 (Ang - 2 ) is The free EPO is removed by means of anion exchange 55 transferred into a reaction buffer ( e . g . 50 mM Hepes, 350 chromatography ( AEC ) . The reaction mixture is diluted with mM sodium chloride , 5 mM calcium chloride, pH 6 . 0 ) and 20 ml Buffer A (50 mM Hepes , pH 7 . 5 ) and loaded onto a diluted to obtain a protein concentration of 1 mg/ ml . To this 20 ml HiPrep QFF 16 /10 column (GE Healthcare, Fairfield , solution , NalO4 is added to give a final concentration of 200 Conn . ) pre - equilibrated with Buffer A . Then the column is UM . The oxidation is carried at RT for 30 min in the dark eluted with Buffer B (50 mM Hepes , 1 M NaCl, pH 7 . 5 ) . 60 under gentle shaking . The reaction is then quenched with Free EPO is eluted by washing the column with 25 % Buffer cysteine ( final concentration : 10 mM ) for 60 min at RT. B and the conjugate at 50 % Buffer B . The conductivity of The solution is next subjected to UF /DF employing the conjugate containing fractions is subsequently raised to Vivaspin centrifugal filtrators to remove excess periodate , ~ 190 mS/ cm with Buffer C ( 50 mM Hepes , 5 M NaCl , pH quencher and the byproducts , or, in the alternative , subjected 6 . 9 ) and loaded onto a 20 mlHiPrep Butyl FF 16 / 10 column 65 to an IEX column with a volume of 20 ml (Merck EMD (GE Healthcare , Fairfield , Conn .) pre -equilibrated with Buf- TMAE ( M ) ) which is equilibrated with Buffer A (20 mM fer D (50 mM Hepes, 3 M NaCl, pH 6 . 9 ). Free PSA - reagent Hepes , 5 mM CaCl2 , pH 7 .0 ). The column is equilibrated US 10 ,414 ,793 B2 95 96 with 5 CV Buffer A . The oxidized Ang - 2 is eluted with The oxidized Ang - 2 is further purified by ion exchange Buffer B ( 20 mM Hepes, 5 mM CaCl2, 1 M NaCl, pH 7 . 0 ) . chromatography . The oxidized Ang - 2 containing fractions of The Ang -2 containing fractions are collected . The protein the eluate are collected and used for the conjugation reac content is determined (Coomassie , Bradford ) and adjusted to tion . 1 mg/ ml with reaction buffer and adjusted to pH 6 .0 by 5 The aminooxy -polysialic acid (PSA -ONH2 ) reagent is dropwise addition of 0 . 5 M HC1. added in a 50 - fold molar excess to the eluate containing the A 50 - fold molar excess of aminooxy - PSA reagent with a purified oxidized Ang - 2 within a maximum time period ( t ) MW of 20 kb (described above ) is added followed by of 15 minutes under gentle stirring . Then an aqueous m - toluidine as a nucleophilic catalyst ( final concentration : m - toluidine solution (50 mM ) is added within 15 minutes to 10 mM ). The coupling reaction is performed for 2 hours inin 10 get a final concentration of 10 mM . The reaction mixture is the dark under gentle shaking at room temperature . The incubated for 120 + / – 10 min . at pH 6 . 0 in the dark at a excess of aminooxy reagent is removed by means of HIC . temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking The conductivity of the reaction mixture is adjusted by (protein concentration : 1 mg/ ml ) . adding a buffer containing ammonium acetate (50 mM 15 jon The-exchange obtained chromat PSA - Ang - 2 conjugate is further purified by Hepes , 350 mM sodium chloride , 5 mM calcium chloride , The PSA -Ang - 2 conjugate containing fractions are col M ammonium acetate , pH 6 . 9 ) and loaded onto a column lected and concentrated by ultra - /diafiltration (UF /DF ) using filled with 80 ml Phenyl Sepharose FF (GE Healthcare , a membrane made of regenerated cellulose with an appro Fairfield , Conn .) pre -equilibrated with 50 mM Hepes, 2 .5 M priate molecular weight cut off (Millipore ) . ammonium acetate , 350 mM sodium chloride, 5 mM cal- 20 The conjugate prepared by use of this procedure is cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted analytically characterized by measuring total protein , bio with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaCl2 . logical activity , and determination of the polysialyation Finally , the PSA - Ang - 2 -containing fractions are collected degree by measuring the PSA content (resorcinol assay ) . and subjected to UF /DF by use of a membrane made of Method 3 : regenerated cellulose (Millipore ) . The preparation is next 25 Angiopoietin - 2 (Ang - 2 ) is transferred into reaction buffer analytically characterized by measuring total protein ( Coo - ( 50 mM Hepes , 350 mM sodium chloride , 5 mM calcium massie , Bradford ) and biological activity according to meth chloride , pH 6 . 0 ) and diluted to obtain a protein concentra tion of 1 mg/ml . A 50 fold molar excess of a PSA aminooxy ods known in the art . reagent with a MW of 20 kD (described above ) is added In an alternative embodiment, Method 1 is carried out as 30 followed by m - toluidine as a nucleophilic catalyst ( 10 mM follows. Angiopoietin - 2 (Ang - 2 ) is transferred into a reac final concentration ) and NalO4 ( final concentration : 400 tion buffer ( e . g ., 50 mM Hepes , 350 mM sodium chloride , UM ). The coupling reaction is performed for 2 hours in the 5 mM calcium chloride, pH 6 . 0 ) and diluted to obtain a dark under gentle shaking at room temperature . Subse protein concentration of 1 mg/ml . To this solution , NalO4 is quently , the reaction is quenched with cysteine for 60 min at added to give a final concentration of 200 uM . The oxidation" 35 RT ( cysteine concentration : 10 mM ). Then the conductivity is carried at RT for 30 min in the dark under gentle shaking . of the reaction mixture is adjusted by adding a buffer The reaction is then quenched with cysteine ( final concen containing ammonium acetate ( 50 mM Hepes , 350 mM tration : 10 mM ) for 60 min at R . T . sodium chloride , 5 mM calcium chloride , 8 M ammonium The solution is next subjected to UF /DF employing acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl Vivaspin centrifugal filtrators to remove excess periodate, 40 Sepharose FF (GE Healthcare , Fairfield , Conn .) pre -equili quencher and the byproducts thereof. brated with 50 mM Hepes , 2 . 5 M ammonium acetate , 350 A 50 - fold molar excess of aminooxy -PSA reagent with a mM sodium chloride , 5 mM calcium chloride, 0 .01 % Tween MW of 20 kD ( described above) is added followed by 80 , pH 6 . 9 . Subsequently , the conjugate is eluted with 50 m - toluidine as a nucleophilic catalyst ( final concentration : mM Hepes , 5 mM calcium chloride , pH 7. 5 . Finally , the 10 mM ) . The coupling reaction is performed for 2 hours in 45 PSA Ang - 2 -containing fractions are collected and subjected the dark under gentle shaking at room temperature . The to UF /DF by use of a membrane made of regenerated excess of aminooxy reagent is removed by means of ion cellulose (Millipore ) . The preparation is analytically char exchange chromatography . The PSA - Ang - 2 conjugate - con - acterized by measuring total protein ( Bradford ) and biologi taining fractions of the eluate are collected and subjected to cal activity according to methods known in the art. UF /DF by use of a membrane made of regenerated cellulose 50 In an alternative embodiment, Method 3 is carried out as (Millipore ) . The preparation is next analytically character follows. Angiopoietin - 2 ( Ang- 2 ) is transferred into reaction ized by measuring total protein (Coomassie , Bradford ) and buffer ( e . g . 50 mM Hepes , 350 mM sodium chloride, 5 mM biological activity according to methods known in the art . calcium chloride , pH 6 . 0 ) and diluted to obtain a protein Method 2 : concentration of 1 mg/ml . A 50 - fold molar excess of a PSA Ang - 2 is transferred or dissolved in reaction buffer ( e . g . 55 aminooxy reagent with a MW of 20 kD ( described above ) is 50 mM Hepes, 350 mM sodium chloride , 5 mM calcium added followed by m - toluidine as a nucleophilic catalyst ( 10 chloride , pH 6 .0 ) to get a final protein concentration of mM final concentration ) and NalO4 ( final concentration : 1 .0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected 400 UM ). The coupling reaction is performed for 2 hours in to 6 . 0 by drop wise addition of a 0 .5 N aqueous HC1 the dark under gentle shaking at room temperature . Subse solution . Subsequently , a 40 mM aqueous sodium periodate 60 quently , the reaction is quenched with cysteine for 60 min at solution is added within 10 minutes to give a concentration RT ( cysteine concentration : 10 mM ) and the conjugate is of 200 uM . The oxidation reaction is carried out for 30 + / - 5 purified by ion exchange chromatography . PSA Ang - 2 min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction containing fractions of the eluate are collected and subjected is stopped by addition of an aqueous L -cysteine solution ( 1 to UF /DF by use of a membrane made of regenerated M ) within 15 minutes at T = + 22 + / - 2° C . to give a final 65 cellulose (Millipore ). The preparation is analytically char concentration of 10 mM in the reaction mixture and incu - acterized by measuring total protein ( Bradford ) and biologi bation for 60 + / - 5 min . cal activity according to methods known in the art . US 10 ,414 ,793 B2 97 98 Method 4 : M ammonium acetate , pH 6 . 9 ) and loaded onto a column Ang - 2 is dissolved in or transferred to a reaction buffer filled with 80 ml Phenyl Sepharose FF (GE Healthcare , ( e. g . 50 mM Hepes, 350 mM sodium chloride, 5 mM Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes, 2 . 5 M calcium chloride , pH 6 .0 ) to get a final protein concentration ammonium acetate , 350 mM sodium chloride , 5 mM cal of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor- 5 cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . solution . Finally the PSA - VEGF - containing fractions are collected Subsequently , the aminooxy - polysialic acid ( PSA -ONH2 ) and subjected to UE/ DE by use of a membrane made of reagent is added in a 50 - fold molar excess to this Ang - 2 regenerated cellulose (Millipore ). The preparation is next solution within a maximum time period (t ) of 15 minutes 10 analytically characterized by measuring total protein (Coo under gentle stirring . Then an aqueous m - toluidine solution massie , Bradford ) and biological activity according to meth ( 50 mM ) is added within 15 minutes to get a final concen tration of 10 mM . Finally a 40 mM aqueous sodium perio ods known in the art . date solution is added to give a concentration of 400 uM . In an alternative embodiment, Method 1 is carried out as The reaction mixture is incubated for 120 + / – 10 min . in 1515 follows. Vascular endothelial growth factor ( VEGF ) is trans the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle ferred into a reaction buffer ( e . g . , 50 mM Hepes, 350 mm shaking . Then the reaction is stopped by the addition of an sodium chloride, 5 mM calcium chloride, pH 6 .0 ) and aqueous L - cysteine solution ( 1 M ) to give a final concen diluted to obtain a protein concentration of 1 mg/ ml . To this tration of 10 mM in the reaction mixture and incubation for solution , NalO4 is added to give a final concentration of 200 60 + / - 5 min . 20 UM . The oxidation is carried at RT for 30 min in the dark The obtained PSA - Ang - 2 conjugate is purified by ion - under gentle shaking . The reaction is then quenched with exchange chromatography . The PSA - Ang - 2 containing frac - cysteine ( final concentration : 10 mM ) for 60 min at RT. tions of the eluate are collected and concentrated by ultra The solution is next subjected to UF /DF employing diafiltration (UF /DF ) using a membrane made of Vivaspin centrifugal filtrators to remove excess periodate , regenerated cellulose (Millipore ) . 25 quencher and the byproducts thereof. The conjugates prepared by use of this procedure are 50 - fold molar excess of aminooxy - PSA reagent with a analytically characterized by measuring total protein , bio - MW of 20 kD ( described above ) is added followed by logical activity according to methods known in the art , and m - toluidine as a nucleophilic catalyst ( final concentration : determination of the polysialyation degree by measuring the 10 mM ) . The coupling reaction is performed for 2 hours in PSA content (resorcinol assay ). 30 the dark under gentle shaking at room temperature . The excess of aminooxy reagent is removed by means of ion Example 20 exchange chromatography. The PSA - VEGF - containing fractions of the eluate are collected and subjected to UF /DF Polysialylation of VEGF Using Aminooxy -PSA and by use of a membrane made of regenerated cellulose (Mil m - Toluidine as a Nucleophilic Catalyst 35 lipore ). The preparation is next analytically characterized by measuring total protein (Coomassie , Bradford ) and biologi Method 1 : cal activity according to methods known in the art . A starting concentration of vascular endothelial growth Method 2 : factor (VEGF ) is transferred into a reaction buffer ( e . g . , 50 VEGF is transferred or dissolved in reaction buffer ( e . g . mM Hepes , 350 mM sodium chloride , 5 mM calcium 40 50 mM Hepes, 350 mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to obtain a protein concentra - chloride , pH 6 . 0 ) to get a final protein concentration of tion of 1 mg/ ml . To this solution , NalO4 is added to give a 1 .0 + / - 0 . 25 mg/ ml . Then the pH of the solution is corrected final concentration of 200 uM . The oxidation is carried at RT to 6 .0 by drop wise addition of a 0 .5 N aqueous HCI for 30 min in the dark under gentle shaking . The reaction is solution . Subsequently a 40 mM aqueous sodium periodate then quenched with cysteine ( final concentration : 10 mM ) 45 solution is added within 10 minutes to give a concentration for 60 min at RT. of 200 uM . The oxidation reaction is carried out for 30 + / - 5 The solution is next subjected to UF /DF employing min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction Vivaspin centrifugal filtrators to remove excess periodate , is stopped by addition of an aqueous L - cysteine solution ( 1 quencher and the byproducts thereof or, in the alternative , to M ) within 15 minutes at T = + 22 + / - 2° C . to give a final an IEX column with a volume of 20 ml (Merck EMD TMAE 50 concentration of 10 mM in the reaction mixture and incu ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes, 5 bation for 60 + / - 5 min . mM CaC12 , pH 7 . 0 ) . The column is equilibrated with 5 CV The oxidized VEGF is further purified by ion exchange Buffer A . The oxidized VEGF is eluted with Buffer B ( 20 chromatography . The oxidized VEGF containing fractions mM Hepes, 5 mM CaC12 , 1 M NaCl, pH 7 . 0 ). The VEGF of the eluate are collected and used for the conjugation containing fractions are collected . The protein content is 55 reaction . determined (Coomassie , Bradford ) and adjusted to 1 mg/ml The aminooxy -polysialic acid (PSA -ONH2 ) reagent is with reaction buffer and adjusted to pH 6 . 0 by dropwise added in a 50 - fold molar excess to the eluate containing the addition of 0 .5M NaOH . purified oxidized VEGF within a maximum time period ( t ) A 50 - fold molar excess of aminooxy - PSA reagent with a of 15 minutes under gentle stirring . Then an aqueous MW of 20 kD (described above ) is added followed by 60 m - toluidine solution (50 mM ) is added within 15 minutes to m - toluidine as a nucleophilic catalyst ( final concentration : get a final concentration of 10 mM . The reaction mixture is 10 mM ) . The coupling reaction is performed for 2 hours in incubated for 120 + / – 10 min . at pH 6 . 0 in the dark at a the dark under gentle shaking at room temperature . The temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking excess of aminooxy reagent is removed by means of HIC . (protein concentration : 1 mg/ ml ) . The conductivity of the reaction mixture is adjusted by 65 The obtained PSA - VEGF conjugate is further purified by adding a buffer containing ammonium acetate (50 mM ion exchange chromatography. The PSA - VEGF conjugate Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 containing fractions are collected and concentrated by ultra -/ US 10 , 414 , 793 B2 99 100 diafiltration (UF /DF ) using a membrane made of regener The reaction mixture is incubated for 120 + / – 10 min . in ated cellulose with an appropriate molecular weight cut off the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle (Millipore ) . shaking . Then the reaction is stopped by the addition of an The conjugate prepared by use of this procedure is aqueous L - cysteine solution ( 1 M ) to give a final concen analytically characterized by measuring total protein , bio - 5 tration of 10 mM in the reaction mixture and incubation for logical activity , and determination of the polysialyation 60 + / - 5 min . degree by measuring the PSA content (resorcinol assay ) . The obtained VEGF- conjugate is purified by ion -ex Method 3 : change chromatography . The PSA - VEGF containing frac Vascular endothelial growth factor (VEGF ) is transferred tions of the eluate are collected and concentrated by ultra - / into reaction buffer (50 mM Hepes , 350 mM sodium chlo - 10 diafiltration (UF / DF ) using a membrane made of ride, 5 mM calcium chloride , pH 6 .0 ) and diluted to obtain regenerated cellulose (Millipore ) . a protein concentration of 1 mg/ml . A 50 - fold molar excess The conjugates prepared by use of this procedure are of a PSA aminooxy reagent with a MW of 20 kD ( described analytically characterized by measuring total protein , bio above ) is added followed by m -toluidine as a nucleophilic logical activity according to methods known in the art , and catalyst (10 mM final concentration ) and NalO4 ( final 15 determination of the polysialyation degree by measuring the concentration : 400 UM ) . The coupling reaction is performed PSA content ( resorcinol assay ) . for 2 hours in the dark under gentle shaking at room temperature . Subsequently, the reaction is quenched with Example 21 cysteine for 60 min at RT ( cysteine concentration : 10 mM ) . Then the conductivity of the reaction mixture is adjusted by 20 Polysialylation of EGF Using Aminooxy -PSA and adding a buffer containing ammonium acetate (50 mM m - Toluidine as a Nucleophilic Catalyst Hepes, 350 mM sodium chloride , 5 mM calcium chloride , 8 M ammonium acetate , pH 6 . 9 ) and loaded onto a column Method 1 : filled with Phenyl Sepharose FF (GE Healthcare , Fairfield , starting concentration of epidermal growth factor Conn . ) pre - equilibrated with 50 mM Hepes , 2 . 5 M ammo - 25 (EGF ) is transferred into a reaction buffer ( e . g ., 50 mM nium acetate , 350 mM sodium chloride, 5 mM calcium Hepes, 350 mM sodium chloride, 5 mM calcium chloride , chloride, 0 .01 % Tween 80 , pH 6 . 9 . Subsequently the con - pH 6 . 0 ) and diluted to obtain a protein concentration of 1 jugate is eluted with 50 mM Hepes , 5 mM calcium chloride , mg/ ml . To this solution , Nal04 is added to give a final pH 7 . 5 . Finally , the PSA - VEGF containing fractions are concentration of 200 UM . The oxidation is carried at RT for collected and subjected to UF /DF by use of a membrane 30 30 min in the dark under gentle shaking . The reaction is then made of regenerated cellulose (Millipore ). The preparation quenched with cysteine ( final concentration : 10 mM ) for 60 is analytically characterized by measuring total protein min at R . T . (Bradford ) and biological activity according to methods The solution is next subjected to UF /DF employing known in the art . Vivaspin centrifugal filtrators to remove excess periodate , In an alternative embodiment, Method 3 is carried out as 35 quencher and the byproducts thereof or, in the alternative , to follows . Vascular endothelial growth factor (VEGF ) is trans- an IEX column with a volume of 20 ml (Merck EMD TMAE ferred into reaction buffer ( e . g . 50 mM Hepes , 350 mM (M ) ) which is equilibrated with Buffer A (20 mM Hepes, 5 sodium chloride, 5 mM calcium chloride , pH 6 . 0 ) and mM CaCl2 , pH 7 . 0 ) . The column is equilibrated with 5 CV diluted to obtain a protein concentration of 1 mg/ml . A Buffer A . The oxidized EGF is eluted with Buffer B (20 mM 50 - fold molar excess of aminooxy - PSA reagent with a MW 40 Hepes , 5 mM CaC12 , 1M NaC1, pH 7 . 0 ) . The EGF contain of 20 kD (described above ) is added followed by m - tolui- ing fractions are collected . The protein content is determined dine as a nucleophilic catalyst (10 mM final concentration ) (Coomassie , Bradford ) and adjusted to 1 mg/ ml with reac and NalO4 ( final concentration : 400 UM ) . The coupling tion buffer and adjusted to pH 6 . 0 by dropwise addition of reaction is performed for 2 hours in the dark under gentle 0 .5M HCl. shaking at room temperature . Subsequently , the reaction is 45 A 50 -fold molar excess of aminooxy - PSA reagent with a quenched with cysteine for 60 min at RT ( cysteine concen - MW of 20 kD (described above ) is added followed by tration : 10 mM ) and the conjugate is purified by ion m - toluidine as a nucleophilic catalyst ( final concentration : exchange chromatography. The PSA -VEGF containing frac - 10 mM ). The coupling reaction is performed for 2 hours in tions of the eluate are collected and subjected to UF /DF by the dark under gentle shaking at room temperature . The use of a membrane made of regenerated cellulose (Milli - 50 excess of aminooxy reagent is removed by means of HIC . pore ) . The preparation is analytically characterized by mea - The conductivity of the reaction mixture is adjusted by suring total protein (Bradford ) and biological activity adding a buffer containing ammonium acetate (50 mM according to methods known in the art. Hepes, 350 mM sodium chloride , 5 mM calcium chloride, 8 Method 4 : M ammonium acetate , pH 6 . 9 ) and loaded onto a column VEGF is dissolved in or transferred to a reaction buffer 55 filled with 80 ml Phenyl Sepharose FF (GE Healthcare , ( e . g . 50 mM Hepes, 350 mM sodium chloride, 5 mM Fairfield , Conn .) pre - equilibrated with 50 mM Hepes, 2 . 5 M calcium chloride, pH 6 .0 ) to get a final protein concentration ammonium acetate , 350 mM sodium chloride, 5 mM cal of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is corar cium chloride, pH 6 . 9 . Subsequently , the conjugate is eluted rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . solution . 60 Finally , the PSA - EGF containing fractions are collected and Subsequently , the aminooxy- polysialic acid (PSA -ONH2 ) subjected to UF /DF by use of a membrane made of regen reagent is added in a 50 -fold molar excess to this VEGF erated cellulose (Millipore ) . The preparation is next analyti solution within a maximum time period ( t) of 15 minutes cally characterized by measuring total protein (Coomassie , under gentle stirring . Then an aqueous m - toluidine solution Bradford ) and biological activity according to methods ( 50 mM ) is added within 15 minutes to get a final concen - 65 known in the art. tration of 10 mM . Finally a 40 mM aqueous sodium perio In an alternative embodiment, Method 1 is carried out as date solution is added to give a concentration of 400 uM . follows. Epidermal growth factor (EGF ) is transferred into US 10 ,414 ,793 B2 101 102 a reaction buffer ( e. g ., 50 mM Hepes, 350 mM sodium 400 uM ). The coupling reaction is performed for 2 hours in chloride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to the dark under gentle shaking at room temperature . Subse obtain a protein concentration of 1 mg/ ml . To this solution , quently , the reaction is quenched with cysteine for 60 min at NalO4 is added to give a final concentration of 200 UM . The RT ( cysteine concentration : 10 mM ) . Then the conductivity oxidation is carried at RT for 30 min in the dark under gentle 5 of the reaction mixture is adjusted by adding a buffer shaking . The reaction is then quenched with cysteine ( final containing ammonium acetate (50 mM Hepes, 350 mM concentration : 10 mM ) for 60 min at R . T . sodium chloride , 5 mM calcium chloride , 8 M ammonium The solution is next subjected to UF /DF employing acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl Vivaspin centrifugal filtrators to remove excess periodate , Sepharose FF (GE Healthcare , Fairfield , Conn .) pre - equili quencher and the byproducts thereof. 10 brated with 50 mM Hepes , 2 . 5 M ammonium acetate , 350 A 50 - fold molar excess of aminooxy - PSA reagent with a mM sodium chloride , 5 mM calcium chloride , 0 .01 % Tween MW of 20 kD (described above ) is added followed by 80 , pH 6 . 9 . Subsequently the conjugate is eluted with 50 m - toluidine as a nucleophilic catalyst ( final concentration : mM Hepes, 5 mM calcium chloride , pH 7 . 5 . Finally the 10 mM ) . The coupling reaction is performed for 2 hours in PSA - EGF containing fractions are collected and subjected to the dark under gentle shaking at room temperature . The 15 UF /DF by use of a membrane made of regenerated cellulose excess of aminooxy reagent is removed by means of ion (Millipore ) . The preparation is analytically characterized by exchange chromatography . The PSA - EGF containing frac - measuring total protein ( Bradford ) and biological activity tions of the eluate are collected and subjected to UF /DF by according to methods known in the art. use of a membrane made of regenerated cellulose (Milli - In an alternative embodiment, Method 3 is carried out as pore ). The preparation is next analytically characterized by 20 follows. Epidermal growth factor ( EGF ) is transferred into measuring total protein (Coomassie , Bradford ) and biologi - reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chlo cal activity according to methods known in the art . ride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to obtain Method 2 : a protein concentration of 1 mg/ml . A 50 - fold molar excess EGF is transferred or dissolved in reaction buffer ( e . g . 50 of a PSA aminooxy reagent with a MW of 20 kD ( described mM Hepes , 350 mM sodium chloride , 5 mM calcium 25 above ) is added followed by m -toluidine as a nucleophilic chloride , pH 6 . 0 ) to get a final protein concentration of catalyst ( 10 mM final concentration ) and NalO4 ( final 1 . 0 + / - 0 . 25 mg/ ml. Then the pH of the solution is corrected concentration : 400 uM ) . The coupling reaction is performed to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 for 2 hours in the dark under gentle shaking at room solution . Subsequently , a 40 mM aqueous sodium periodate temperature . Subsequently , the reaction is quenched with solution is added within 10 minutes to give a concentration 30 cysteine for 60 min at RT ( cysteine concentration : 10 mM ) of 200 uM . The oxidation reaction is carried out for 30 + / - 5 and the conjugate is purified by ion exchange chromatog min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction raphy . The conjugate containing fractions of the eluate are is stopped by addition of an aqueous L -cysteine solution ( 1 collected and subjected to UF /DF by use of a membrane M ) within 15 minutes at T = + 22 + / - 2° C . to give a final made of regenerated cellulose (Millipore ) . The preparation concentration of 10 mM in the reaction mixture and incu - 35 is analytically characterized by measuring total protein bation for 60 + / – 5 min . (Bradford ) and biological activity according to methods The oxidized EGF is further purified by ion exchange known in the art . chromatography . The oxidized EGF containing fractions of Method 4 : the eluate are collected and used for the conjugation reac - EGF is dissolved in or transferred to a reaction buffer (e . g . tion . 40 50 mM Hepes , 350 mM sodium chloride , 5 mM calcium The aminooxy -polysialic acid ( PSA -ONH2 ) reagent is chloride , pH 6 . 0 ) to get a final protein concentration of added in a 50 - fold molar excess to the eluate containing the 1 . 0 + / - 0 . 25 mg/ml . Then the pH of the solution is corrected purified oxidized EGF within a maximum time period ( t ) of to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 15 minutes under gentle stirring . Then an aqueous m - tolui - solution . dine solution (50 mM ) is added within 15 minutes to get a 45 Subsequently the aminooxy - polysialic acid (PSA -ONH2 ) final concentration of 10 mM . The reaction mixture is reagent is added in a 50 - fold molar excess to this EGF incubated for 120 + / - 10 min . at pH 6 . 0 in the dark at a solution within a maximum time period ( t ) of 15 minutes temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking under gentle stirring . Then an aqueous m - toluidine solution (protein concentration : 1 mg/ ml ). ( 50 mm ) is added within 15 minutes to get a final concen The obtained PSA - EGF conjugate is further purified by 50 tration of 10 mM . Finally a 40 mM aqueous sodium perio ion exchange chromatography . The PSA - EGF conjugate date solution is added to give a concentration of 400 UM . containing fractions are collected and concentrated by ultra - / The reaction mixture is incubated for 120 + / – 10 min . in diafiltration (UF /DF ) using a membrane made of regener - the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle ated cellulose with an appropriate molecular weight cut off shaking . Then the reaction is stopped by the addition of an (Millipore ) . 55 aqueous L -cysteine solution ( 1 M ) to give a final concen The conjugate prepared by use of this procedure is tration of 10 mM in the reaction mixture and incubation for analytically characterized by measuring total protein , bio - 60 + / - 5 min . logical activity , and determination of the polysialyation The obtained EGF - conjugate is purified by ion - exchange degree by measuring the PSA content ( resorcinol assay ) . chromatography. The PSA - EGF containing fractions of the Method 3 : 60 eluate are collected and concentrated by ultra - / diafiltration Epidermal growth factor ( EGF) is transferred into reac - (UF /DF ) using a membrane made of regenerated cellulose tion buffer ( 50 mM Hepes , 350 mM sodium chloride , 5 mM (Millipore ) . calcium chloride, pH 6 . 0 ) and diluted to obtain a protein The conjugates prepared by use of this procedure are concentration of 1 mg/ ml . A 50 - fold molar excess of a PSA analytically characterized by measuring total protein , bio aminooxy reagentwith a MW of 20 kD (described above ) is 65 logical activity according to methods known in the art, and added followed by m - toluidine as a nucleophilic catalyst ( 10 determination of the polysialyation degree by measuring the mM final concentration ) and NalO4 ( final concentration : PSA content ( resorcinol assay ) . US 10 ,414 ,793 B2 103 104 Example 22 use of a membrane made of regenerated cellulose (Milli pore ). The preparation is next analytically characterized by Polysialylation of NGF Using Aminooxy - PSA and measuring total protein (Coomassie , Bradford ) and biologi m - Toluidine as a Nucleophilic Catalyst cal activity according to methods known in the art . 5 Method 2 : Method 1 : NGF is transferred or dissolved in reaction buffer ( e . g . 50 A starting concentration of nerve growth factor (NGF ) is mM Hepes , 350 mM sodium chloride , 5 mM calcium transferred into a reaction buffer ( e . g . , 50 mM Hepes , 350 chloride , pH 6 . 0 ) to get a final protein concentration of mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is corrected diluted to obtain a protein concentration of 1 mg/ ml . To this 10 to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 solution , NalO4 is added to give a final concentration of 200 solution . Subsequently , a 40 mM aqueous sodium periodate uM . The oxidation is carried at RT for 30 min in the dark solution is added within 10 minutes to give a concentration under gentle shaking . The reaction is then quenched with of 200 uM . The oxidation reaction is carried out for 30 + / - 5 cysteine ( final concentration : 10 mM ) for 60 min at RT. min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction The solution is next subjected to UF/ DF employing 15 is stopped by addition of an aqueous L - cysteine solution ( 1 Vivaspin centrifugal filtrators to remove excess periodate, M ) within 15 minutes at T = + 22 + / - 2° C . to give a final quencher and the byproducts thereof or , in the alternative , to concentration of 10 mM in the reaction mixture and incu an IEX column with a volume of 20 ml (Merck EMD TMAE bation for 60 + / - 5 min . ( M ) which is equilibrated with Buffer A ( 20 mM Hepes , 5 The oxidized NGF is further purified by ion exchange mM CaCl2 , pH 7 .0 ) . The column is equilibrated with 5 CV 20 chromatography . The oxidized NGFcontaining fractions of Buffer A . The oxidized NGF is eluted with Buffer B ( 20 mM the eluate are collected and used for the conjugation reac Hepes, 5 mM CaC12 , 1M NaCl, pH 7 . 0 ) . The NGF contain - tion . ing fractions are collected . The protein content is determined The aminooxy - polysialic acid (PSA - ONH2) reagent is (Coomassie , Bradford ) and adjusted to 1 mg/ml with reac - added in a 50 - fold molar excess to the eluate containing the tion buffer and adjusted to pH 6 . 0 by dropwise addition of 25 purified oxidized NGF within a maximum time period ( t ) of 0 .5M HC1. 15 minutes under gentle stirring . Then an aqueous m - tolui A 50 - fold molar excess of aminooxy - PSA reagent with a dine solution (50 mM ) is added within 15 minutes to get a MW of 20 kD ( described above ) is added followed by final concentration of 10 mM . The reaction mixture is m - toluidine as a nucleophilic catalyst ( final concentration : incubated for 120 + / – 10 min . at pH 6 . 0 in the dark at a 10 mM ). The coupling reaction is performed for 2 hours in 30 temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking the dark under gentle shaking at room temperature . The (protein concentration : 1 mg/ ml ) . excess of aminooxy reagent is removed by means of HIC . The obtained PSA -NGF conjugate is further purified by The conductivity of the reaction mixture is adjusted by ion exchange chromatography . The PSA - NGF conjugate adding a buffer containing ammonium acetate (50 mM containing fractions are collected and concentrated by ultra - / Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 35 diafiltration (UF / DF ) using a membrane made of regener M ammonium acetate , pH 6 . 9 ) and loaded onto a column a ted cellulose with an appropriate molecular weight cut off filled with 80 ml Phenyl Sepharose FF (GE Healthcare , (Millipore ) . Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes , 2 . 5 M The conjugate prepared by use of this procedure is ammonium acetate , 350 mM sodium chloride , 5 mM cal analytically characterized by measuring total protein , bio cium chloride, pH 6 . 9 . Subsequently , the conjugate is eluted 40 logical activity , and determination of the polysialyation with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . degree by measuring the PSA content ( resorcinol assay ) . Finally , the PSA - NGF containing fractions are collected and Method 3 : subjected to UF /DF by use of a membrane made of regen - Nerve growth factor ( NGF ) is transferred into reaction erated cellulose (Millipore ) . The preparation is next analyti - buffer (50 mM Hepes , 350 mM sodium chloride , 5 mM cally characterized by measuring total protein (Coomassie , 45 calcium chloride , pH 6 . 0 ) and diluted to obtain a protein Bradford ) and biological activity according to methods concentration of 1 mg/ml . A 50 - fold molar excess of ami known in the art . nooxy -PSA reagent with a MW of 20 kD (described above ) In an alternative embodiment, Method 1 is carried out as is added followed by m - toluidine as a nucleophilic catalyst follows. Nerve growth factor (NGF ) is transferred into a ( 10 mM final concentration ) and NalO4 ( final concentration : reaction buffer ( e . g ., 50 mM Hepes, 350 mM sodium chlo - 50 400 uM ) . The coupling reaction is performed for 2 hours in ride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to obtain the dark under gentle shaking at room temperature . Subse a protein concentration of 1 mg/ ml . To this solution , NalO4 quently , the reaction is quenched with cysteine for 60 min at is added to give a final concentration of 200 UM . The RT ( cysteine concentration : 10 mM ) . Then the conductivity oxidation is carried at RT for 30 min in the dark under gentle of the reaction mixture is adjusted by adding a buffer shaking . The reaction is then quenched with cysteine ( final 55 containing ammonium acetate (50 mM Hepes, 350 mM concentration : 10 mM ) for 60 min at RT. sodium chloride , 5 mM calcium chloride , 8 M ammonium The solution is next subjected to UF /DF employing acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl Vivaspin centrifugal filtrators to remove excess periodate , Sepharose FF (GE Healthcare , Fairfield , Conn . ) pre - equili quencher and the byproducts thereof. brated with 50 mM Hepes , 2 . 5 M ammonium acetate , 350 A 50 - fold molar excess of aminooxy - PSA reagent with a 60 mM sodium chloride , 5 mM calcium chloride , 0 .01 % Tween MW of 20 kD (described above ) is added followed by 80 , pH 6 . 9 . Subsequently the conjugate is eluted with 50 m - toluidine as a nucleophilic catalyst ( final concentration : mM Hepes, 5 mM calcium chloride , pH 7 . 5 . Finally , the 10 mM ). The coupling reaction is performed for 2 hours in PSA NGF -containing fractions are collected and subjected the dark under gentle shaking at room temperature . The to UF / DF by use of a membrane made of regenerated excess of aminooxy reagent is removed by means of ion 65 cellulose (Millipore ) . The preparation is analytically char exchange chromatography . The PSA - NGF containing frac - acterized by measuring total protein (Bradford ) and biologi tions of the eluate are collected and subjected to UF /DF by cal activity according to methods known in the art. US 10 ,414 ,793 B2 105 106 In an alternative embodiment, Method 3 is carried out as 30 min in the dark under gentle shaking . The reaction is then follows . Nerve growth factor (NGF ) is transferred into quenched with cysteine ( final concentration : 10 mM ) for 60 reaction buffer ( e . g . 50 mM Hepes, 350 mM sodium chlo - min at RT. ride, 5 mM calcium chloride , pH 6 . 0 ) and diluted to obtain The solution is next subjected to UF /DF employing a protein concentration of 1 mg/ ml . A 50 - fold molar excess 5 Vivaspin centrifugal filtrators to remove excess periodate , of aminooxy -PSA reagent with a MW of 20 kb ( described quencher and the byproducts thereof or, in the alternative , to above ) is added followed by m - toluidine as a nucleophilic an IEX column with a volume of 20 ml (Merck EMD TMAE ( M )) which is equilibrated with Buffer A (20 mM Hepes, 5 catalyst (10 mM final concentration ) and Nal04 ( final mM CaC12 , pH 7 . 0 ) . The column is equilibrated with 5 CV concentration : 400 UM ). The coupling reaction is performed 10 Buffer A . The oxidized HGH is eluted with Buffer B (20 mM for 2 hours in the dark under gentle shaking at room Hepes, 5 mM CaCl2 , 1 M NaCl, pH 7 . 0 ) . The HGH temperature . Subsequently , the reaction is quenched with containing fractions are collected . The protein content is cysteine for 60 min at RT ( cysteine concentration : 10 mm ) determined (Coomassie , Bradford ) and adjusted to 1 mg/ ml and the conjugate is purified by ion exchange chromatog with reaction buffer and adjusted to pH 6 . 0 by dropwise raphy . Then the PSA - NGF containing fractionsTractions of the eluateeluate 15 addition of 0 . 5 M HCl. are collected and subjected to UF /DF by use of a membrane A 50 - fold molar excess of aminooxy - PSA reagent with a made of regenerated cellulose (Millipore ). The preparation MW of 20 kD (described above) is added followed by is analytically characterized by measuring total protein m -toluidine as a nucleophilic catalyst ( final concentration : (Bradford ) and biological activity according to methods 10 mM ) . The coupling reaction is performed for 2 hours in known in the art . 20 the dark under gentle shaking at room temperature . The Method 4 : excess of aminooxy reagent is removed by means of HIC . NGF is dissolved in or transferred to a reaction buffer ( e . g . The conductivity of the reaction mixture is adjusted by 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium adding a buffer containing ammonium acetate (50 mM chloride , pH 6 . 0 ) to get a final protein concentration of Hepes , 350 mM sodium chloride, 5 mM calcium chloride , 8 1. 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected 25 M ammonium acetate , pH 6 .9 ) and loaded onto a column to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 filled with 80 ml Phenyl Sepharose FF (GE Healthcare , solution . Fairfield , Conn . ) pre - equilibrated with 50 mM Hepes , 2 . 5 M Subsequently , the aminooxy -polysialic acid ( PSA -ONH2 ) ammonium acetate , 350 mM sodium chloride , 5 mM cal reagent is added in a 50 - fold molar excess to this NGF - cium chloride, pH 6 . 9 . Subsequently the conjugate is eluted solution within a maximum time period (t ) of 15 minutes 30 with 50 mM Hepes buffer pH 7 .5 containing 5 mM CaC12 . under gentle stirring . Then an aqueous m - toluidine solution Finally , the PSA -HGH containing fractions are collected and (50 mM ) is added within 15 minutes to get a final concen - subjected to UF /DF by use of a membrane made of regen tration of 10 mM . Finally a 40 mM aqueous sodium perio erated cellulose (Millipore ) . The preparation is next analyti date solution is added to give a concentration of 400 uM . cally characterized by measuring total protein (Coomassie , The reaction mixture is incubated for 120 + / – 10 min . in 35 Bradford ) and biological activity according to methods the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle known in the art. shaking . Then the reaction is stopped by the addition of an In an alternative embodiment, Method 1 is carried out as aqueous L - cysteine solution ( 1 M ) to give a final concen - follows. As described herein , the amino acid sequence of tration of 10 mM in the reaction mixture and incubation for human growth hormone (HGH ) is first modified to incor 60 + / - 5 min . 40 porate at least one glycosylation site . Following purification , The obtained NGF - conjugate is purified by ion - exchange HGH is glycosylated in vitro according to methods known chromatography . The PSA -NGF containing fractions of the in the art . HGH is transferred into a reaction buffer (e . g ., 50 eluate are collected and concentrated by ultra - /diafiltration mM Hepes, 350 mM sodium chloride , 5 mM calcium (UF /DF ) using a membrane made of regenerated cellulose chloride , pH 6 . 0 ) and diluted to obtain a protein concentra (Millipore ) . 45 tion of 1 mg/ ml . To this solution , NalO4 is added to give a The conjugates prepared by use of this procedure are final concentration of 200 uM . The oxidation is carried at RT analytically characterized by measuring total protein , bio for 30 min in the dark under gentle shaking . The reaction is logical activity according to methods known in the art , and then quenched with cysteine ( final concentration : 10 mM ) determination of the polysialyation degree by measuring the for 60 min at RT. PSA content ( resorcinol assay ) . 50 The solution is next subjected to UF /DF employing Vivaspin centrifugal filtrators to remove excess periodate , Example 23 quencher and the byproducts thereof . A 50 -fold molar excess of aminooxy -PSA reagent with a Polysialylation of HGH Using Aminooxy - PSA and MW of 20 kD (described above ) is added followed by m - Toluidine as a Nucleophilic Catalyst 55 m - toluidine as a nucleophilic catalyst ( final concentration : 10 mM ) . The coupling reaction is performed for 2 hours in Method 1 : the dark under gentle shaking at room temperature . The As described herein , the amino acid sequence of human excess of aminooxy reagent is removed by means of ion growth hormone (HGH ) is first modified to incorporate at exchange chromatography . The PSA -HGH containing frac least one glycosylation site . Following purification , HGH is 60 tions of the eluate are collected and subjected to UF /DF by glycosylated in vitro according to methods known in the art . use of a membrane made of regenerated cellulose (Milli A starting concentration of human growth hormone pore ) . The preparation is next analytically characterized by ( HGH ) is transferred into a reaction buffer ( e . g ., 50 mM measuring total protein ( Coomassie , Bradford ) and biologi Hepes , 350 mM sodium chloride , 5 mM calcium chloride , cal activity according to methods known in the art . pH 6 . 0 ) and diluted to obtain a protein concentration of 1 65 Method 2 : mg/ml . To this solution , NalO4 is added to give a final As described herein , the amino acid sequence of human concentration of 200 uM . The oxidation is carried at RT for growth hormone (HGH ) is first modified to incorporate at US 10 ,414 ,793 B2 107 108 least one glycosylation site . Following purification , HGH is is analytically characterized by measuring total protein glycosylated in vitro according to methods known in the art. (Bradford ) and biological activity according to methods HGH is transferred or dissolved in reaction buffer (e .g . 50 known in the art. mM Hepes, 350 mM sodium chloride, 5 mM calcium In an alternative embodiment, Method 3 is carried out as chloride. pH 6 . 0 ) to get a final protein concentration of 5 follows. As described herein , the amino acid sequence of human growth hormone (HGH ) is first modified to incor 1 . 0 + / - 0 . 25 mg/ ml. Then the pH of the solution is corrected porate at least one glycosylation site . Following purification , to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 HGH is glycosylated in vitro according to methods known solution . Subsequently , a 40 mM aqueous sodium periodate in the art . HGH is transferred into reaction buffer ( e . g . 50 solution is added within 10 minutes to give a concentration mM Hepes, 350 mM sodium chloride , 5 mM calcium of 200 uM . The oxidation reaction is carried out for 30 + / - 5 " chloride , pH 6 . 0 ) and diluted to obtain a protein concentra min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction tion of 1 mg/ ml . A 50 fold molar excess of aminooxy - PSA is stopped by addition of an aqueous L -cysteine solution ( 1 reagent with a MW of 20 kD (described above ) is added M ) within 15 minutes at T = + 22 + / - 2° C . to give a final followed by m - toluidine as a nucleophilic catalyst ( 10 mM final concentration ) and NaI04 ( final concentration : 400 concentration of 10 mM in the reaction mixture and incu - 1516 UM ) . The coupling reaction is performed for 2 hours in the bation for 60 + / – 5 min . dark under gentle shaking at room temperature . Subse The oxidized HGH is further purified by ion exchange quently, the reaction is quenched with cysteine for 60 min at chromatography. The oxidized HGH containing fractions of RT ( cysteine concentration : 10 mm ) and the conjugate is the eluate are collected and used for the conjugation reac purified by ion exchange chromatography . Then the PSA tion . 20 HGH - containing fractions of the eluate are collected and The aminooxy -polysialic acid ( PSA -ONH2 ) reagent is subjected to UF /DF by use of a membrane made of regen added in a 50 - fold molar excess to the eluate containing the erated cellulose (Millipore ) . The preparation is analytically purified oxidized HGH within a maximum time period ( t ) of characterized by measuring total protein (Bradford ) and 15 minutes under gentle stirring . Then an aqueous m -tolui - biological activity according to methods known in the art. dine solution (50 mM ) is added within 15 minutes to get a 25 Method 4 : final concentration of 10 mM . The reaction mixture is As described herein , the amino acid sequence of human incubated for 120 + / – 10 min . at pH 6 . 0 in the dark at a growth hormone (HGH ) is first modified to incorporate at temperature ( 1 ) of 1 = + 22 + / - 2° C . under gentle shaking least one glycosylation site . Following purification , HGH is ( protein concentration : 1 mg/ ml ) . glycosylated in vitro according to methods known in the art . The obtained PSA -HGH conjugate is further purified by 30 HGH is dissolved in or transferred to a reaction buffer ion exchange chromatography. The PSA -HGH conjugate ( e. g . 50 mM Hepes, 350 mM sodium chloride, 5 mM containing fractions are collected and concentrated by ultra - calcium chloride, pH 6 . 0 ) to get a final protein concentration diafiltration (UF / DF ) using a membrane made of regener - of 1 .0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor ated cellulose with an appropriate molecular weight cut off rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HCI (Millipore ). 35 solution . The conjugate prepared by use of this procedure is Subsequently , the aminooxy -polysialic acid ( PSA - ONH2 ) analytically characterized by measuring total protein , bio - reagent is added in a 50 - fold molar excess to this HGH logical activity , and determination of the polysialyation solution within a maximum time period (t ) of 15 minutes degree by measuring the PSA content ( resorcinol assay ) . under gentle stirring . Then an aqueous m - toluidine solution Method 3 : 40 (50 mM ) is added within 15 minutes to get a final concen As described herein , the amino acid sequence of human tration of 10 mM . Finally a 40 mM aqueous sodium perio growth hormone (HGH ) is first modified to incorporate at date solution is added to give a concentration of 400 UM . least one glycosylation site . Following purification , HGH is The reaction mixture is incubated for 120 + / – 10 min . in glycosylated in vitro according to methods known in the art . the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle Human growth hormone (HGH ) is transferred into reac - 45 shaking. Then the reaction is stopped by the addition of an tion buffer (50 mM Hepes , 350 mM sodium chloride , 5 mM aqueous L -cysteine solution ( 1 M ) to give a final concen calcium chloride , pH 6 .0 ) and diluted to obtain a protein tration of 10 mM in the reaction mixture and incubation for concentration of 1 mg/ ml . A 50 fold molar excess of 60 + / - 5 min . aminooxy - PSA reagent with a MW of 20 kD ( described The obtained HGH - conjugate is purified by ion - exchange above ) is added followed by m - toluidine as a nucleophilic 50 chromatography . The PSA - HGH containing fractions of the catalyst ( 10 mM final concentration ) and NalO4 ( final eluate are collected and concentrated by ultra - / diafiltration concentration : 400 uM ) . The coupling reaction is performed (UF /DF ) using a membrane made of regenerated cellulose for 2 hours in the dark under gentle shaking at room (Millipore ) . temperature . Subsequently, the reaction is quenched with The conjugates prepared by use of this procedure are cysteine for 60 min at RT ( cysteine concentration : 10 mM ) . 55 analytically characterized by measuring total protein , bio Then the conductivity of the reaction mixture is adjusted by logical activity according to methods known in the art, and adding a buffer containing ammonium acetate (50 mM determination of the polysialyation degree by measuring the Hepes , 350 mM sodium chloride, 5 mM calcium chloride , 8 PSA content (resorcinol assay ) . M ammonium acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl Sepharose FF (GE Healthcare , Fairfield , 60 Example 24 Conn .) pre- equilibrated with 50 mM Hepes , 2 . 5 M ammo nium acetate , 350 mM sodium chloride , 5 mM calcium Polysialylation of TNF - Alpha Using chloride, 0 .01 % Tween 80 , pH 6 .9 . Subsequently the con Aminooxy - PSA and m - Toluidine as a Nucleophilic jugate is eluted with 50 mM Hepes , 5 mM calcium chloride , Catalyst pH 7 . 5 . Finally, the PSA HGH - containing fractions are 65 collected and subjected to UF /DF by use of a membrane A starting concentration of tumor necrosis factor- alpha made of regenerated cellulose (Millipore ). The preparation ( TNF- alpha ) is transferred into a reaction buffer (e . g. , 50 US 10 , 414 ,793 B2 109 110 mM Hepes , 350 mM sodium chloride , 5 mM calcium calcium chloride, pH 6 . 0 ) to get a final protein concentration chloride , pH 6 . 0 ) and diluted to obtain a protein concentra - of 1 . 0 + / - 0 . 25 mg /ml . Then the pH of the solution is cor tion of 1 mg/ ml . To this solution , NalO4 is added to give a rected to 6 .0 by drop wise addition of a 0 . 5 N aqueous HCI final concentration of 200 uM . The oxidation is carried at RT solution . Subsequently , a 40 mM aqueous sodium periodate for 30 min in the dark under gentle shaking . The reaction is 5 solution is added within 10 minutes to give a concentration then quenched with cysteine ( final concentration : 10 mM of 200 uM . The oxidation reaction is carried out for 30 + / - 5 for 60 min at RT. min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction The solution is next subjected to UF /DF employing is stopped by addition of an aqueous L - cysteine solution ( 1 Vivaspin centrifugal filtrators to remove excess periodate , M ) within 15 minutes at T = + 22 + / - 2° C . to give a final quencher and the byproducts thereof or , in the alternative , to 10 concentration of 10 mM in the reaction mixture and incu an IEX column with a volume of 20 ml (Merck EMD TMAE bation for 60 + / - 5 min . ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes , 5 The oxidized TNF - alpha is further purified by ion mM CaCl2 , pH 7 .0 ) . The column is equilibrated with 5 CV exchange chromatography. The oxidized TNF - alpha con Buffer A . The oxidized TNF -alpha is eluted with Buffer B taining fractions of the eluate are collected and used for the ( 20 mM Hepes , 5 mM CaC12 , IM NaCl, pH 7 . 0 ) . The 15 conjugation reaction . TNF - alpha containing fractions are collected . The protein The aminooxy -polysialic acid ( PSA -ONH2 ) reagent is content is determined (Coomassie , Bradford ) and adjusted to added in a 50 - fold molar excess to the eluate containing the 1 mg/ ml with reaction buffer and adjusted to pH 6 . 0 by purified oxidized TNF -alpha within a maximum time period dropwise addition of 0 .5M HC1. ( t ) of 15 minutes under gentle stirring . Then an aqueous A 50 - fold molar excess of aminooxy - PSA reagent with a 20 m - toluidine solution (50 mM ) is added within 15 minutes to MW of 20 kD (described above ) is added followed by get a final concentration of 10 mM . The reaction mixture is m - toluidine as a nucleophilic catalyst ( final concentration : incubated for 120 + / – 10 min . at pH 6 . 0 in the dark at a 10 mM ). The coupling reaction is performed for 2 hours in temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking the dark under gentle shaking at room temperature . The (protein concentration : 1 mg/ ml ) . excess of aminooxy reagent is removed by means of HIC . 25 The obtained PSA - TNF -alpha conjugate is further puri The conductivity of the reaction mixture is adjusted by fied by ion exchange chromatography . The PSA - TNF - alpha adding a buffer containing ammonium acetate (50 mM conjugate containing fractions are collected and concen Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 trated by ultra - / diafiltration (UF /DF ) using a membrane M ammonium acetate , pH 6 . 9 ) and loaded onto a column made of regenerated cellulose with an appropriate molecular filled with 80 ml Phenyl Sepharose FF (GE Healthcare , 30 weight cut off (Millipore ). Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes, 2 . 5 M The conjugate prepared by use of this procedure is ammonium acetate , 350 mM sodium chloride , 5 mM cal analytically characterized by measuring total protein , bio cium chloride, pH 6 . 9 . Subsequently , the conjugate is eluted logical activity , and determination of the polysialyation with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . degree by measuring the PSA content ( resorcinol assay ) . Finally the PSA - TNF -alpha -containing fractions are col- 35 Method 3 : lected and subjected to UF/ DF by use of a membrane made Tumor necrosis factor -alpha ( TNF -alpha ) is transferred of regenerated cellulose (Millipore ) . The preparation is next into reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium analytically characterized by measuring total protein ( Coo chloride, 5 mM calcium chloride, pH 6 . 0 ) and diluted to massie , Bradford ) and biological activity according to meth - obtain a protein concentration of 1 mg/ ml . A 50 - fold molar ods known in the art. 40 excess of aminooxy - PSA reagent with a MW of 20 kD In an alternative embodiment, Method 1 is carried out as described above ) is added followed by m - toluidine as a follows. Tumor necrosis factor -alpha ( TNF - alpha ) is trans - nucleophilic catalyst ( 10 mM final concentration ) and ferred into a reaction buffer (e .g . 50 mM Hepes , 350 mM Nal04 ( final concentration : 400 UM ) . The coupling reaction sodium chloride, 5 mM calcium chloride , pH 6 . 0 ) and is performed for 2 hours in the dark under gentle shaking at diluted to obtain a protein concentration of 1 mg /ml . To this 45 room temperature . Subsequently , the reaction is quenched solution , NalO4 is added to give a final concentration of 200 with cysteine for 60 min at RT (cysteine concentration : 10 uM . The oxidation is carried at RT for 30 min in the dark mM ). Then the conductivity of the reaction mixture is under gentle shaking . The reaction is then quenched with adjusted by adding a buffer containing ammonium acetate cysteine ( final concentration : 10 mM ) for 60 min at RT. (50 mM Hepes , 350 mM sodium chloride , 5 mM calcium The solution is next subjected to UF /DF employing 50 chloride , 8 M ammonium acetate , pH 6 . 9 ) and loaded onto Vivaspin centrifugal filtrators to remove excess periodate , a column filled with Phenyl Sepharose FF (GE Healthcare , quencher and the byproducts thereof. A 50 - fold molar excess Fairfield , Conn .) pre - equilibrated with 50 mM Hepes, 2 . 5 M of aminooxy - PSA reagent with a MW of 20 kD ( described ammonium acetate , 350 mM sodium chloride , 5 mM cal above ) is added followed by m -toluidine as a nucleophilic cium chloride , 0 . 01 % Tween 80 , pH 6 . 9 . Subsequently the catalyst ( final concentration : 10 mM ) . The coupling reaction 55 conjugate is eluted with 50 mM Hepes , 5 mM calcium is performed for 2 hours in the dark under gentle shaking at chloride , pH 7 . 5 . Finally the PSA - TNF -alpha -containing room temperature . The excess of aminooxy reagent is fractions are collected and subjected to UF/ DF by use of a removed by means of ion exchange chromatography. The membrane made of regenerated cellulose (Millipore ) . The PSA - TNF -alpha containing fractions of the eluate are col preparation is analytically characterized by measuring total lected and subjected to UF /DF by use of a membrane made 60 protein ( Bradford ) and biological activity according to meth of regenerated cellulose (Millipore ) . The preparation is next ods known in the art . analytically characterized by measuring total protein ( Coo - In an alternative embodiment, Method 3 is carried out as massie , Bradford ) and biological activity according to meth follows. Tumor necrosis factor - alpha ( TNF -alpha ) is trans ods known in the art . ferred into reaction buffer (e . g . 50 mM Hepes , 350 mM Method 2 : 65 sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and TNF - alpha is transferred or dissolved in reaction buffer diluted to obtain a protein concentration of 1 mg/ ml . A ( e. g . 50 mM Hepes, 350 mM sodium chloride , 5 mM 50 - fold molar excess of aminooxy -PSA reagent with a MW US 10 ,414 ,793 B2 111 112 of 20 kD (described above ) is added followed by m -tolui - (M ) ) which is equilibrated with Buffer A (20 mM Hepes, 5 dine as a nucleophilic catalyst ( 10 mM final concentration ) mM CaCl2 , pH 7 . 0 ) . The column is equilibrated with 5 CV and NalO4 ( final concentration : 400 UM ) . The coupling Buffer A . The oxidized insulin is eluted with Buffer B ( 20 reaction is performed for 2 hours in the dark under gentle mM Hepes , 5 mM CaCl2 , 1 M NaCl, pH 7 . 0 ) . The insulin shaking at room temperature . Subsequently , the reaction is 5 containing fractions are collected . The protein content is quenched with cysteine for 60 min at RT (cysteine concen - determined (Coomassie , Bradford ) and adjusted to 1 mg/ ml tration : 10 mM ). and the conjugate is purified by ion with reaction buffer and adjusted to pH 6 . 0 by dropwise exchange chromatography . The PSA - TNF - alpha containing addition of 0 . 5 M HCl. fractions of the eluate are collected and subjected to UF /DF A 50 -fold molar excess of aminooxy - PSA reagent with a by use of a membrane made of regenerated cellulose (Mil - 10 MW of 20 kD (described above ) is added followed by lipore ) . The preparation is analytically characterized by m - toluidine as a nucleophilic catalyst ( final concentration : measuring total protein (Bradford ) and biological activity 10 mM ) . The coupling reaction is performed for 2 hours in according to methods known in the art. the dark under gentle shaking at room temperature . The Method 4 : excess of aminooxy reagent is removed by means of HIC . TNF - alpha is dissolved in or transferred to a reaction 15 The conductivity of the reaction mixture is adjusted by buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride, 5 mM adding a buffer containing ammonium acetate (50 mM calcium chloride , pH 6 . 0 ) to get a final protein concentration Hepes , 350 mM sodium chloride , 5 mM calcium chloride, 8 of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is cor M ammonium acetate , pH 6 . 9 ) and loaded onto a column rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 filled with 80 ml Phenyl Sepharose FF (GE Healthcare , solution . 20 Fairfield , Conn . ) pre - equilibrated with 50 mM Hepes, 2 . 5 M Subsequently the aminooxy - polysialic acid ( PSA -ONH2 ) ammonium acetate , 350 mM sodium chloride , 5 mM cal reagent is added in a 50 - fold molar excess to this TNF cium chloride, pH 6 . 9. Subsequently the conjugate is eluted alpha - solution within a maximum time period ( t ) of 15 with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . minutes under gentle stirring . Then an aqueous m - toluidine Finally the PSA - insulin containing fractions are collected solution (50 mM ) is added within 15 minutes to get a final 25 and subjected to UF /DF by use of a membrane made of concentration of 10 mM . Finally a 40 mM aqueous sodium regenerated cellulose (Millipore ). The preparation is next periodate solution is added to give a concentration of 400 analytically characterized by measuring total protein ( Coo UM . massie, Bradford ) and biological activity according to meth The reaction mixture is incubated for 120 + / - 10 min . in ods known in the art . the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle 30 In an alternative embodiment, Method 1 is carried out as shaking . Then the reaction is stopped by the addition of an follows. As described herein , the amino acid sequence of aqueous L -cysteine solution ( 1 M ) to give a final concen insulin is first modified to incorporate at least one glycosy tration of 10 mM in the reaction mixture and incubation for lation site . Following purification, insulin is glycosylated in 60 + / - 5 min . vitro according to methods known in the art. Insulin is The obtained TNF - alpha conjugate is purified by ion - 35 transferred into a reaction buffer ( e . g ., 50 mM Hepes, 350 exchange chromatography . The PSA - TNF - alpha containing mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and fractions of the eluate are collected and concentrated by diluted to obtain a protein concentration of 1 mg/ml . To this ultra - / diafiltration (UF / DF ) using a membrane made of solution , NalO4 is added to give a final concentration of 200 regenerated cellulose (Millipore ) . UM . The oxidation is carried at RT for 30 min in the dark The conjugates prepared by use of this procedure are 40 under gentle shaking . The reaction is then quenched with analytically characterized by measuring total protein , bio - cysteine ( final concentration : 10 mM ) for 60 min at RT. logical activity according to methods known in the art , and The solution is next subjected to UF /DF employing determination of the polysialyation degree by measuring the Vivaspin centrifugal filtrators to remove excess periodate , PSA content ( resorcinol assay ) . quencher and the byproducts thereof. A 50 - fold molar excess of aminooxy - PSA reagent with a Example 25 MW of 20 kD ( described above ) is added followed by m - toluidine as a nucleophilic catalyst ( final concentration : Polysialylation of Insulin Using Aminooxy -PSA 10 mM ). The coupling reaction is performed for 2 hours in and m - Toluidine as a Nucleophilic Catalyst the dark under gentle shaking at room temperature . The 50 excess of aminooxy reagent is removed by means of ion Method 1 : exchange chromatography . The PSA - insulin containing As described herein , the amino acid sequence of insulin is fractions of the eluate are collected and subjected to UF /DF first modified to incorporate at least one glycosylation site . by use of a membrane made of regenerated cellulose (Mil Following purification , insulin is glycosylated in vitro lipore ) . The preparation is next analytically characterized by according to methods known in the art . A starting concen - 55 measuring total protein (Coomassie , Bradford ) and biologi tration of insulin is transferred into a reaction buffer ( e. g . , 50 cal activity according to methods known in the art. mM Hepes , 350 mM sodium chloride , 5 mM calcium Method 2 : chloride, pH 6 . 0 ) and diluted to obtain a protein concentra As described herein , the amino acid sequence of insulin is tion of 1 mg/ml . To this solution , NalO4 is added to give a first modified to incorporate at least one glycosylation site . final concentration of 200 uM . The oxidation is carried at RT 60 Following purification , insulin is glycosylated in vitro for 30 min in the dark under gentle shaking . The reaction is according to methods known in the art . then quenched with cysteine ( final concentration : 10 mM ) Insulin is transferred or dissolved in reaction buffer ( e . g . for 60 min at RT. 50 mM Hepes, 350 mM sodium chloride , 5 mM calcium The solution is next subjected to UF /DF employing chloride , pH 6 . 0 ) to get a final protein concentration of Vivaspin centrifugal filtrators to remove excess periodate , 65 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is corrected quencher and the byproducts thereof or, in the alternative , to t o 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 an IEX column with a volume of 20 ml (Merck EMD TMAE solution . Subsequently , a 40 mM aqueous sodium periodate US 10 ,414 ,793 B2 113 114 solution is added within 10 minutes to give a concentration pH 6 . 0 ) and diluted to obtain a protein concentration of 1 of 200 uM . The oxidation reaction is carried out for 30 + / - 5 mg/ ml . A 50 - fold molar excess of aminooxy -PSA reagent min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction with a MW of 20 kD (described above ) is added followed by is stopped by addition of an aqueous L -cysteine solution ( 1 m - toluidine as a nucleophilic catalyst ( 10 mM final concen M ) within 15 minutes at T = + 22 + / - 2° C . to give a final 5 tration ) and NaI04 ( final concentration : 400 UM ) . The concentration of 10 mM in the reaction mixture and incu - coupling reaction is performed for 2 hours in the dark under bation for 60 + / - 5 min . gentle shaking at room temperature . Subsequently , the reac The oxidized insulin is further purified by ion exchange tion is quenched with cysteine for 60 min at RT (cysteine chromatography . The oxidized insulin containing fractions concentration : 10 mM ) and the conjugate is purified by ion of the eluate are collected and used for the conjugation 10 exchange chromatography . PSA - insulin containing fractions reaction . of the eluate are collected and subjected to UF /DF by use of The aminooxy -polysialic acid (PSA -ONH2 ) reagent is a membrane made of regenerated cellulose (Millipore ) . The added in a 50 - fold molar excess to the eluate containing the preparation is analytically characterized by measuring total purified oxidized insulin within a maximum time period (t ) protein (Bradford ) and biological activity according to meth of 15 minutes under gentle stirring . Then an aqueous 15 ods known in the art . m - toluidine solution ( 50 mM ) is added within 15 minutes to Method 4 : get a final concentration of 10 mM . The reaction mixture is As described herein , the amino acid sequence of insulin is incubated for 120 + / – 10 min . at pH 6 . 0 in the dark at a first modified to incorporate at least one glycosylation site . temperature ( T ) of T = + 22 +/ - 2° C . under gentle shaking Following purification , insulin is glycosylated in vitro ( protein concentration : 1 mg/ ml ) . 20 according to methods known in the art . The obtained PSA - insulin conjugate is further purified by Insulin is dissolved in or transferred to a reaction buffer ion exchange chromatography . The PSA -insulin conjugate (e .g . 50 mM Hepes, 350 mM sodium chloride , 5 mM containing fractions are collected and concentrated by ultra - / calcium chloride, pH 6 . 0 ) to get a final protein concentration diafiltration (UF /DF ) using a membrane made of regener - of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is cor ated cellulose with an appropriate molecular weight cut off 25 rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 (Millipore ). solution . The conjugate prepared by use of this procedure is Subsequently , the aminooxy - polysialic acid (PSA -ONH2 ) analytically characterized by measuring total protein , bio - reagent is added in a 50 - fold molar excess to this insulin logical activity, and determination of the polysialyation solution within a maximum time period ( t ) of 15 minutes degree by measuring the PSA content ( resorcinol assay ). 30 under gentle stirring . Then an aqueous m - toluidine solution Method 3 : (50 mM ) is added within 15 minutes to get a final concen As described herein , the amino acid sequence of insulin is tration of 10 mM . Finally a 40 mM aqueous sodium perio first modified to incorporate at least one glycosylation site . date solution is added to give a concentration of 400 UM . Following purification , insulin is glycosylated in vitro The reaction mixture is incubated for 120 + / – 10 min . in according to methods known in the art. 35 the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle Insulin is transferred into reaction buffer (50 mM Hepes , shaking . Then the reaction is stopped by the addition of an 350 mM sodium chloride , 5 mM calcium chloride, pH 6 . 0 ) aqueous L - cysteine solution ( 1 M ) to give a final concen and diluted to obtain a protein concentration of 1 mg /ml . A tration of 10 mM in the reaction mixture and incubation for 50 - fold molar excess of aminooxy -PSA reagent with a MW 60 + / – 5 min . of 20 kD (described above ) is added followed by m - tolui- 40 The obtained insulin conjugate is purified by ion - ex dine as a nucleophilic catalyst ( 10 mM final concentration ) change chromatography. The PSA -insulin containing frac and NalO4 ( final concentration : 400 M ). The coupling tions of the eluate are collected and concentrated by ultra -/ reaction is performed for 2 hours in the dark under gentle diafiltration (UF /DF ) using a membrane made of shaking at room temperature . Subsequently , the reaction is regenerated cellulose (Millipore ) . quenched with cysteine for 60 min at RT ( cysteine concen - 45 The conjugates prepared by use of this procedure are tration : 10 mM ) . Then the conductivity of the reaction analytically characterized by measuring total protein , bio mixture is adjusted by adding a buffer containing ammo logical activity according to methods known in the art , and nium acetate (50 mM Hepes, 350 mM sodium chloride , 5 determination of the polysialyation degree by measuring the mM calcium chloride , 8 M ammonium acetate , pH 6 . 9 ) and PSA content (resorcinol assay ). loaded onto a column filled with Phenyl Sepharose FF (GE 50 Healthcare , Fairfield , Conn . ) pre - equilibrated with 50 mM Example 26 Hepes, 2 .5 M ammonium acetate , 350 mM sodium chloride , 5 mM calcium chloride, 0 .01 % Tween 80 , pH 6 .9 . Subse Polysialylation of Interferon - Alpha Using quently the conjugate is eluted with 50 mM Hepes, 5 mM Aminooxy - PSA and m - Toluidine as a Nucleophilic calcium chloride , pH 7 . 5 . Finally , the PSA - insulin contain - 55 Catalyst ing fractions are collected and subjected to UF /DF by use of a membrane made of regenerated cellulose (Millipore ) . The Method 1 : preparation is analytically characterized by measuring total A starting concentration of interferon - alpha is transferred protein (Bradford ) and biological activity according to meth - into a reaction buffer ( e . g ., 50 mM Hepes, 350 mM sodium ods known in the art . 60 chloride , 5 mM calcium chloride, pH 6 . 0 ) and diluted to In an alternative embodiment, Method 3 is carried out as obtain a protein concentration of 1 mg/ ml . To this solution , follows. As described herein , the amino acid sequence of NalO4 is added to give a final concentration of 200 uM . The insulin is first modified to incorporate at least one glycosy - oxidation is carried at RT for 30 min in the dark under gentle lation site . Following purification , insulin is glycosylated in shaking . The reaction is then quenched with cysteine ( final vitro according to methods known in the art . 65 concentration : 10 mM ) for 60 min at RT . Insulin is transferred into reaction buffer ( e . g . 50 mM The solution is next subjected to UF /DF employing Hepes, 350 mM sodium chloride, 5 mM calcium chloride , Vivaspin centrifugal filtrators to remove excess periodate , US 10 ,414 ,793 B2 115 116 quencher and the byproducts thereof or, in the alternative , to M ) within 15 minutes at T = + 22 + / - 2° C . to give a final an IEX column with a volumeof 20 ml( Merck EMD TMAE concentration of 10 mM in the reaction mixture and incu ( M ) ) which is equilibrated with Buffer A (20 mM Hepes, 5 bation for 60 + / - 5 min . mM CaC12 , pH 7 .0 ). The column is equilibrated with 5 CV The oxidized interferon - alpha is further purified by ion Buffer A . The oxidized interferon - alpha is eluted with Buffer 5 exchange chromatography . The oxidized interferon -alpha B ( 20 mM Hepes, 5 mM CaC12 , 1M NaCl. pH 7 . 0 ) . The containing fractions of the eluate are collected and used for the conjugation reaction . interferon - alpha containing fractions are collected . The pro The aminooxy -polysialic acid (PSA -ONH2 ) reagent is tein content is determined (Coomassie , Bradford ) and added in a 50 - fold molar excess to the eluate containing the adjusted to 1 mg/ ml with reaction buffer and adjusted to pH 10 purified oxidized interferon - gamma within a maximum time 6 . 0 by dropwise addition of 0 . 5 M HCl. period (t ) of 15 minutes under gentle stirring. Then an A 50 - fold molar excess of aminooxy - PSA reagent with a aqueous m - toluidine solution (50 mM ) is added within 15 MW of 20 kD ( described above ) is added followed by minutes to get a final concentration of 10 mM . The reaction m - toluidine as a nucleophilic catalyst ( final concentration : mixture is incubated for 120 + / – 10 min . at pH 6 . 0 in the dark 10 mM ) . The coupling reaction is performed for 2 hours iin 15 at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking the dark under gentle shaking at room temperature . The (protein concentration : 1 mg/ ml ) . excess of aminooxy reagent is removed by means of HIC . The obtained PSA - interferon - alpha conjugate is further The conductivity of the reaction mixture is adjusted by purified by ion exchange chromatography. The PSA - inter adding a buffer containing ammonium acetate (50 mM feron -alpha conjugate containing fractions are collected and Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 20 concentrated by ultra - /diafiltration (UF /DF ) using a mem M ammonium acetate , pH 6 . 9 ) and loaded onto a column brane made of regenerated cellulose with an appropriate filled with 80 ml Phenyl Sepharose FF (GE Healthcare , molecular weight cut off (Millipore ) . Fairfield , Conn .) pre- equilibrated with 50 mM Hepes, 2 .5 M Method 3 : ammonium acetate , 350 mM sodium chloride , 5 mM cal Interferon - alpha is transferred into reaction buffer ( 50 cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted 25 mM Hepes, 350 mM sodium chloride , 5 mM calcium with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . chloride , pH 6 . 0 ) and diluted to obtain a protein concentra Finally the PSA - interferon - alpha containing fractions are tion of 1 mg/ml . A 50 -fold molar excess of a PSA aminooxy collected and subjected to UF /DF by use of a membrane reagent with a MW of 20 kD ( described above ) is added made of regenerated cellulose (Millipore ) . The preparation followed by m - toluidine as a nucleophilic catalyst ( 10 mM is next analytically characterized by measuring total protein 30 final concentration ) and NalO4 ( final concentration : 400 ( Coomassie , Bradford ) and biological activity according to JM ) . The coupling reaction is performed for 2 hours in the methods known in the art . dark under gentle shaking at room temperature . Subse In an alternative embodiment, Method 1 is carried out as quently , the reaction is quenched with cysteine for 60 min at follows . Interferon - alpha is transferred into a reaction buffer RT ( cysteine concentration : 10 mM ) . Then the conductivity ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 mM 35 of the reaction mixture is adjusted by adding a buffer calcium chloride , pH 6 . 0 ) and diluted to obtain a protein containing ammonium acetate (50 mM Hepes, 350 mm concentration of 1 mg/ ml . To this solution , NalO4 is added sodium chloride , 5 mM calcium chloride , 8 M ammonium to give a final concentration of 200 UM . The oxidation is acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl carried at RT for 30 min in the dark under gentle shaking . Sepharose FF (GE Healthcare , Fairfield , Conn . ) pre - equili The reaction is then quenched with cysteine ( final concen - 40 brated with 50 mM Hepes, 2 . 5 M ammonium acetate , 350 tration : 10 mM ) for 60 min at RT. mM sodium chloride, 5 mM calcium chloride , 0 .01 % Tween The solution is next subjected to UF /DF employing 80 , pH 6 . 9. Subsequently the conjugate is eluted with 50 Vivaspin centrifugal filtrators to remove excess periodate , mM Hepes, 5 mM calcium chloride , pH 7 . 5 . Finally , the quencher and the byproducts thereof. PSA - interferon -alpha containing fractions are collected and A 50 - fold molar excess of aminooxy - PSA reagent with a 45 subjected to UF/ DF by use of a membrane made of regen MW of 20 kD ( described above) is added followed by erated cellulose (Millipore ). The preparation is analytically m - toluidine as a nucleophilic catalyst ( final concentration : characterized by measuring total protein ( Bradford ) and 10 mM ). The coupling reaction is performed for 2 hours in biological activity according to methods known in the art . the dark under gentle shaking at room temperature . The In an alternative embodiment, Method 3 is carried out as excess of aminooxy reagent is removed by means of ion - 50 follows. Interferon - alpha is transferred into reaction buffer exchange chromatography . The PSA - interferon -alpha con - ( e . g . 50 mM Hepes, 350 mM sodium chloride , 5 mM taining fractions of the eluate are collected and subjected to calcium chloride , pH 6 . 0 ) and diluted to obtain a protein UF /DF by use of a membrane made of regenerated cellulose concentration of 1 mg/ ml . A 50 - fold molar excess of ami (Millipore ) . The preparation is next analytically character nooxy -PSA reagent with a MW of 20 kD (described above ) ized by measuring total protein ( Coomassie , Bradford ) and 55 is added followed by m -toluidine as a nucleophilic catalyst biological activity according to methods known in the art . ( 10 mM final concentration ) and NaI04 ( final concentration : Method 2 : 400 UM ). The coupling reaction is performed for 2 hours in Interferon - alpha is transferred or dissolved in reaction the dark under gentle shaking at room temperature . Subse buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride, 5 mM quently , the reaction is quenched with cysteine for 60 min at calcium chloride , pH 6 . 0 ) to get a final protein concentration 60 RT ( cysteine concentration : 10 mM ) and the conjugate is of 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is cor - purified by ion exchange chromatography . The PSA - inter rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 feron - alpha containing fractions of the eluate are collected solution . Subsequently , a 40 mM aqueous sodium periodate and subjected to UF/ DF by use of a membrane made of solution is added within 10 minutes to give a concentration regenerated cellulose (Millipore ). The preparation is ana of 200 uM . The oxidation reaction is carried out for 30 + / - 5 65 lytically characterized by measuring total protein (Bradford ) min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction and biological activity according to methods known in the is stopped by addition of an aqueous L -cysteine solution (1 art . US 10 ,414 ,793 B2 117 118 Method 4 : ml HiPrep Butyl FF 16 / 10 column (GE Healthcare, Fairfield , Interferon - alpha is dissolved in or transferred to a reaction Conn . ) pre - equilibrated with Buffer D (50 mM Hepes , 3 M buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride, 5 mM NaCl, pH 6 . 9 ) . Free PSA - reagent is washed out within 5 CV calcium chloride , pH 6 . 0 ) to get a final protein concentration Buffer D . Subsequently, the conjugate is eluted with 100 % of 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is cor - 5 Buffer E (50 mM Hepes , pH 6 . 9 ) . The conjugate containing rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 fractions are concentrated by UF /DF using a 10 kD mem solution . brane made of regenerated cellulose (88 cm2, cutoff 10 kD , Subsequently , the aminooxy -polysialic acid ( PSA -ONH2 ) Millipore ) . The final diafiltration step is performed against reagent is added in a 50 - fold molar excess to this interferon - histidine buffer , pH 6 . 9 containing 150 mM NaCl. The alpha solution within a maximum time period ( t ) of 15 10 preparation is analytically characterized by measuring total minutes under gentle stirring . Then an aqueous m - toluidine protein (Bradford ) and biological activity according to meth solution (50 mM ) is added within 15 minutes to get a final ods known in the art. For the PSA - Interferon - gamma con concentration of 10 mM . Finally , a 40 mM aqueous sodium jugate a specific activity of > 50 % in comparison to native periodate solution is added to give a concentration of 400 Interferon - gamma is determined . The conjugate is addition uM . 15 ally analytically characterized by Size Exclusion HPLC The reaction mixture is incubated for 120 + / – 10 min . in using a Agilent 1200 HPLC system equipped with a Shodex the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle KW 803 column under conditions as previously described shaking . Then the reaction is stopped by the addition of an (Kolarich et al, Transfusion 2006 ; 46 : 1959- 77 ) . It is shown aqueous L -cysteine solution ( 1 M ) to give a final concen - that the preparation contains no free Interferon gamma . tration of 10 mM in the reaction mixture and incubation for 20 Method 2 : 60 + / - 5 min . 10 mg interferon -gamma is dissolved in 8 ml histidine The obtained interferon - alpha conjugate is purified by buffer, pH 6 . 0 ( 20 mM L -histidine , 150 mM NaCl) . 200 ul ion - exchange chromatography . The PSA -interferon -alpha of an aqueous sodium periodate solution (5 mM ) and 2 ml containing fractions of the eluate are collected and concen - of an aqueous m - toluidine solution (50 mm ) are then added . trated by ultra - / diafiltration (UF /DF ) using a membrane 25 Subsequently the aminooxy - PSA reagent with a MW of 20 made of regenerated cellulose (Millipore ) . kD (described above ) is added to give a 5 - fold molar reagent The conjugates prepared by use of this procedure are excess. The mixture is incubated for 2 h in the dark at room analytically characterized by measuring total protein , bio temperature under gentle stirring and quenched for 15 min logical activity according to methods known in the art , and at room temperature by the addition of 100 ul of 1 M determination of the polysialyation degree by measuring the 30 aqueous cysteine solution . PSA content ( resorcinol assay ) . The free interferon gamma is removed by means of cation exchange chromatography (CEC ) . The reaction mixture is Example 27 diluted with 20 ml Buffer A (50 mM Hepes, pH 6 .5 ) and loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE Polysialylation of Interferon -Gamma Using 35 Healthcare , Fairfield , Conn . ) pre - equilibrated with Buffer A . Aminooxy -PSA and m - Toluidine as a Nucleophilic Then the column is eluted with Buffer B (50 mM Hepes, 1 Catalyst M NaCl, pH 6 . 5 ) . Free interferon - gamma is eluted by washing the column with 25 % Buffer B and the conjugate at Method 1 : 50 % Buffer B . The conductivity of the conjugate containing 10 mg interferon - gamma is dissolved in 5 ml histidine 40 fractions is subsequently raised to - 190 mS/ cm with Buffer buffer , pH 6 . 0 (20 mM L -histidine , 150 mM NaCl) . 100 ul C (50 mM Hepes, 5 M NaCl, pH 6 . 9 ) and loaded onto a 20 of an aqueous sodium periodate solution ( 5 mm ) is then ml HiPrep Butyl FF 16 /10 column (GE Healthcare, Fairfield , added and the reaction mixture is incubated for 1 h in the Conn .) pre -equilibrated with Buffer D (50 mM Hepes, 3 M dark at 4° C . under gentle stirring and quenched for 15 min NaCl, pH 6 . 9 ) . Free PSA -reagent is washed out within 5 CV at room temperature by the addition of 50 ul of a 1 M 45 Buffer D . Subsequently, the conjugate is eluted with 100 % aqueous cysteine solution . The mixture is subsequently Buffer E (50 mM Hepes , pH 6 . 9 ) . The conjugate containing subjected to UF /DF employing Vivaspin 15R 10 kD cen - fractions are concentrated by UF /DF using a 10 kD mem trifugal filtrators to remove excess periodate , quencher and brane made of regenerated cellulose (88 cm2, cut -off 10 the byproducts thereof. kD /Millipore ) . The final diafiltration step is performed The retentate ( approx . 7 ml) , containing oxidized inter - 50 against histidine buffer, pH 6 . 9 containing 150 mM NaCl. feron - gamma, is mixed with 2 ml of an aqueous m - toluidine The preparation is analytically characterized by measuring solution (50 mM ) and incubated for 30 min at room tem total protein ( Bradford ) and biological activity according to perature . Then aminooxy -PSA reagent with a MW of 20 kD methods known in the art. For the PSAinterferon - gamma ( described above ) is added to give a 5 - fold molar reagent conjugate a specific activity of > 50 % in comparison to excess . This mixture is incubated for 2 . 5 h at RT in the dark 55 native interferon - gamma is determined . The conjugate is under gentle stirring . additionally analytically characterized by Size Exclusion The free Interferon - gamma is removed by means of cation HPLC using a Agilent 1200 HPLC system equipped with a exchange chromatography (CEC ). The reaction mixture is Shodex KW 803 column under conditions as previously diluted with 20 ml Buffer A (50 mM Hepes , pH 6 . 5 ) and described (Kolarich et al , Transfusion 2006 ; 46 : 1959 -77 ) . It loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE 60 is shown that the preparation contains no free interferon Healthcare, Fairfield , Conn . ) pre -equilibrated with Buffer A . gamma . Then the column is eluted with Buffer B (50 mM Hepes , 1 Method 3 : M NaCl, pH 6 . 5 ) . Free interferon - gamma is eluted by 10 mg interferon - gamma is dissolved in 8 ml histidine washing the column with 25 % Buffer B and the conjugate at buffer, pH 6 . 0 (20 mM L -histidine , 150 mM NaCl) . 200 ul 50 % Buffer B . The conductivity of the conjugate containing 65 of an aqueous sodium periodate solution ( 5 mM ) and 2 ml fractions is subsequently raised to - 190 mS/ cm with Buffer of an aqueous m - toluidine solution ( 50 mm ) are then added . C (50 mM Hepes, 5 M NaCl, pH 6 . 9 ) and loaded onto a 20 Subsequently the aminooxy - PSA reagent with a MW of 20 US 10 ,414 ,793 B2 119 120 kD (described above ) is added to give a 5 - fold molar reagent Example 28 excess . The mixture is incubated for 2 h in the dark at room temperature under gentle stirring and quenched for 15 min Polysialylation of G -CSF Using Aminooxy -PSA at room temperature by the addition of 100 ul of 1 M and m - Toluidine as a Nucleophilic Catalyst aqueous cysteine solution . The free interferon gamma is removed by means of cation Method 1 : exchange chromatography (CEC ) . The reaction mixture is starting concentration of granulocyte - colony stimulat diluted with 20 ml Buffer A (50 mM Hepes, pH 6 .5 ) and ing factor (G - CSF) is transferred into a reaction buffer ( e. g ., loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE 50 mM Hepes, 350 mM sodium chloride , 5 mM calcium Healthcare , Fairfield , Conn . ) pre -equilibrated with Buffer A . 10 chloride , pH 6 .0 ) and diluted to obtain a protein concentra Then the column is eluted with Buffer B ( 50 mM Hepes , 1 tion of 1 mg/ml . To this solution , NalO4 is added to give a M NaCl, pH 6 . 5 ) . Free interferon - gamma is eluted by final concentration of 200 uM . The oxidation is carried at RT washing the column with 25 % Buffer B and the conjugate at for 30 min in the dark under gentle shaking . The reaction is 50 % Buffer B . The conductivity of the conjugate containing then quenched with cysteine ( final concentration : 10 mM ) fractions is subsequently raised to - 190 mS/ cm with Buffer 15 for 60 min at RT. C (50 mM Hepes, 5 M NaCl, pH 6 . 9 ) and loaded onto a 20 The solution is next subjected to UF /DF employing mlHiPrep Butyl FF 16 / 10 column (GE Healthcare, Fairfield , Vivaspin centrifugal filtrators to remove excess periodate , Conn . ) pre -equilibrated with Buffer D (50 mM Hepes, 3 M quencher and the byproducts thereof or, in the alternative, to NaCl, pH 6 . 9 ) . Free PSA - reagent is washed out within 5 CV an IEX column with a volumeof 20 ml (Merck EMD TMAE Buffer D . Subsequently the conjugate is eluted with 100 % 20 ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes , 5 Buffer E ( 50 mM Hepes , pH 6 . 9 ) . The conjugate containing mM CaCl2 , pH 7 . 0 ) . The column is equilibrated with 5 CV fractions are concentrated by UF /DF using a 10 kD mem - Buffer A . The oxidized G -CSF is eluted with Buffer B (20 brane made of regenerated cellulose ( 88 cm2, cutoff 10 mM Hepes , 5 mM CaCl2 , 1 M NaCl, pH 7 . 0 ). The G -CSF kD /Millipore ) . The final diafiltration step is performed containing fractions are collected . The protein content is against histidine buffer , pH 6 . 9 containing 150 mM NaCl. 25 determined (Coomassie , Bradford ) and adjusted to 1 mg/ ml The preparation is analytically characterized by measuring with reaction buffer and adjusted to pH 6 . 0 by dropwise total protein ( Bradford ) and biological activity according to addition of 0 . 5 M HC1. methods known in the art . For the PSAinterferon - gamma A 50 - fold molar excess of aminooxy - PSA reagent with a conjugate a specific activity of > 50 % in comparison to MW of 20 kD ( described above ) is added followed by native interferon - gamma is determined . The conjugate is 30 m - toluidine as a nucleophilic catalyst ( final concentration : additionally analytically characterized by Size Exclusion 10 mM ) . The coupling reaction is performed for 2 hours in HPLC using a Agilent 1200 HPLC system equipped with a the dark under gentle shaking at room temperature . The Shodex KW 803 column under conditions as previously excess of aminooxy reagent is removed by means of HIC . described (Kolarich et al, Transfusion 2006 ; 46 : 1959 - 77 ) . It The conductivity of the reaction mixture is adjusted by is shown that the preparation contains no free interferon - 35 adding a buffer containing ammonium acetate ( 50 mM gamma. Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 Method 4 : M ammonium acetate , pH 6 . 9 ) and loaded onto a column Interferon - gamma is dissolved in or transferred to a filled with 80 ml Phenyl Sepharose FF (GE Healthcare , reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chlo - Fairfield , Conn . ) pre - equilibrated with 50 mM Hepes , 2 . 5 M ride, 5 mM calcium chloride , pH 6 . 0 ) to get a final protein 40 ammonium acetate , 350 mM sodium chloride , 5 mM cal concentration of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted solution is corrected to 6 . 0 by drop wise addition of a 0 . 5 N with 50 mM Hepes buffer pH 7 .5 containing 5 mM CaC12 . aqueous HCl solution . Finally the PSA -G -CSF -containing fractions are collected Subsequently , the aminooxy -polysialic acid ( PSA -ONH2 ) and subjected to UF /DF by use of a membrane made of reagent is added in a 50 - fold molar excess to this interferon - 45 regenerated cellulose (Millipore ) . The preparation is next gamma solution within a maximum time period ( t) of 15 analytically characterized by measuring total protein ( Coo minutes under gentle stirring . Then an aqueous m - toluidine massie , Bradford ) and biological activity according to meth solution (50 mM ) is added within 15 minutes to get a final ods known in the art . concentration of 10 mM . Finally , a 40 mM aqueous sodium In an alternative embodiment, Method 1 is carried out as periodate solution is added to give a concentration of 400 50 follows. Granulocyte - colony stimulating factor ( G -CSF ) is UM . transferred into a reaction buffer ( e . g ., 50 mM Hepes, 350 The reaction mixture is incubated for 120 + / - 10 min . in mM sodium chloride, 5 mM calcium chloride, pH 6 . 0 ) and the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle diluted to obtain a protein concentration of 1 mg/ ml . To this shaking . Then the reaction is stopped by the addition of an solution , NalO4 is added to give a final concentration of 200 aqueous L -cysteine solution ( 1 M ) to give a final concen - 55 UM . The oxidation is carried at RT for 30 min in the dark tration of 10 mM in the reaction mixture and incubation for under gentle shaking . The reaction is then quenched with 60 + / - 5 min . cysteine ( final concentration : 10 mM ) for 60 min at RT. The obtained interferon - gamma conjugate is purified by The solution is next subjected to UF /DF employing ion - exchange chromatography . The PSA - interferon - gamma Vivaspin centrifugal filtrators to remove excess periodate , containing fractions of the eluate are collected and concen - 60 quencher and the byproducts thereof. A 50 - fold molar excess trated by ultra - /diafiltration (UF / DF ) using a membrane of aminooxy -PSA reagent with a MW of 20 kD (described made of regenerated cellulose (Millipore ) . above ) is added followed by m -toluidine as a nucleophilic The conjugates prepared by use of this procedure are catalyst ( final concentration : 10 mM ) . The coupling reaction analytically characterized by measuring total protein , bio - is performed for 2 hours in the dark under gentle shaking at logical activity according to methods known in the art , and 65 room temperature . The excess of aminooxy reagent is determination of the polysialyation degree by measuring the removed by means of ion exchange chromatography . The PSA content ( resorcinol assay) . PSA -G -CSF containing fractions of the eluate are collected US 10 , 414 , 793 B2 121 122 and subjected to UF /DF by use of a membrane made of preparation is analytically characterized by measuring total regenerated cellulose (Millipore ) . The preparation is next protein (Bradford ) and biological activity according to meth analytically characterized by measuring total protein (Coo ods known in the art . massie , Bradford ) and biological activity according to meth In an alternative embodiment , Method 3 is carried out as ods known in the art . 5 follows. Granulocyte -colony stimulating factor ( G -CSF ) is Method 2 : transferred into reaction buffer (e . g . 50 mM Hepes, 350 mM G - CSF is transferred or dissolved in reaction buffer ( e . g . sodium chloride , 5 mM calcium chloride, pH 6 . 0 ) and 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium diluted to obtain a protein concentration of 1 mg/ml . A chloride, pH 6 . 0 ) to get a final protein concentration of 50 - fold molar excess of aminooxy -PSA reagent with a MW 1. 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected 10 of 20 kD ( described above) is added followed by m - tolui to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 dine as a nucleophilic catalyst ( 10 mM final concentration ) solution . Subsequently , a 40 mM aqueous sodium periodate and NalO4 ( final concentration : 400 UM ) . The coupling solution is added within 10 minutes to give a concentration reaction is performed for 2 hours in the dark under gentle of 200 uM . The oxidation reaction is carried out for 30 + / - 5 15 shaking at room temperature . Subsequently , the reaction is min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction quenched with cysteine for 60 min at RT ( cysteine concen is stopped by addition of an aqueous L -cysteine solution (1 tration : 10 mM ) and the conjugate is purified by ion M ) within 15 minutes at T = + 22 + / - 2° C . to give a final exchange chromatography. The PSA - G - CSF containing concentration of 10 mM in the reaction mixture and incu fractions of the eluate are collected and subjected to UF /DF bation for 60 + / - 5 min . 20 by use of a membrane made of regenerated cellulose (Mil The oxidized G - CSF is further purified by ion exchange lipore ) . The preparation is analytically characterized by chromatography. The oxidized G - CSF containing fractions measuring total protein (Bradford ) and biological activity of the eluate are collected and used for the conjugation according to methods known in the art . reaction . Method 4 : The aminooxy -polysialic acid (PSA - ONH2) reagent is 25 G -CSF is dissolved in or transferred to a reaction buffer added in a 50 - fold molar excess to the eluate containing the ( e . g . 50 mM Hepes, 350 mM sodium chloride , 5 mM purified oxidized G -CSF within a maximum time period ( t ) calcium chloride, pH 6 . 0 ) to get a final protein concentration of 15 minutes under gentle stirring . Then an aqueous of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is cor m - toluidine solution (50 mM ) is added within 15 minutes to rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 get a final concentration of 10 mM . The reaction mixture is 30 solution . incubated for 120 + / – 10 min . at pH 6 .0 in the dark at a Subsequently , the aminooxy -polysialic acid (PSA -ONH2 ) temperature ( T) of T = + 22 + / - 2° C . under gentle shaking reagent is added in a 50 - fold molar excess to this G - CSF (protein concentration : 1 mg/ml ) . solution within a maximum time period ( t ) of 15 minutes The obtained PSA - G - CSF conjugate is further purified by under gentle stirring . Then an aqueous m - toluidine solution ion exchange chromatography . The PSA - G -CSF conjugate 35 (50 mM ) is added within 15 minutes to get a final concen containing fractions are collected and concentrated by ultra - tration of 10 mM . Finally , a 40 mM aqueous sodium diafiltration (UF /DF ) using a membrane made of regener- periodate solution is added to give a concentration of 400 ated cellulose with an appropriate molecular weight cut off UM . (Millipore ) . The reaction mixture is incubated for 120 + / – 10 min . in The conjugate prepared by use of this procedure is 40 the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle analytically characterized by measuring total protein , bio - shaking . Then the reaction is stopped by the addition of an logical activity , and determination of the polysialyation aqueous L -cysteine solution (1 M ) to give a final concen degree by measuring the PSA content ( resorcinol assay ) . tration of 10 mM in the reaction mixture and incubation for Method 3 : 60 + / - 5 min . Granulocyte -colony stimulating factor ( G -CSF ) is trans - 45 The obtained G -CSF conjugate is purified by ion - ex ferred into reaction buffer ( 50 mM Hepes , 350 mM sodium change chromatography . The PSA - G -CSF containing frac chloride, 5 mM calcium chloride , pH 6 . 0 ) and diluted to tions of the eluate are collected and concentrated by ultra - / obtain a protein concentration of 1 mg/ml . A 50 - fold molar diafiltration (UF /DF ) using a membrane made of excess of aminooxy -PSA reagent with a MW of 20 kD regenerated cellulose (Millipore ) . ( described above ) is added followed by m - toluidine as a 50 The conjugates prepared by use of this procedure are nucleophilic catalyst ( 10 mM final concentration ) and analytically characterized by measuring total protein , bio NaI04 ( final concentration : 400 uM ) . The coupling reaction logical activity according to methods known in the art , and is performed for 2 hours in the dark under gentle shaking at determination of the polysialyation degree by measuring the room temperature. Subsequently , the reaction is quenched PSA content ( resorcinol assay ) . with cysteine for 60 min at RT ( cysteine concentration : 10 55 mM ) . Then the conductivity of the reaction mixture is Example 29 adjusted by adding a buffer containing ammonium acetate ( 50 mM Hepes , 350 mM sodium chloride , 5 mM calcium Polysialylation of Humira Using Aminooxy - PSA chloride , 8 M ammonium acetate , pH 6 . 9 ) and loaded onto and m - Toluidine as a Nucleophilic Catalyst a column filled with Phenyl Sepharose FF (GE Healthcare , 60 Fairfield , Conn .) pre -equilibrated with 50 mM Hepes, 2 .5 M Method 1 : ammonium acetate, 350 mM sodium chloride , 5 mM cal- A starting concentration of Humira is transferred into a cium chloride, 0 .01 % Tween 80 , pH 6 . 9. Subsequently the reaction buffer (e . g . 50 mM Hepes , 350 mM sodium chlo conjugate is eluted with 50 mM Hepes, 5 mM calcium ride , 5 mM calcium chloride, pH 6 . 0 ) and diluted to obtain chloride, pH 7 . 5 . Finally , the PSA - G - CSF- containing frac - 65 a protein concentration of 1 mg/ ml . To this solution , Nal04 tions are collected and subjected to UF/ DF by use of a is added to give a final concentration of 200 uM . The membrane made of regenerated cellulose (Millipore ). The oxidation is carried at RT for 30 min in the dark under gentle US 10 ,414 ,793 B2 123 124 shaking . The reaction is then quenched with cysteine ( final of 200 uM . The oxidation reaction is carried out for 30 + / - 5 concentration : 10 mM ) for 60 min at RT . min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction The solution is next subjected to UF /DF employing is stopped by addition of an aqueous L -cysteine solution (1 Vivaspin centrifugal filtrators to remove excess periodate , M ) within 15 minutes at T = + 22 + / - 2° C . to give a final quencher and the byproducts thereof or, in the alternative , to 5 concentration of 10 mM in the reaction mixture and incu an IEX column with a volume of 20 ml (Merck EMD TMAE bation for 60 + / - 5 min . ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes , 5 The oxidized Humira is further purified by ion exchange mM CaC12 , pH 7 . 0 ) . The column is equilibrated with 5 CV chromatography . The oxidized Humira containing fractions Buffer A . The oxidized Humira is eluted with Buffer B ( 20 of the eluate are collected and used for the conjugation mM Hepes , 5 mM CaCl2 , 1M NaCl, pH 7 . 0 ) . The Humira 10 reaction . containing fractions are collected . The protein content is The aminooxy -polysialic acid ( PSA - ONH2) reagent is determined (Coomassie , Bradford ) and adjusted to 1 mg/ml added in a 50 - fold molar excess to the eluate containing the with reaction buffer and adjusted to pH 6 . 0 by dropwise purified oxidized Humira within a maximum time period ( t ) addition of 0 . 5M HCl. of 15 minutes under gentle stirring . Then an aqueous A 50 - fold molar excess of aminooxy - PSA reagent with a 15 m - toluidine solution ( 50 mM ) is added within 15 minutes to MW of 20 kD (described above ) is added followed by get a final concentration of 10 mM . The reaction mixture is m - toluidine as a nucleophilic catalyst ( final concentration : incubated for 120 + / – 10 min . at pH 6 . 0 in the dark at a 10 mM ). The coupling reaction is performed for 2 hours in temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking the dark under gentle shaking at room temperature . The (protein concentration : 1 mg/ml ) . excess of aminooxy reagent is removed by means of HIC . 20 The obtained PSA -Humira conjugate is further purified by The conductivity of the reaction mixture is adjusted by ion exchange chromatography . The PSA -Humira conjugate adding a buffer containing ammonium acetate ( 50 mM containing fractions are collected and concentrated by ultra Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 diafiltration (UF /DF ) using a membrane made of regener M ammonium acetate , pH 6 . 9 ) and loaded onto a column a ted cellulose with an appropriate molecular weight cut off filled with 80 ml Phenyl Sepharose FF (GE Healthcare , 25 (Millipore ). Fairfield , Conn .) pre- equilibrated with 50 mM Hepes, 2 .5 M The conjugate prepared by use of this procedure is ammonium acetate , 350 mM sodium chloride , 5 mM cal analytically characterized by measuring total protein , bio cium chloride , pH 6 . 9 . Subsequently the conjugate is eluted logical activity , and determination of the polysialyation with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . degree by measuring the PSA content ( resorcinol assay ) . Finally , the PSA -Humira containing fractions are collected 30 Method 3 : and subjected to UF /DF by use of a membrane made of Humira is transferred into reaction buffer (50 mM Hepes , regenerated cellulose (Millipore ). The preparation is next 350 mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) analytically characterized by measuring total protein (Coo - and diluted to obtain a protein concentration of 1 mg/ ml . A massie , Bradford ) and biological activity according to meth - 50 - fold molar excess of aminooxy - PSA reagent with a MW ods known in the art. 35 of 20 kD ( described above ) is added followed by m - tolui In an alternative embodiment, Method 1 is carried out as dine as a nucleophilic catalyst ( 10 mM final concentration ) follows. Humira is transferred into a reaction buffer ( e . g . 50 and NalO4 ( final concentration : 400 uM ). The coupling mM Hepes , 350 mM sodium chloride , 5 mM calcium reaction is performed for 2 hours in the dark under gentle chloride, pH 6 . 0 ) and diluted to obtain a protein concentra - shaking at room temperature . Subsequently, the reaction is tion of 1 mg/ml . To this solution , NalO4 is added to give a 40 quenched with cysteine for 60 min at RT ( cysteine concen final concentration of 200 uM . The oxidation is carried at RT tration : 10 mM ) . Then the conductivity of the reaction for 30 min in the dark under gentle shaking . The reaction is mixture is adjusted by adding a buffer containing ammo then quenched with cysteine ( final concentration : 10 mM ) nium acetate (50 mM Hepes, 350 mM sodium chloride, 5 for 60 min at RT . mM calcium chloride , 8 M ammonium acetate , pH 6 . 9 ) and The solution is next subjected to UF /DF employing 45 loaded onto a column filled with Phenyl Sepharose FF (GE Vivaspin centrifugal filtrators to remove excess periodate , Healthcare , Fairfield , Conn .) pre - equilibrated with 50 mM quencher and the byproducts thereof. A 50 - fold molar excess Hepes , 2 . 5 Mammonium acetate , 350 mM sodium chloride , of aminooxy - PSA reagent with a MW of 20 kD ( described 5 mM calcium chloride , 0 .01 % Tween 80 , pH 6 . 9 . Subse above ) is added followed by m - toluidine as a nucleophilic quently the conjugate is eluted with 50 mM Hepes , 5 mM catalyst ( final concentration : 10 mM ) . The coupling reaction 50 calcium chloride , pH 7 . 5 . Finally the PSA -Humira contain is performed for 2 hours in the dark under gentle shaking at ing fractions are collected and subjected to UF /DF by use of room temperature . The excess of aminooxy reagent is a membrane made of regenerated cellulose (Millipore ). The removed by means of ion exchange chromatography The preparation is analytically characterized by measuring total PSA -Humira containing fractions of the elutae are collected protein ( Bradford ) and biological activity according to meth and subjected to UF/ DF by use of a membrane made of 55 ods known in the art. regenerated cellulose (Millipore ). The preparation is next I n an alternative embodiment, Method 3 is carried out as analytically characterized by measuring total protein (Coo follows. Humira is transferred into reaction buffer ( e . g . 50 massie , Bradford ) and biological activity according to meth mM Hepes, 350 mM sodium chloride, 5 mM calcium ods known in the art . chloride , pH 6 . 0 ) and diluted to obtain a protein concentra Method 2 : 60 tion of 1 mg/ ml . A 50 - fold molar excess of aminooxy - PSA Humira is transferred or dissolved in reaction buffer ( e .g . reagent with a MW of 20 kD (described above ) is added 50 mM Hepes , 350 mM sodium chloride , 5 mM calcium followed by m - toluidine as a nucleophilic catalyst ( 10 mM chloride , pH 6 . 0 ) to get a final protein concentration of final concentration ) and Nal04 ( final concentration : 400 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected UM ) . The coupling reaction is performed for 2 hours in the to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC165 dark under gentle shaking at room temperature . Subse solution . Subsequently , a 40 mM aqueous sodium periodate quently , the reaction is quenched with cysteine for 60 min at solution is added within 10 minutes to give a concentration RT (cysteine concentration : 10 mM ) and the conjugate is US 10 ,414 ,793 B2 125 126 purified by ion exchange chromatography . The PSA - Humira the dark under gentle shaking at room temperature . The containing fractions of the eluate are collected and subjected excess of aminooxy reagent is removed by means of HIC . to UF/ DF by use of a membrane made of regenerated The conductivity of the reaction mixture is adjusted by cellulose (Millipore ). The preparation is analytically char adding a buffer containing ammonium acetate (50 mM acterized by measuring total protein ( Bradford ) and biologi - 5 Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 cal activity according to methods known in the art. M ammonium acetate , pH 6 . 9 ) and loaded onto a column Method 4 : filled with 80 ml Phenyl Sepharose FF (GE Healthcare, Humira is dissolved in or transferred to a reaction buffer Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes, 2 . 5 M ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 mM ammonium acetate , 350 mM sodium chloride , 5 mM cal calcium chloride , pH 6 . 0 ) to get a final protein concentration 10 cium chloride, pH 6 . 9 . Subsequently the conjugate is eluted of 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is cor - with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 Finally , the PSA - Prolia containing fractions are collected solution . and subjected to UF /DF by use of a membrane made of Subsequently , the aminooxy -polysialic acid (PSA -ONH2 ) regenerated cellulose (Millipore ). The preparation is next reagent is added in a 50 - fold molar excess to this Humira 15 analytically characterized by measuring total protein ( Coo solution within a maximum time period ( t ) of 15 minutes massie , Bradford ) and biological activity according to meth under gentle stirring . Then an aqueous m - toluidine solution ods known in the art . (50 mM ) is added within 15 minutes to get a final concen In an alternative embodiment, Method 1 is carried out as tration of 10 mM . Finally a 40 mM aqueous sodium perio - follows. 10 mg Prolia is dissolved in 5 ml histidine buffer, date solution is added to give a concentration of 400 uM . 20 pH 6 . 0 (20 mM L -histidine , 150 mM NaCl) . 100 ul of an The reaction mixture is incubated for 120 + / – 10 min . in aqueous sodium periodate solution (5 mM ) is then added the dark at a temperature ( T) of T = + 22 + /- 2° C . under gentle and the reaction mixture is incubated for 1 h in the dark at shaking . Then the reaction is stopped by the addition of an 4° C . under gentle stirring and quenched for 15 min at room aqueous L - cysteine solution ( 1 M ) to give a final concen - temperature by the addition of 50 ul of a 1 M aqueous tration of 10 mM in the reaction mixture and incubation for 25 cysteine solution . The mixture is subsequently subjected to 60 + / - 5 min . UF /DF employing Vivaspin 15R 10 kD centrifugal filtrators The obtained Humira -conjugate is purified by ion - ex - to remove excess periodate , quencher and the byproducts change chromatography . The PSA -Humira containing frac thereof. tions of the eluate are collected and concentrated by ultra - / The retentate (approx . 7 ml) , containing oxidized Prolia , diafiltration (UF /DF ) using a membrane made of 30 is mixed with 2 ml of an aqueous m - toluidine solution (50 regenerated cellulose (Millipore ) . mM ) and incubated for 30 min at room temperature . Then The conjugates prepared by use of this procedure are aminooxy -PSA reagent with a MW of 20 kD ( described analytically characterized by measuring total protein , bio above ) is added to give a 5 - fold molar reagent excess . This logical activity according to methods known in the art , and mixture is incubated for 2 . 5 h at RT in the dark under gentle determination of the polysialyation degree by measuring the 35 stirring . PSA content (resorcinol assay ) . The free Prolia is removed by means of cation exchange chromatography (CEC ) . The reaction mixture is diluted with Example 30 20 ml Buffer A (50 mM Hepes , pH 6 . 5 ) and loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE Healthcare , Fairfield , Polysialylation of Prolia Using Aminooxy - PSA and 40 Conn .) pre - equilibrated with Buffer A . Then the column is m - Toluidine as a Nucleophilic Catalyst eluted with Buffer B (50 mM Hepes , 1 M NaCl, pH 6 . 5 ) . Free Prolia is eluted by washing the column with 25 % Buffer Method 1 : B and the conjugate at 50 % Buffer B . The conductivity of A starting concentration of Prolia is transferred into a the conjugate containing fractions is subsequently raised to reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chlo - 45 - 190 mS/ cm with Buffer C (50 mM Hepes, 5 M NaCl, pH ride , 5 mM calcium chloride, pH 6 . 0 ) and diluted to obtain 6 . 9 ) and loaded onto a 20 ml HiPrep Butyl FF 16 / 10 column a protein concentration of 1 mg/ ml . To this solution , NalO4 (GE Healthcare , Fairfield , Conn . ) pre -equilibrated with Buf is added to give a final concentration of 200 UM . The fer D (50 mM Hepes, 3 M NaCl, pH 6 .9 ). Free PSA -reagent oxidation is carried at RT for 30 min in the dark under gentle is washed out within 5 CV Buffer D . Subsequently , the shaking . The reaction is then quenched with cysteine ( final 50 conjugate is eluted with 100 % Buffer E (50 mM Hepes , pH concentration : 10 mM ) for 60 min at RT. 6 . 9 ) . The conjugate containing fractions are concentrated by The solution is next subjected to UF /DF employing UF /DF using a 10 kD membrane made of regenerated Vivaspin centrifugal filtrators to remove excess periodate , cellulose (88 cm2, cutoff 10 kD , Millipore ) . The final quencher and the byproducts thereof or, in the alternative, to diafiltration step is performed against histidine buffer, pH an IEX column with a volume of 20 ml( Merck EMD TMAE 55 6 . 9 containing 150 mM NaCl. The preparation is analyti ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes, 5 cally characterized by measuring total protein (Bradford ) mM CaCl2 , pH 7 .0 ) . The column is equilibrated with 5 CV and biological activity according to methods known in the Buffer A . The oxidized Prolia is eluted with Buffer B ( 20 art . For the PSA -Prolia conjugate a specific activity of > 50 % mM Hepes , 5 mM CaCl2 , 1M NaCl, pH 7 . 0 ) . The Prolia in comparison to native Prolia is determined . The conjugate containing fractions are collected . The protein content is 60 is additionally analytically characterized by Size Exclusion determined ( Coomassie , Bradford ) and adjusted to 1 mg/ ml HPLC using a Agilent 1200 HPLC system equipped with a with reaction buffer and adjusted to pH 6 . 0 by dropwise Shodex KW 803 column under conditions as previously addition of 0 . 5 M HCl. described ( Kolarich et al, Transfusion 2006 ; 46 : 1959 - 77 ) . It A 50 - fold molar excess of aminooxy - PSA reagent with a is shown that the preparation contains no free Prolia . MW of 20 kD (described above ) is added followed by 65 Method 2 : m - toluidine as a nucleophilic catalyst ( final concentration : Prolia is transferred or dissolved in reaction buffer ( e . g . 50 10 mM ) . The coupling reaction is performed for 2 hours in mM Hepes, 350 mM sodium chloride, 5 mM calcium US 10 ,414 ,793 B2 127 128 chloride , pH 6 . 0 ) to get a final protein concentration of excess . The mixture is incubated for 2 h in the dark at room 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected temperature under gentle stirring and quenched for 15 min to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 at room temperature by the addition of 100 ul of 1 M solution . Subsequently , a 40 mM aqueous sodium periodate aqueous cysteine solution . solution is added within 10 minutes to give a concentration 5 The free Prolia is removed by means of cation exchange of 200 uM . The oxidation reaction is carried out for 30 + / - 5 chromatography (CEC ) . The reaction mixture is diluted with min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction 20 ml Buffer A (50 mM Hepes , pH 6 . 5 ) and loaded onto a is stopped by addition of an aqueous L - cysteine solution ( 1 20 ml HiPrep SPFF 16 / 10 column (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with Buffer A . Then the column is M ) within 15 minutes at T = + 22 + / - 2° C . to give a final eluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 6 . 5 ) . concentration of 10 mM in the reaction mixture and incu - 10 Free Prolia is eluted by washing the column with 25 % Buffer bation for 60 + / - 5 min . B and the conjugate at 50 % Buffer B . The conductivity of The oxidized Prolia is further purified by ion exchange the conjugate containing fractions is subsequently raised to chromatography. The oxidized Prolia containing fractions of - 190 mS/ cm with Buffer C (50 mM Hepes, 5 M NaC1, pH the eluate are collected and used for the conjugation reac 6 . 9 ) and loaded onto a 20 mlHiPrep Butyl FF 16 / 10 column tion . 15 (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with Buf The aminooxy -polysialic acid (PSA -ONH2 ) reagent is fer D ( 50 mM Hepes, 3 M NaCl, pH 6 .9 ) . Free PSA -reagent added in a 50 - fold molar excess to the eluate containing the is washed out within 5 CV Buffer D . Subsequently the purified oxidized Prolia within a maximum time period ( t ) of conjugate is eluted with 100 % Buffer E (50 mM Hepes, pH 15 minutes under gentle stirring . Then an aqueous m - tolui- 6 . 9 ) . The conjugate containing fractions are concentrated by dine solution (50 mM ) is added within 15 minutes to get a 20 UF /DF using a 10 kD membrane made of regenerated final concentration of 10 mM . The reaction mixture is cellulose ( 88 cm², cutoff 10 kD /Millipore ). The final dia incubated for 120 + / - 10 min . at pH 6 . 0 in the dark at a filtration step is performed against histidine buffer, pH 6 . 9 temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking containing 150 mM NaCl. The preparation is analytically (protein concentration : 1 mg/ml ) . characterized by measuring total protein (Bradford ) and The obtained Prolia conjugate is further purified by ion 25 biological activity according to methods known in the art. exchange chromatography. The Prolia conjugate containing For the PSA - Prolia conjugate a specific activity of > 50 % in comparison to native Prolia is determined . The conjugate is fractions are collected and concentrated by ultra - / diafiltra additionally analytically characterized by Size Exclusion tion (UF / DF ) using a membrane made of regenerated cel HPLC using a Agilent 1200 HPLC system equipped with a lulose with an appropriate molecular weight cut off (Milli Shodex KW 803 column under conditions as previously pore ) . 30 described (Kolarich et al, Transfusion 2006 ; 46 : 1959- 77 ) . It The conjugate prepared by use of this procedure is is shown that the preparation contains no free Prolia . analytically characterized by measuring total protein , bio Method 4 : logical activity , and determination of the polysialyation Prolia is dissolved in or transferred to a reaction buffer degree by measuring the PSA content (resorcinol assay ). ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 mM Method 3 : 35 calcium chloride , pH 6 . 0 ) to get a final protein concentration Prolia is transferred into reaction buffer (50 mM Hepes , of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor 350 mM sodium chloride, 5 mM calcium chloride, pH 6 .0 ) rected to 6 .0 by drop wise addition of a 0 . 5 N aqueous HC1 and diluted to obtain a protein concentration of 1 mg/ ml. A solution . 50 - fold molar excess of aminooxy - PSA reagent with a MW Subsequently the aminooxy -polysialic acid ( PSA -ONH , ) of 20 kD (described above ) is added followed by m - tolui - 40 reagent is added in a 50 - fold molar excess to this Prolia dine as a nucleophilic catalyst ( 10 mM final concentration solution within a maximum time period ( t ) of 15 minutes and Nalo , ( final concentration : 400 UM ) . The coupling under gentle stirring . Then an aqueous m - toluidine solution reaction is performed for 2 hours in the dark under gentle ( 50 mM ) is added within 15 minutes to get a final concen shaking at room temperature . Subsequently , the reaction is tration of 10 mm . Finally a 40 mM aqueous sodium perio quenched with cysteine for 60 min at RT (cysteine concen - 45 date solution is added to give a concentration of 400 uM . The reaction mixture is incubated for 120 + / – 10 min . in tration : 10 mM ) . Then the conductivity of the reaction the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle mixture is adjusted by adding a buffer containing ammo shaking . Then the reaction is stopped by the addition of an nium acetate (50 mM Hepes, 350 mM sodium chloride , 5 aqueous L -cysteine solution (1 M ) to give a final concen mM calcium chloride , 8 M ammonium acetate , pH 6 . 9 ) and tration of 10 mM in the reaction mixture and incubation for loaded onto a column filled with Phenyl Sepharose FF (GE 50 60 + / - 5 min . Healthcare, Fairfield , Conn . ) pre - equilibrated with 50 mM The obtained Prolia conjugate is purified by ion - exchange Hepes, 2 . 5 M ammonium acetate , 350 mM sodium chloride , chromatography. The PSA -Prolia containing fractions of the 5 mM calcium chloride, 0 .01 % Tween 80 , pH 6 . 9 . Subse eluate are collected and concentrated by ultra - / diafiltration quently the conjugate is eluted with 50 mM Hepes, 5 mM (UF /DF ) using a membrane made of regenerated cellulose calcium chloride, pH 7 .5 . Finally the PSA Prolia - containing 55 Millipore ). fractions are collected and subjected to UF /DF by use of a The conjugates prepared by use of this procedure are membrane made of regenerated cellulose (Millipore ) . The analytically characterized by measuring total protein , bio preparation is analytically characterized by measuring total logical activity according to methods known in the art , and protein (Bradford ) and biological activity according to meth - determination of the polysialyation degree by measuring the ods known in the art. 60 PSA content (resorcinol assay ) . In an alternative embodiment, Method 3 is carried out as follows. 10 mg Prolia is dissolved in 8 ml histidine buffer, Example 31 pH 6 . 0 ( 20 mM L -histidine , 150 mM NaCl) . 200 ul of an aqueous sodium periodate solution ( 5 mM ) and 2 ml of an Polysialylation of Other Therapeutic Proteins aqueous m - toluidine solution ( 50 mm ) are then added . 65 Subsequently the aminooxy -PSA reagent with a MW of 20 Polysialylation reactions performed in the presence of kD (described above ) is added to give a 5 fold molar reagent alternative nucleophilic catalysts like m - toluidine or o -amin US 10 , 414 , 793 B2 129 130 obenzoic acid as described herein may be extended to other mixture is diluted with 20 ml Buffer A (50 mM Hepes, pH therapeutic proteins . For example , in various aspects of the 7 . 5 ) and loaded onto a 20 mlHiPrep QFF 16 / 10 column (GE invention , the above polysialylation or PEGylation reactions Healthcare , Fairfield , Conn . ) pre -equilibrated with Buffer A . as described herein with PSA aminooxy or PEG aminooxy Then the column is eluted with Buffer B (50 mM Hepes , 1 reagents is repeated with therapeutic proteins such as those 5 M NaC1, pH 7 . 5 ) . Free EPO is eluted by washing the column proteins described herein . with 25 % Buffer B and the conjugate at 50 % Buffer B . The conjugate containing fractions are concentrated by UF/ DF Example 32 using a 10 kD membrane made of regenerated cellulose ( 88 cm2, cut - off 10 kD /Millipore ) . The final diafiltration step is PEGylation of EPO Using an Aminooxy - PEG 10 performed against histidine buffer, pH 7 . 2 containing 150 Reagent and m - Toluidine as a Nucleophilic mM NaCl. The preparation is analytically characterized by Catalyst measuring total protein (Bradford ) and biological activity biological activity according to methods known in the art . Method 1 : For the PEG -EPO conjugate a specific activity of > 50 % in Erythropoietin ( EPO ) is PEGylated by use of a linear 20 15 comparison to native EPO is determined . The conjugate is KD PEGylation reagent containing an aminooxy group . An additionally analytically characterized by Size Exclusion example of this type of reagent is the Sunbright® CA series HPLC using a Agilent 1200 HPLC system equipped with a from NOF (NOF Corp ., Tokyo , Japan ). EPO is dissolved in Shodex KW 803 column under conditions as previously 7 . 0 ml histidine buffer , pH 6 . 0 ( 20 mM L - histidine, 150 mM described (Kolarich et al , Transfusion 2006 ; 46 : 1959 -77 ) . It NaC1, 5 mM CaC12 ) . An aqueous sodium periodate solution 20 is shown that the preparation contains no free EPO . ( 5 mM ) is then added and the reaction mixture is incubated Method 2 : for 1 h in the dark at 4° C . under gentle stirring and quenched EPO is PEGylated by use of a linear 20 kD PEGylation for 15 min at room temperature by the addition of 7 .5 al of reagent containing an aminooxy group . An example of this a 1 M aqueous cysteine solution . The mixture is subse - type of reagent is the Sunbright® CA series from NOF (NOF quently subjected to UF/ DF employing Vivaspin centrifugal 25 Corp ., Tokyo , Japan ). filtrators to remove excess periodate , quencher and the EPO is transferred or dissolved in reaction buffer ( e. g. 50 byproducts thereof. mM Hepes , 350 mM sodium chloride , 5 mM calcium The retentate containing oxidized EPO is nextmixed with chloride , pH 6 . 0 ) to get a final protein concentration of an aqueous m - toluidine solution (50 mM ) and incubated for 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is corrected 30 min at room temperature . aminooxy - PEG reagent with a 30 to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 MW of 20 kD is then added to give a 5 - fold molar reagent solution . Subsequently a 40 mM aqueous sodium periodate excess . This mixture is incubated for 2 .5 h at room tem - solution is added within 10 minutes to give a concentration perature in the dark under gentle stirring . of 200 uM . The oxidation reaction is carried out for 30 + / - 5 Finally , the PEG -EPO conjugate is purified by ion -ex min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction change chromatography ( e . g . on Sepharose FF ). For 35 is stopped by addition of an aqueous L - cysteine solution ( 1 example , 1 . 5 mg protein /ml gel is loaded on the column M ) within 15 minutes at T = + 22 + / - 2° C . to give a final equilibrated with 50 mM Hepes buffer, pH 7 . 4 containing 5 concentration of 10 mM in the reaction mixture and incu mM CaCl2 . The conjugate is eluted with 50 mM Hepes bation for 60 + / - 5 min . buffer containing 5 mM CaCl, and 500 mM sodium chlo - The oxidized EPO is further purified by ion exchange ride, pH 7 . 4 and is then subjected to UF /DF using an 40 chromatography. The oxidized EPO containing fractions of appropriate MW cutoff membrane . The preparation is next the eluate are collected and used for the conjugation reac analytically characterized by measuring total protein (Coo - tion . massie , Bradford ) and biological activity according to meth The aminooxy -PEG reagent with a MW of 20 kD reagent ods known in the art . is added in a 50 - fold molar excess to the eluate containing In an alternative embodiment, Method 1 is carried out as 45 the purified oxidized EPO within a maximum time period (t ) follows. EPO is PEGylated by use of a linear 20 kD of 15 minutes under gentle stirring . Then an aqueous PEGylation reagent containing an aminooxy group . An m - toluidine solution (50 mM ) is added within 15 minutes to example of this type of reagent is the Sunbright® CA series get a final concentration of 10 mM . The reaction mixture is from NOF (NOF Corp ., Tokyo , Japan ) . 10 mg EPO is incubated for 120 + / – 10 min . in the dark at a temperature ( T ) dissolved in 5 ml histidine buffer, pH 6 . 0 (20 mM L - histi - 50 of T = + 22 + / - 2° C . under gentle shaking . dine , 150 mM NaCl) . 100 ul of an aqueous sodium periodate The obtained PEG - EPO conjugate is further purified by solution ( 5 mm ) is then added and the reaction mixture is ion exchange chromatography . The PEG - EPO conjugate incubated for 1 h in the dark at 4° C . under gentle stirring containing fractions are collected and concentrated by ultra -/ and quenched for 15 min at room temperature by the diafiltration (UF /DF ) using a membrane made of regener addition of 50 ul of a 1 M aqueous cysteine solution . The 55 ated cellulose with an appropriate molecular weight cut off mixture is subsequently subjected to UF /DF employing (Millipore ). Vivaspin 15R 10 kD centrifugal filtrators to remove excess The conjugate prepared by use of this procedure are periodate , quencher and the byproducts thereof. analytically characterized by measuring total protein and The retentate ( approx . 7 ml) , containing oxidized EPO , is biological activity according to methods known in the art. mixed with 2 ml of an aqueous m - toluidine solution (50 60 Method 3 : mM ) and incubated for 30 min at room temperature . Then EPO is PEGylated by use of a linear 20 kD PEGylation aminooxy - PEG reagent with a MW of 20 kD (described reagent containing an aminooxy group . An example of this above) is added to give a 5 - fold molar reagent excess. This type of reagent is the Sunbright® CA series from NOF (NOF mixture is incubated for 2 .5 h at RT in the dark under gentle Corp ., Tokyo, Japan ). EPO is dissolved in Hepes buffer (50 stirring . 65 mM Hepes , 150 mM sodium chloride , 5 mM calcium Finally, the PEG - EPO conjugate is purified by ion -ex - chloride , pH 6 .0 ) and mixed with an aqueous sodium change chromatography on Q Sepharose FF . The reaction periodate solution ( 10 mm ) , and an aqueous m -toluidine US 10 ,414 ,793 B2 131 132 solution ( 50 mM ) . Subsequently , the aminooxy reagent is under gentle stirring and quenched for 15 min at room added to give a 20 - fold molar reagent excess. The mixture temperature by the addition of a 1 M aqueous L - cysteine is incubated for 2 h in the dark at room temperature under solution to give a final concentration of 10 mM . gentle stirring and quenched for 15 min at room temperature The PEG -EPO conjugate is purified by means of ion by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . 5 exchange chromatography ( IEC ) . The conjugate containing Finally , the PEG - EPO conjugate is purified by ion - ex - fractions of the eluate are concentrated by UF /DF using a 10 change chromatography on Sepharose FF . 1. 5 mg protein kD membrane made of regenerated cellulose (88 cm², ml gel is loaded on the column pre equilibrated with 50 mM cutoff 10 kD /Millipore ) . The final diafiltration step is per Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The conjugate formed against Hepes buffer (50 mM Hepes, 5 mM CaCl2 , is eluted with 50 mM Hepes buffer containing 5 mM CaC12 10 pH 7 . 5 ) . and 500 mM sodium chloride , pH 7 . 4 and is then subjected The preparation is analytically characterized by measur to UF /DF using a membrane . The preparation is analytically ing total protein ( Bradford and BCA procedure ) and bio characterized by measuring total protein (Bradford ) and logical activity according to known methods. biological activity according to methods known in the art . In an alternative embodiment , Method 3 is carried out as 15 Example 33 follows . EPO is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An PEGylation of Ang - 2 Using an Aminooxy -PEG example of this type of reagent is the Sunbright® CA series Reagent and m - Toluidine as a Nucleophilic from NOF (NOF Corp . , Tokyo , Japan ) . 10 mg EPO is Catalyst dissolved in ~ 8 ml histidine buffer , pH 6 . 0 ( 20 mM L - his - 20 tidine, 150 mM NaCl) . 200 ul of an aqueous sodium Method 1 : periodate solution ( 5 mM ) and 2 ml of an aqueous m -tolui - Ang -2 is PEGylated by use of a linear 20 kD PEGylation dine solution (50 mM ) are then added . Subsequently , the reagent containing an aminooxy group . An example of this aminooxy -PEG reagent with a MW of 20 kD ( described type of reagent is the Sunbright® CA series from NOF (NOF above ) is added to give a 5 - fold molar reagent excess . The 25 Corp ., Tokyo , Japan ). Ang - 2 is dissolved in 7 . 0 ml histidine mixture is incubated for 2 h in the dark at room temperature buffer , pH 6 . 0 (20 mM L -histidine , 150 mM NaCl, 5 mM under gentle stirring and quenched for 15 min at room CaCl2 ). An aqueous sodium periodate solution ( 5 mM ) is temperature by the addition of 100 ul of 1 M aqueous then added and the reaction mixture is incubated for 1 h in cysteine solution . the dark at 4° C . under gentle stirring and quenched for 15 Finally, the PEG -EPO conjugate is purified by ion - ex - 30 min at room temperature by the addition of 7 . 5 ul of a 1 M change chromatography on Q Sepharose FF . The reaction aqueous cysteine solution . The mixture is subsequently mixture is diluted with 20 ml Buffer A (50 mM Hepes, pH subjected to UF /DF employing Vivaspin centrifugal filtra 7 .5 ) and loaded onto a 20 ml HiPrep QFF 16 / 10 column (GE tors to remove excess periodate , quencher and the byprod Healthcare, Fairfield , Conn . ) pre - equilibrated with BufferA u cts thereof. Then the column is eluted with Buffer B (50 mM Hepes , 1 35 The retentate containing oxidized Ang - 2 is next mixed M NaC1, pH 7 . 5 ). Free EPO is eluted by washing the column with an aqueous m - toluidine solution ( 50 mm ) and incu with 25 % Buffer B and the conjugate at 50 % Buffer B . The bated for 30 min at room temperature . Aminooxy - PEG conjugate containing fractions are concentrated by UF/ DF reagent with a MW of 20 kD is then added to give a 5 - fold using a 10 kD membrane made of regenerated cellulose (88 molar reagent excess . This mixture is incubated for 2 . 5 h at cm2, cutoff 10 kD /Millipore ) . The final diafiltration step is 40 room temperature in the dark under gentle stirring. performed against histidine buffer , pH 7 . 2 containing 150 Finally, the PEG - Ang - 2 conjugate is purified by ion mM NaCl. The preparation is analytically characterized by exchange chromatography ( e . g . on Q Sepharose FF ). For measuring total protein (Bradford ) and biological activity example , 1 . 5 mg protein /ml gel is loaded on the column according to methods known in the art. For the PEG - EPO equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 conjugate a specific activity of > 50 % in comparison to 45 mM CaC12 . The conjugate is eluted with 50 mM Hepes native EPO is determined . The conjugate is additionally buffer containing 5 mM CaC12 and 500 mM sodium chlo analytically characterized by Size Exclusion HPLC using a ride, pH 7 . 4 and is then subjected to UF /DF using an Agilent 1200 HPLC system equipped with a Shodex KW appropriate MW cutoff membrane . The preparation is next 803 column under conditions as previously described (Kola - analytically characterized by measuring total protein (Coo rich et al , Transfusion 2006 ; 46 : 1959 - 77 ) . It is shown that 50 massie , Bradford ) and biological activity according to meth the preparation contains no free EPO . ods known in the art . Method 4 : In an alternative embodiment, Method 1 is carried out as EPO is PEGylated by use of a linear 20 kD PEGylation follows . Ang - 2 is PEGylated by use of a linear 20 kD reagent containing an aminooxy group . An example of this PEGylation reagent containing an aminooxy group . An type of reagent is the Sunbright® CA series from NOF (NOF 55 example of this type of reagent is the Sunbright® CA series Corp . , Tokyo , Japan ) . An initial concentration or weight of from NOF ( NOF Corp . , Tokyo , Japan ). Ang- 2 is dissolved EPO is transferred or dissolved in Hepes buffer (50 mM in 7 .0 ml histidine buffer , pH 6 .0 (20 mM L -histidine , 150 Hepes , 150 mM sodium chloride, 5 mM calcium chloride , mM NaCl, 5 mM CaCl2 ). An aqueous sodium periodate pH 6 . 0 ) to get a final protein concentration of 2 mg EPO /ml . solution ( 5 mM ) is then added and the reaction mixture is Subsequently an 5 mM aqueous sodium periodate solution is 60 incubated for 1 h in the dark at 4° C . under gentle stirring added within 15 minutes to give a final concentration of 100 and quenched for 15 min at room temperature by the UM , followed by addition of an 50 mM aqueous m - toluidine addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The solution to get a final concentration of 10 mM within a time mixture is subsequently subjected to UF /DF employing period of 30 minutes . Then the aminooxy - PEG reagent with Vivaspin centrifugal filtrators to remove excess periodate , a MW of 20 kD (described above ) is added to give a 20 - fold 65 quencher and the byproducts thereof. molar reagent excess . After correction of the pH to 6 . 0 the The retentate containing oxidized Ang - 2 is next mixed mixture is incubated for 2 h in the dark at room temperature with an aqueous m - toluidine solution ( 50 mm ) and incu US 10 ,414 ,793 B2 133 134 bated for 30 min at room temperature . Aminooxy - PEG Finally , the PEG - Ang - 2 conjugate is purified by ion reagent with a MW of 20 kD is then added to give a 5 - fold exchange chromatography on Sepharose FF . 1 . 5 mg molar reagent excess . This mixture is incubated for 2 . 5 h at protein /ml gel is loaded on the column pre equilibrated with room temperature in the dark under gentle stirring . 50 mM Hepes buffer , pH 7 . 4 containing 5 mM CaCl2 . The Finally , the PEG - Ang - 2 conjugate is purified by ion - conjugate is eluted with 50 mM Hepes buffer containing 5 exchange chromatography . The conjugate containing frac mM CaC12 and 500 mM sodium chloride , pH 7 . 4 and is then tion of the eluate are collected and then subjected to UF /DF subjected to UF /DF using a membrane . The preparation is using an appropriate MW cutoff membrane . The preparation analytically characterized by measuring total protein (Brad is next analytically characterized by measuring total protein ford ) and biological activity according to methods known in (Coomassie , Bradford ) and biological activity according to. 10 the art. methods known in the art . In an alternative embodiment, Method 3 is carried out as follows. Ang - 2 is PEGylated by use of a linear 20 kD Method 2 : PEGylation reagent containing an aminooxy group . An Ang - 2 is PEGylated by use of a linear 20 kD PEGylation 15 example of this type of reagent is the Sunbright® CA series reagent containing an aminooxy group . An example of this from NOF (NOF Corp ., Tokyo , Japan ). Ang -2 is dissolved type of reagent is the Sunbright® CA series from NOF (NOF in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, Corp . , Tokyo , Japan ) . 5 mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous Ang - 2 is transferred or dissolved in reaction buffer ( e . g . sodium periodate solution (10 mm ) , and an aqueous 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium 20 m - toluidine solution (50 mm ). Subsequently the aminooxy chloride, pH 6 . 0 ) to get a final protein concentration of reagent is added to give a 20 - fold molar reagent excess. The 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected mixture is incubated for 2 h in the dark at room temperature to 6 . 0 by drop wise addition of a 0 . 5N aqueous HCl solution under gentle stirring and quenched for 15 min at room Subsequently a 40 mM aqueous sodium periodate solution is temperature by the addition of 8 ul of aqueous cysteine added within 10 minutes to give a concentration of 200 uM . 25 solution ( 1 M . The oxidation reaction is carried out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction is Finally the PEG - Ang - 2 conjugate is purified by ion stopped by addition of an aqueous L - cysteine solution ( 1 M ) exchange chromatography The conjugate containg freac within 15 minutes at T = + 22 + / - 2° C . to give a final concen tions of the eluate are collected and then subjected to UF /DF . tration of 10 mM in the reaction mixture and incubation for 30 The preparation is analytically characterized by measuring 60 + / - 5 min . total protein (Bradford ) and biological activity according to The oxidized Ang - 2 is further purified by ion exchange methods known in the art . chromatography . The oxidized Ang - 2 containing fractions of Method 4 : the eluate are collected and used for the conjugation reac - 26 Ang - 2 is PEGylated by use of a linear 20 kD PEGylation tion . reagent containing an aminooxy group . An example of this The aminooxy - PEG reagent with a MW of 20 kD reagent type of reagent is the Sunbright® CA series from NOF (NOF is added in a 50 - fold molar excess to the eluate containing Corp ., Tokyo, Japan ). An initial concentration or weight of the purified oxidized Ang - 2 within a maximum time period Ang - 2 is transferred or dissolved in Hepes buffer (50 mM ( t ) of 15 minutes under gentle stirring . Then an aqueous 40 Hedes . 150 mM sodium chloride , 5 mM calcium chloride , m - toluidine solution (50 mM ) is added within 15 minutes to pH 6 . 0 ) to get a final protein concentration of 2 mg Ang - 2 / get a final concentration of 10 mM . The reaction mixture is ml. Subsequently an 5 mM aqueous sodium periodate solu incubated for 120 + / – 10 min . in the dark at a temperature ( T) tion is added within 15 minutes to give a final concentration of T = + 22 + / - 2° C . under gentle shaking . of 100 UM , followed by addition of an 50 mM aqueous The obtained PEG - Ang - 2 conjugate is further purified by 45 m - toluidine solution to get a final concentration of 10 mm ion exchange chromatography . The PEG -Ang - 2 conjugate within a time period of 30 minutes . Then the aminooxy - PEG containing fractions are collected and concentrated by ultra - / reagent with a MW of 20 kD (described above ) is added to diafiltration (UF /DF ) using a membrane made of regener give a 20 - fold molar reagent excess. After correction of the ated cellulose with an appropriate molecular weight cut off pH to 6 .0 the mixture is incubated for 2 h in the dark at room (Millipore ). 50 temperature under gentle stirring and quenched for 15 min The conjugate prepared by use of this procedure are at room temperature by the addition of an 1 M aqueous analytically characterized by measuring total protein and L - cysteine solution to give a final concentration of 10 mM . biological activity according to methods known in the art. The PEG -Ang - 2 conjugate is purified by means of ion Method 3 : 55 exchange chromatography ( IEC ) . The conjugate containing Ang- 2 is PEGylated by use of a linear 20 kD PEGylation fractions of the eluate are concentrated by UF/ DF using a 10 reagent containing an aminooxy group . An example of this kD membrane made of regenerated cellulose ( 88 cm2, type of reagent is the Sunbright® CA series from NOF (NOF cutoff 10 kD /Millipore ). The final diafiltration step is per Corp . , Tokyo , Japan ) . Ang - 2 is dissolved in Hepes buffer (50 formed against Hepes buffer ( 50 mM Hepes, 5 mM CaC12 , mM Hepes , 150 mM sodium chloride , 5 mM calcium 60 pH 7 5 ). chloride , pH 6 . 0 ) and mixed with an aqueous sodium periodate solution ( 10 mM ) , and an aqueous m - toluidine The preparation is analytically characterized by measur solution (50 mm ). Subsequently the aminooxy reagent is inginglo total protein ( Bradford and BCA procedure ) and bio added to give a 20 - fold molar reagent excess. The mixture logical activity according to known methods. is incubated for 2 h in the dark at room temperature under 65 Subsequently , the free Ang - 2 is removed by means of ion gentle stirring and quenched for 15 min at room temperature exchange chromatography (IEC ) . The conjugate containing by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . fractions of the eluate are concentrated by UF/ DF . US 10 ,414 ,793 B2 135 136 Example 34 type of reagent is the Sunbright® CA series from NOF (NOF Corp ., Tokyo , Japan ). VEGF is transferred or dissolved in PEGylation of VEGF Using an Aminooxy - PEG reaction buffer (e .g . 50 mM Hepes, 350 mM sodium chlo Reagent and m - Toluidine as a Nucleophilic ride, 5 mM calcium chloride, pH 6 . 0 ) to get a final protein Catalyst concentration of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is corrected to 6 . 0 by drop wise addition of a 0 . 5 N Method 1 : aqueous HCl solution . Subsequently , a 40 mM aqueous VEGF is PEGylated by use of a linear 20 kD PEGylation sodium periodate solution is added within 10 minutes to give reagent containing an aminooxy group . An example of this a concentration of 200 uM . The oxidation reaction is carried type of reagent is the Sunbright® CA series from NOF (NOF 10 out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . Corp . , Tokyo , Japan ) . VEGF is dissolved in 7 . 0 mlhistidine Then the reaction is stopped by addition of an aqueous buffer, pH 6 . 0 ( 20 mM L -histidine , 150 mM NaC1, 5 mM L -cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° CaCl2 ). An aqueous sodium periodate solution (5 mM ) is C . to give a final concentration of 10 mM in the reaction then added and the reaction mixture is incubated for 1 h in mixture and incubation for 60 + / - 5 min . the dark at 4° C . under gentle stirring and quenched for 15 15 The oxidized VEGF is further purified by ion exchange min at room temperature by the addition of 7 . 5 ul of a 1 M chromatography . The oxidized VEGF containing fractions aqueous cysteine solution . The mixture is subsequently of the eluate are collected and used for the conjugation subjected to UF/ DF employing Vivaspin centrifugal filtra - reaction . tors to remove excess periodate , quencher and the byprod - The aminooxy -PEG reagent with a MW of 20 kD reagent ucts thereof. 20 is added in a 50 - fold molar excess to the eluate containing The retentate containing oxidized VEGF is next mixed the purified oxidized VEGF within a maximum time period with an aqueous m - toluidine solution (50 mM ) and incu - ( t ) of 15 minutes under gentle stirring . Then an aqueous bated for 30 min at room temperature . Aminooxy - PEG m - toluidine solution ( 50 mM ) is added within 15 minutes to reagent with a MW of 20 kD is then added to give a 5 - fold get a final concentration of 10 mM . The reaction mixture is molar reagent excess. This mixture is incubated for 2 . 5 h at 25 incubated for 120 + / – 10 min . in the dark at a temperature ( T ) room temperature in the dark under gentle stirring . of T = + 22 + / - 2° C . under gentle shaking . Finally , the PEG -VEGF conjugate is purified by ion - The obtained PEG -VEGF conjugate is further purified by exchange chromatography ( e . g . , on Sepharose FF ) . For ion exchange chromatography. The PEG - VEGF conjugate example , 1 .5 mg protein /ml gel is loaded on the column containing fractions are collected and concentrated by ultra - / equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 30 diafiltration (UF /DF ) using a membrane made of regener mM CaC12 . The conjugate is eluted with 50 mM Hepes ated cellulose with an appropriate molecular weight cut off buffer containing 5 mM CaCl2 and 500 mM sodium chlo (Millipore ). ride, pH 7 . 4 and is then subjected to UF /DF using an The conjugate prepared by use of this procedure are appropriate MW cutoff membrane . The preparation is next analytically characterized by measuring total protein and analytically characterized by measuring total protein (Coo - 35 biological activity according to methods known in the art . massie , Bradford ) and biological activity according to meth - Method 3 : ods known in the art . VEGF is PEGylated by use of a linear 20 kD PEGylation In an alternative embodiment, Method 1 is carried out as reagent containing an aminooxy group . An example of this follows. VEGF is PEGylated by use of a linear 20 kD type of reagent is the Sunbright® CA series from NOF (NOF PEGylation reagent containing an aminooxy group . An 40 Corp . , Tokyo , Japan ) . VEGF is dissolved in Hepes buffer (50 example of this type of reagent is the Sunbright® CA series mM Hepes, 150 mM sodium chloride , 5 mM calcium from NOF (NOF Corp . , Tokyo , Japan ). VEGF is dissolved chloride, pH 6 .0 ) and mixed with an aqueous sodium in 7 .0 ml histidine buffer, pH 6 . 0 (20 mM L - histidine, 150 periodate solution ( 10 mM ), and an aqueous m - toluidine mM NaCl, 5 mM CaCl2 ) . An aqueous sodium periodate solution (50 mm ) . Subsequently , the aminooxy reagent is solution (5 mm ) is then added and the reaction mixture is 45 added to give a 20 - fold molar reagent excess. The mixture incubated for 1 h in the dark at 4° C . under gentle stirring is incubated for 2 h in the dark at room temperature under and quenched for 15 min at room temperature by the gentle stirring and quenched for 15 min at room temperature addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . mixture is subsequently subjected to UF /DF employing Finally, the PEG -VEGF conjugate is purified by ion Vivaspin centrifugal filtrators to remove excess periodate , 50 exchange chromatography on Sepharose FF . 1 . 5 mg quencher and the byproducts thereof. protein /ml gel is loaded on the column pre equilibrated with The retentate containing oxidized VEGF is next mixed 50 mM Hepes buffer, pH 7 .4 containing 5 mM CaC12 . The with an aqueous m - toluidine solution (50 mM ) and incu - conjugate is eluted with 50 mM Hepes buffer containing 5 bated for 30 min at room temperature . Aminooxy -PEG mM CaC12 and 500 mM sodium chloride , pH 7 . 4 and is then reagent with a MW of 20 kD is then added to give a 5 - fold 55 subjected to UF /DF using a membrane. The preparation is molar reagent excess. This mixture is incubated for 2 . 5 h at analytically characterized by measuring total protein (Brad room temperature in the dark under gentle stirring . ford ) and biological activity according to methods known in Finally , the PEG - VEGF conjugate is purified by ion the art . exchange chromatography The conjugate containg fractions In an alternative embodiment, Method 3 is carried out as of the eluate are collected and then subjected to UF /DF using 60 follows. VEGF is PEGylated by use of a linear 20 kD an appropriate MW cutoff membrane . The preparation is PEGylation reagent containing an aminooxy group . An next analytically characterized by measuring total protein example of this type of reagent is the Sunbright® CA series ( Coomassie , Bradford ) and biological activity according to from NOF (NOF Corp ., Tokyo , Japan ). VEGF is dissolved methods known in the art . in Hepes buffer ( 50 mM Hepes , 150 mM sodium chloride , Method 2 : 65 5 mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous VEGF is PEGylated by use of a linear 20 kD PEGylation sodium periodate solution ( 10 mm ) , and an aqueous reagent containing an aminooxy group . An example of this m - toluidine solution ( 50 mm ). Subsequently , the aminooxy US 10 ,414 ,793 B2 137 138 reagent is added to give a 20 - fold molar reagent excess . The excess . This mixture is incubated for 2 . 5 h at room tem mixture is incubated for 2 h in the dark at room temperature perature in the dark under gentle stirring . under gentle stirring and quenched for 15 min at room Finally , the PEG - EGF conjugate is purified by ion -ex temperature by the addition of 8 ul of aqueous cysteine change chromatography ( e . g . , on Q Sepharose FF ) . For solution ( 1 M ) . 5 example , 1 . 5 mg protein /ml gel is loaded on the column Finally, the PEG - VEGF conjugate is purified by ion equilibrated with 50 mM Hepes buffer, pH 7 . 4 containing 5 exchange chromatography. The conjugate fractions of the mM CaC12 . The conjugate is eluted with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium chlo eluate are collected and then subjected to UF /DF . The ride , pH 7 . 4 and is then subjected to UF /DF using an preparation is analytically characterized by measuring total 10 appropriate MW cutoff membrane . The preparation is next protein (Bradford ) and biological activity according to meth analytically characterized by measuring total protein (Coo ods known in the art. massie , Bradford ) and biological activity according to meth Method 4 : ods known in the art . VEGF is PEGylated by use of a linear 20 kD PEGylation In an alternative embodiment, Method 1 is carried out as reagent containing an aminooxy group . An example of this 15 follows. EGF is PEGylated by use of a linear 20 kD type of reagent is the Sunbright® CA series from NOF (NOF PEGylation reagent containing an aminooxy group . An Corp ., Tokyo , Japan ). An initial concentration or weight of example of this type of reagent is the Sunbrighte CA series VEGF is transferred or dissolved in Hepes buffer ( 50 mM from NOF (NOF Corp ., Tokyo , Japan ). EGF is dissolved in Hepes , 150 mM sodium chloride , 5 mM calcium chloride , 7 . 0 mlhistidine buffer , pH 6 . 0 ( 20 mM L - histidine , 150 mM pH 6 . 0 ) to get a final protein concentration of 2 mg VEGF/ 20 NaCl, 5 mM CaC12 ) . An aqueous sodium periodate solution ml. Subsequently , an 5 mM aqueous sodium periodate (5 mM ) is then added and the reaction mixture is incubated solution is added within 15 minutes to give a final concen - for 1 h in the dark at 4° C . under gentle stirring and quenched tration of 100 uM , followed by addition of an 50 mM for 15 min at room temperature by the addition of 7 . 5 ul of aqueous m - toluidine solution to get a final concentration of a 1 M aqueous cysteine solution . The mixture is subse 10 mM within a time period of 30 minutes . Then the 25 quently subjected to UF /DF employing Vivaspin centrifugal aminooxy -PEG reagent with a MW of 20 kD ( described filtrators to remove excess periodate , quencher and the above ) is added to give a 20 - fold molar reagent excess. After byproducts thereof. correction of the pH to 6 .0 the mixture is incubated for 2 h. The retentate containing oxidized EGF is next mixed with in the dark at room temperature under gentle stirring and an aqueous m - toluidine solution (50 mM ) and incubated for quenched for 15 min at room temperature by the addition of 30 30 min at room temperature . Aminooxy - PEG reagent with a an 1 M aqueous L - cysteine solution to give a final concen - MW of 20 kD is then added to give a 5 - fold molar reagent tration of 10 mM . excess . This mixture is incubated for 2 . 5 h at room tem The PEG - VEGF conjugate is purified by means of ion perature in the dark under gentle stirring . exchange chromatography ( IEC ) . The conjugate containing Finally , the PEG - EGF conjugate is purified by ion - ex fractions of the eluate are concentrated by UF /DF using a 10 35 change chromatography. The conjugate containg fractions of kD membrane made of regenerated cellulose ( 88 cm2, the eluate are collected and then subjected to UF /DF using cut -off 10 kD /Millipore ) . The final diafiltration step is per- an appropriate MW cutoff membrane . The preparation is formed against Hepes buffer ( 50 mM Hepes , 5 mM CaC12 , next analytically characterized by measuring total protein pH 7 . 5 ) . ( Coomassie , Bradford ) and biological activity according to The preparation is analytically characterized by measur- 40 methods known in the art . ing total protein ( Bradford and BCA procedure ) and bio - Method 2 : logical activity according to known methods . EGF is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An example of this Example 35 type of reagent is the Sunbright® CA series from NOF (NOF 45 Corp ., Tokyo , Japan ) . EGF is transferred or dissolved in PEGylation of EGF Using an Aminooxy - PEG reaction buffer ( e . g . 50 mM Hepes, 350 mM sodium chlo Reagent and m - Toluidine as a Nucleophilic ride, 5 mM calcium chloride , pH 6 . 0 ) to get a final protein Catalyst concentration of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected to 6 . 0 by drop wise addition of a 0 . 5 N Method 1 : 50 aqueous HCl solution . Subsequently , a 40 mM aqueous EGF is PEGylated by use of a linear 20 kD PEGylation sodium periodate solution is added within 10 minutes to give reagent containing an aminooxy group . An example of this a concentration of 200 uM . The oxidation reaction is carried type of reagent is the Sunbright® CA series from NOF (NOF out for 30 + / – 5 min at a temperature ( T ) of T = + 22 + / - 2° C . Corp ., Tokyo , Japan ) . EGF is dissolved in 7 . 0 ml histidine Then the reaction is stopped by addition of an aqueous buffer , pH 6 . 0 ( 20 mM L -histidine , 150 mM NaCl, 5 mM 55 L -cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° CaC12 ) . An aqueous sodium periodate solution ( 5 mm ) is C . to give a final concentration of 10 mM in the reaction then added and the reaction mixture is incubated for 1 h in mixture and incubation for 60 + / - 5 min . the dark at 4° C . under gentle stirring and quenched for 15 The oxidized EGF is further purified by ion exchange min at room temperature by the addition of 7 . 5 ul of a 1 M chromatography . The oxidized EGF containing fractions of aqueous cysteine solution . The mixture is subsequently 60 the eluate are collected and used for the conjugation reac subjected to UF/ DF employing Vivaspin centrifugal filtra tion . tors to remove excess periodate , quencher and the byprod - The aminooxy -PEG reagent with a MW of 20 kD reagent ucts thereof. is added in a 50 - fold molar excess to the eluate containing The retentate containing oxidized EGF is next mixed with the purified oxidized NGF within a maximum time period ( t ) an aqueous m - toluidine solution ( 50 mM ) and incubated for 65 of 15 minutes under gentle stirring . Then an aqueous 30 min at room temperature . Aminooxy - PEG reagent with a m - toluidine solution (50 mM ) is added within 15 minutes to MW of 20 kD is then added to give a 5 - fold molar reagent get a final concentration of 10 mM . The reaction mixture is US 10 ,414 ,793 B2 139 140 incubated for 120 + / - 10 min . in the dark at a temperature ( T ) molar reagent excess. After correction of the pH to 6 . 0 the of T = + 22 + / - 2° C . under gentle shaking . mixture is incubated for 2 h in the dark at room temperature The obtained PEG - EGF conjugate is further purified by under gentle stirring and quenched for 15 min at room ion exchange chromatography. The PEG - EGF conjugate temperature by the addition of an 1 M aqueous L - cysteine containing fractions are collected and concentrated by ultra -/ 5 solution to give a final concentration of 10 mM . diafiltration (UF /DF ) using a membrane made of regener - The PEG - EGF conjugate is purified by means of ion ated cellulose with an appropriate molecular weight cut off exchange chromatography (IEC ). The conjugate containing (Millipore ) . fractions of the eluate are concentrated by UF /DF using a 10 The conjugate prepared by use of this procedure are kD membrane made of regenerated cellulose (88 cm2 , analytically characterized by measuring total protein and 10 cutoff 10 kD /Millipore ) . The final diafiltration step is per biological activity according to methods known in the art . formed against Hepes buffer ( 50 mM Hepes , 5 mM CaC12 , Method 3 : pH 7 . 5 ) . EGF is PEGylated by use of a linear 20 kD PEGylation The preparation is analytically characterized by measur reagent containing an aminooxy group . An example of this ing total protein (Bradford and BCA procedure ) and bio type of reagent is the Sunbright® CA series from NOF (NOF 15 logical activity according to known methods. Corp . , Tokyo , Japan ). EGF is dissolved in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , 5 mM calcium Example 36 chloride , pH 6 . 0 ) and mixed with an aqueous sodium periodate solution ( 10 mM ) , and an aqueous m -toluidine PEGylation of NGF Using an Aminooxy - PEG solution (50 mM ) . Subsequently the aminooxy reagent is 20 Reagent and m - Toluidine as a Nucleophilic added to give a 20 - fold molar reagent excess . The mixture Catalyst is incubated for 2 h in the dark at room temperature under gentle stirring and quenched for 15 min at room temperature Method 1 : by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . NGF is PEGylated by use of a linear 20 kD PEGylation Finally, the PEG - EGF conjugate is purified by ion -ex - 25 reagent containing an aminooxy group . An example of this change chromatography on Q -Sepharose FF . 1 . 5 mg protein type of reagent is the Sunbright® CA series from NOF (NOF ml gel is loaded on the column pre equilibrated with 50 mM Corp ., Tokyo , Japan ). NGF is dissolved in 7 . 0 ml histidine Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The conjugate buffer, pH 6 . 0 (20 mM L -histidine , 150 mM NaC1, 5 mM is eluted with 50 mM Hepes buffer containing 5 mM CaC12 CaC12 ). An aqueous sodium periodate solution ( 5 mm ) is and 500 mM sodium chloride , pH 7 .4 and is then subjected 30 then added and the reaction mixture is incubated for 1 h in to UF /DF using a membrane . The preparation is analytically the dark at 4° C . under gentle stirring and quenched for 15 characterized by measuring total protein ( Bradford ) and min at room temperature by the addition of 7 . 5 ul of a 1 M biological activity according to methods known in the art . aqueous cysteine solution . The mixture is subsequently In an alternative embodiment, Method 3 is carried out as subjected to UF / DF employing Vivaspin centrifugal filtra follows. EGF is PEGylated by use of a linear 20 kD 35 tors to remove excess periodate , quencher and the byprod PEGylation reagent containing an aminooxy group . An u cts thereof. example of this type of reagent is the Sunbright® CA series The retentate containing oxidized NGF is nextmixed with from NOF (NOF Corp ., Tokyo , Japan ). EGF is dissolved in an aqueous m - toluidine solution ( 50 mM ) and incubated for Hepes buffer (50 mM Hepes , 150 mM sodium chloride , 5 30 min at room temperature . Aminooxy - PEG reagent with a mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous 40 MW of 20 kD is then added to give a 5 - fold molar reagent sodium periodate solution ( 10 mm ) , and an aqueous excess. This mixture is incubated for 2 . 5 h at room tem m - toluidine solution (50 mM ) . Subsequently the aminooxy perature in the dark under gentle stirring. reagent is added to give a 20 - fold molar reagent excess. The Finally, the PEG - NGF conjugate is purified by ion - ex mixture is incubated for 2 h in the dark at room temperature change chromatography ( e . g ., on Q -Sepharose FF ) . For under gentle stirring and quenched for 15 min at room 45 example , 1 . 5 mg protein / ml gel is loaded on the column temperature by the addition of 8 ul of aqueous cysteine equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 solution ( 1 M ) . mM CaC12 . The conjugate is eluted with 50 mM Hepes Finally , the PEG - EGF conjugate is purified by ion -ex buffer containing 5 mM CaCl2 and 500 mM sodium chlo change chromatography . The conjugate containing fractions ride, pH 7 . 4 and is then subjected to UF /DF using an of the eluate are collected and then subjected to UF /DF . The 50 appropriate MW cutoff membrane . The preparation is next preparation is analytically characterized by measuring total analytically characterized by measuring total protein ( Coo protein (Bradford ) and biological activity according to meth massie , Bradford ) and biological activity according to meth ods known in the art . ods known in the art . Method 4 : In an alternative embodiment, Method 1 is carried out as EGF is PEGylated by use of a linear 20 kD PEGylation 55 follows. NGF is PEGylated by use of a linear 20 kD reagent containing an aminooxy group . An example of this PEGylation reagent containing an aminooxy group . An type of reagent is the Sunbright® CA series from NOF (NOF example of this type of reagent is the Sunbright® CA series Corp ., Tokyo , Japan ). An initial concentration or weight of from NOF (NOF Corp ., Tokyo , Japan ) . NGF is dissolved in EGF is transferred or dissolved in Hepes buffer ( 50 mM 7 . 0 mlhistidine buffer, pH 6 . 0 ( 20 mM L -histidine , 150 mM Hepes , 150 mM sodium chloride , 5 mM calcium chloride , 60 NaC1, 5 mM CaC12 ) . An aqueous sodium periodate solution pH 6 . 0 ) to get a final protein concentration of 2 mg EGF /ml . ( 5 mM ) is then added and the reaction mixture is incubated Subsequently an 5 mM aqueous sodium periodate solution is for 1 h in the dark at 4° C . under gentle stirring and quenched added within 15 minutes to give a final concentration of 100 for 15 min at room temperature by the addition of 7 . 5 ul of uM , followed by addition of an 50 mM aqueous m -toluidine a 1 M aqueous cysteine solution . The mixture is subse solution to get a final concentration of 10 mM within a time 65 quently subjected to UF /DF employing Vivaspin centrifugal period of 30 minutes . Then the aminooxy - PEG reagent with filtrators to remove excess periodate, quencher and the a MW of 20 kD (described above ) is added to give a 20 - fold byproducts thereof. US 10 , 414 , 793 B2 141 142 The retentate containing oxidized NGF is next mixed with Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The conjugate an aqueous m - toluidine solution (50 mM ) and incubated for is eluted with 50 mM Hepes buffer containing 5 mM CaC12 30 min at room temperature . Aminooxy - PEG reagent with a and 500 mM sodium chloride , pH 7 . 4 and is then subjected MW of 20 kD is then added to give a 5 - fold molar reagent to UF/ DF using a membrane. The preparation is analytically excess . This mixture is incubated for 2 . 5 h at room tem - 5 characterized by measuring total protein (Bradford ) and perature in the dark under gentle stirring . biological activity according to methods known in the art . Finally , the PEG -NGF conjugate is purified by ion -ex - In an alternative embodiment , Method 3 is carried out as change chromatography ( The conjugate containing fractions follows. NGF is PEGylated by use of a linear 20 kD of the eluate are collected and then subjected to UF /DF using PEGylation reagent containing an aminooxy group . An an appropriate MW cutoff membrane. The preparation is 10 example of this type of reagent is the Sunbright® CA series next analytically characterized by measuring total protein from NOF ( NOF Corp . , Tokyo , Japan ) . NGF is dissolved in (Coomassie , Bradford ) and biological activity according to Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 methods known in the art . mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous Method 2 : sodium periodate solution (10 mm ), and an aqueous NGF is PEGylated by use of a linear 20 kD PEGylation 15 m - toluidine solution ( 50 mm ) . Subsequently the aminooxy reagent containing an aminooxy group . An example of this reagent is added to give a 20 - fold molar reagent excess . The type of reagent is the Sunbright® CA series from NOF (NOF mixture is incubated for 2 h in the dark at room temperature Corp . , Tokyo , Japan ). NGF is transferred or dissolved in under gentle stirring and quenched for 15 min at room reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chlo - temperature by the addition of 8 ul of aqueous cysteine ride , 5 mM calcium chloride, pH 6 . 0 ) to get a final protein 20 solution ( 1 M ) . concentration of 1 . 0 + / - 0 .25 mg/ml . Then the pH of the Finally , the PEG -NGF conjugate is purified by ion - ex solution is corrected to 6 . 0 by drop wise addition of a 0 . 5N change chromatography . The conjugate containg fractions aqueous HC1 solution . Subsequently a 40 mM aqueous are collected and then subjected to UF /DF . The preparation sodium periodate solution is added within 10 minutes to give is analytically characterized by measuring total protein a concentration of 200 uM . The oxidation reaction is carried 25 (Bradford ) and biological activity according to methods out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . known in the art . Then the reaction is stopped by addition of an aqueous Method 4 : L -cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° NGF is PEGylated by use of a linear 20 kD PEGylation C . to give a final concentration of 10 mM in the reaction reagent containing an aminooxy group . An example of this mixture and incubation for 60 + / - 5 min . 30 type of reagent is the Sunbright® CA series from NOF (NOF The oxidized NGF is further purified by ion exchange Corp ., Tokyo , Japan ). An initial concentration or weight of chromatography . The oxidized NGF containing fractions of NGF is transferred or dissolved in Hepes buffer ( 50 mM the eluate are collected and used for the conjugation reac Hepes, 150 mM sodium chloride , 5 mM calcium chloride , tion . pH 6 . 0 ) to get a final protein concentration of 2 mg NGF /ml . The aminooxy -PEG reagent with a MW of 20 kD reagent 35 Subsequently an 5 mM aqueous sodium periodate solution is is added in a 50 - fold molar excess to the eluate containing added within 15 minutes to give a final concentration of 100 the purified oxidized NGF within a maximum time period ( t ) uM , followed by addition of an 50 mM aqueous m - toluidine of 15 minutes under gentle stirring . Then an aqueous solution to get a final concentration of 10 mM within a time m - toluidine solution (50 mM ) is added within 15 minutes to period of 30 minutes. Then the aminooxy -PEG reagent with get a final concentration of 10 mM . The reaction mixture is 40 a MW of 20 kD (described above ) is added to give a 20 - fold incubated for 120 + / - 10 min . in the dark at a temperature ( T ) molar reagent excess . After correction of the pH to 6 .0 the of T = + 22 + / - 2° C . under gentle shaking . mixture is incubated for 2 h in the dark at room temperature The obtained PEG -NGF conjugate is further purified by under gentle stirring and quenched for 15 min at room ion exchange chromatography. The PEG -NGF conjugate temperature by the addition of an 1 M aqueous L - cysteine containing fractions are collected and concentrated by ultra - / 45 solution to give a final concentration of 10 mM . diafiltration (UF /DF ) using a membrane made of regener - The PEG - NGF conjugate is purified by means of ion ated cellulose with an appropriate molecular weight cut off exchange chromatography (IEC ) . The conjugate containing (Millipore ) . fractions of the eluate are concentrated by UF /DF using a 10 The conjugate prepared by use of this procedure are kD membrane made of regenerated cellulose (88 cm2 , analytically characterized by measuring total protein and 50 cutoff 10 kD /Millipore ) . The final diafiltration step is per biological activity according to methods known in the art . formed against Hepes buffer ( 50 mM Hepes , 5 mM CaC12 , Method 3 : pH 7 . 5 ) . NGF is PEGylated by use of a linear 20 kD PEGylation The preparation is analytically characterized by measur reagent containing an aminooxy group . An example of this ing total protein (Bradford and BCA procedure ) and bio type of reagent is the Sunbright® CA series from NOF (NOF 55 logical activity according to known methods. Corp . , Tokyo , Japan ) . NGF is dissolved in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , 5 mM calcium Example 37 chloride, pH 6 . 0 ) and mixed with an aqueous sodium periodate solution ( 10 mM ) , and an aqueous m -toluidine PEGylation of HGH Using an Aminooxy - PEG solution (50 mm ) . Subsequently the aminooxy reagent is 60 Reagent and m - Toluidine as a Nucleophilic added to give a 20 -fold molar reagent excess. The mixture Catalyst is incubated for 2 h in the dark at room temperature under gentle stirring and quenched for 15 min at room temperature Method 1 : by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . As described herein , the amino acid sequence of human Finally , the PEG - NGF conjugate is purified by ion -ex - 65 growth hormone (HGH ) is first modified to incorporate at change chromatography on Q Sepharose FF . 1 . 5 mg protein / least one glycosylation site . Following purification , HGH is ml gel is loaded on the column pre equilibrated with 50 mM glycosylated in vitro according to methods known in the art . US 10 ,414 ,793 B2 143 144 HGH is PEGylated by use of a linear 20 kD PEGylation HGH is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An example of this reagent containing an aminooxy group . An example of this type of reagent is the Sunbright® CA series from NOF (NOF type of reagent is the Sunbright® CA series from NOF (NOF Corp . , Tokyo , Japan ) . HGH is dissolved in 7 . 0 ml histidine Corp . , Tokyo , Japan ) . HGH is transferred or dissolved in buffer , pH 6 . 0 ( 20 mM L -histidine , 150 mM NaCl, 5 mM 5 reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chlo CaC12 ) . An aqueous sodium periodate solution ( 5 mm ) is ride , 5 mM calcium chloride , pH 6 . 0 ) to get a final protein then added and the reaction mixture is incubated for 1 h in concentration of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the the dark at 4° C . under gentle stirring and quenched for 15 solution is corrected to 6 . 0 by drop wise addition of a 0 .5N min at room temperature by the addition of 7 . 5 ul of a 1 M aqueous HCl solution . Subsequently , a 40 mM aqueous aqueous cysteine solution . The mixture is subsequently 10 sodium periodate solution is added within 10 minutes to give subjected to UF /DF employing Vivaspin centrifugal filtra - a concentration of 200 uM . The oxidation reaction is carried tors to remove excess periodate , quencher and the byprod out for 30 + / – 5 min at a temperature ( T ) of T = + 22 + / - 2° C . ucts thereof . Then the reaction is stopped by addition of an aqueous The retentate containing oxidized HGH is next mixed L - cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° with an aqueous m -toluidine solution (50 mM ) and incu - 15 C . to give a final concentration of 10 mM in the reaction bated for 30 min at room temperature . Aminooxy - PEG mixture and incubation for 60 + / - 5 min . reagent with a MW of 20 kD is then added to give a 5 - fold The oxidized HGH is further purified by ion exchange molar reagent excess. This mixture is incubated for 2 . 5 h at chromatography . The oxidized HGH containing fractions of room temperature in the dark under gentle stirring . the eluate are collected and used for the conjugation reac Finally , the PEG - HGH conjugate is purified by ion - 20 tion . exchange chromatography (e .g ., on Q Sepharose FF ) . For The aminooxy -PEG reagent with a MW of 20 kD reagent example, 1 . 5 mg protein /ml gel is loaded on the column is added in a 50 - fold molar excess to the eluate containing equilibrated with 50 mM Hepes buffer , pH 7 .4 containing 5 the purified oxidized HGH within a maximum time period mM CaC12 . The conjugate is eluted with 50 mM Hepes (t ) of 15 minutes under gentle stirring . Then an aqueous buffer containing 5 mM CaC12 and 500 mM sodium chlo - 25 m - toluidine solution (50 mM ) is added within 15 minutes to ride , pH 7 . 4 and is then subjected to UF / DF using an get a final concentration of 10 mM . The reaction mixture is appropriate MW cutoff membrane . The preparation is next incubated for 120 + / – 10 min . in the dark at a temperature ( T ) analytically characterized by measuring total protein ( Coo - of T = + 22 + / - 2° C . under gentle shaking . massie , Bradford ) and biological activity according to meth - The obtained PEG -HGH conjugate is further purified by ods known in the art . 30 ion exchange chromatography . The PEG - NGF conjugate In n alternative embodiment, Method 1 is carried out as containing fractions are collected and concentrated by ultra follows. As described herein , the amino acid sequence of diafiltration (UF / DF ) using a membrane made of regener human growth hormone (HGH ) is first modified to incor - ated cellulose with an appropriate molecular weight cut off porate at least one glycosylation site . Following purification , (Millipore ) . HGH is glycosylated in vitro according to methods known 35 The conjugate prepared by use of this procedure are in the art . analytically characterized by measuring total protein and HGH is PEGylated by use of a linear 20 kD PEGylation biological activity according to methods known in the art. reagent containing an aminooxy group . An example of this Method 3 : type of reagent is the Sunbright® CA series from NOF (NOF As described herein , the amino acid sequence of human Corp . , Tokyo , Japan ) . HGH is dissolved in 7 . 0 ml histidine 40 growth hormone (HGH ) is first modified to incorporate at buffer , pH 6 . 0 (20 mM L -histidine , 150 mM NaCl, 5 mM least one glycosylation site . Following purification , HGH is CaC12 ) . An aqueous sodium periodate solution (5 mM ) is glycosylated in vitro according to methods known in the art . then added and the reaction mixture is incubated for 1 h in HGH is PEGylated by use of a linear 20 kD PEGylation the dark at 4° C . under gentle stirring and quenched for 15 reagent containing an aminooxy group . An example of this min at room temperature by the addition of 7 .5 ul of a 1 M 45 type of reagent is the Sunbright® CA series from NOF (NOF aqueous cysteine solution . The mixture is subsequently Corp ., Tokyo , Japan ) . HGH is dissolved in Hepes buffer (50 subjected to UF/ DF employing Vivaspin centrifugal filtra - mM Hepes , 150 mM sodium chloride, 5 mM calcium tors to remove excess periodate , quencher and the byprod chloride, pH 6 . 0 ) and mixed with an aqueous sodium ucts thereof. periodate solution ( 10 mM ) , and an aqueous m - toluidine The retentate containing oxidized HGH is next mixed 50 solution ( 50 mM ) . Subsequently the aminooxy reagent is with an aqueous m - toluidine solution (50 mM ) and incu - added to give a 20 -fold molar reagent excess . The mixture bated for 30 min at room temperature . Aminooxy - PEG is incubated for 2 h in the dark at room temperature under reagentwith a MW of 20 kD is then added to give a 5 - fold gentle stirring and quenched for 15 min at room temperature molar reagent excess . This mixture is incubated for 2 . 5 h at by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . room temperature in the dark under gentle stirring . 55 Finally , the PEG - HGH conjugate is purified by ion Finally, the PEG - HGH conjugate is purified by ion exchange chromatography on Q - Sepharose FF . 1 . 5 mg pro exchange chromatography ( The conjugate containg frac - tein /ml gel is loaded on the column pre equilibrated with 50 tions of the eluate are collected and then subjected to UF /DF mM Hepes buffer, pH 7 .4 containing 5 mM CaC12 . The using an appropriate MW cutoff membrane . The preparation conjugate is eluted with 50 mM Hepes buffer containing 5 is next analytically characterized by measuring total protein 60 mM CaCl2 and 500 mM sodium chloride , pH 7. 4 and is then ( Coomassie , Bradford ) and biological activity according to subjected to UF /DF using a membrane . The preparation is methods known in the art . analytically characterized by measuring total protein (Brad Method 2 : ford ) and biological activity according to methods known in As described herein , the amino acid sequence of human the art . growth hormone (HGH ) is first modified to incorporate at 65 In an alternative embodiment, Method 3 is carried out as least one glycosylation site . Following purification , HGH is follows. As described herein , the amino acid sequence of glycosylated in vitro according to methods known in the art. human growth hormone (HGH ) is first modified to incor US 10 , 414 , 793 B2 145 146 porate at least one glycosylation site . Following purification , NaCl, 5 mM CaCl2 ). An aqueous sodium periodate solution HGH is glycosylated in vitro according to methods known ( 5 mm ) is then added and the reaction mixture is incubated in the art. HGH is PEGylated by use of a linear 20 kD for 1 h in the dark at 4° C . under gentle stirring and quenched PEGylation reagent containing an aminooxy group . An for 15 min at room temperature by the addition of 7 . 5 ul of example of this type of reagent is the Sunbright® CA series 5 a 1 M aqueous cysteine solution . The mixture is subse from NOF (NOF Corp . , Tokyo , Japan ) . HGH is dissolved in quently subjected to UF /DF employing Vivaspin centrifugal Hepes buffer (50 mM Hepes, 150 mM sodium chloride , 5 filtrators to remove excess periodate , quencher and the mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous byproducts thereof. sodium periodate solution ( 10 mm ) , and an aqueous The retentate containing oxidized TNF -alpha is next m - toluidine solution (50 mM ) . Subsequently the aminooxy 10 mixed with an aqueous m - toluidine solution (50 mM ) and reagent is added to give a 20 - fold molar reagent excess . The incubated for 30 min at room temperature . Aminooxy - PEG mixture is incubated for 2 h in the dark at room temperature reagent with a MW of 20 kD is then added to give a 5 - fold under gentle stirring and quenched for 15 min at room molar reagent excess . This mixture is incubated for 2 . 5 h at temperature by the addition of 8 ul of aqueous cysteine room temperature in the dark under gentle stirring . solution ( 1 M ) . 15 Finally , the PEG - TNF -alpha conjugate is purified by Finally , the PEG -HGH conjugate is purified by ion - ion -exchange chromatography (e .g ., on Q -Sepharose FF ). exchange chromatography . The conjugate containg fractions For example , 1 . 5 mg protein /ml gel is loaded on the column are collected and then subjected to UF /DF . The preparation equilibrated with 50 mM Hepes buffer, pH 7 .4 containing 5 is analytically characterized by measuring total protein mM CaC12 . The conjugate is eluted with 50 mM Hepes (Bradford ) and biological activity according to methods 20 buffer containing 5 mM CaCl2 and 500 mM sodium chlo known in the art. ride, pH 7 . 4 and is then subjected to UF /DF using an Method 4 : appropriate MW cutoff membrane. The preparation is next As described herein , the amino acid sequence of human analytically characterized by measuring total protein (Coo growth hormone (HGH ) is first modified to incorporate at massie , Bradford ) and biological activity according to meth least one glycosylation site . Following purification , HGH is 25 ods known in the art . glycosylated in vitro according to methods known in the art . In an alternative embodiment, Method 1 is carried out as HGH is PEGylated by use of a linear 20 kD PEGylation follows. TNF -alpha is PEGylated by use of a linear 20 kD reagent containing an aminooxy group . An example of this PEGylation reagent containing an aminooxy group . An type of reagent is the Sunbright® CA series from NOF (NOF example of this type of reagent is the Sunbright® CA series Corp . , Tokyo , Japan ). An initial concentration or weight of 30 from NOF (NOF Corp ., Tokyo , Japan ). TNF- alpha is dis HGH is transferred or dissolved in Hepes buffer ( 50 mM solved in 7 . 0 mlhistidine buffer, pH 6 . 0 ( 20 mM L -histidine , Hepes, 150 mM sodium chloride , 5 mM calcium chloride , 150 mM NaC1, 5 mM CaC12 ) . An aqueous sodium periodate pH 6 . 0 ) to get a final protein concentration of 2 mg HGH /ml . solution ( 5 mM ) is then added and the reaction mixture is Subsequently an 5 mM aqueous sodium periodate solution is incubated for 1 h in the dark at 4° C . under gentle stirring added within 15 minutes to give a final concentration of 100 35 and quenched for 15 min at room temperature by the UM , followed by addition of an 50 mM aqueous m - toluidine addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The solution to get a final concentration of 10 mM within a time mixture is subsequently subjected to UF /DF employing period of 30 minutes . Then the aminooxy - PEG reagent with Vivaspin centrifugal filtrators to remove excess periodate , a MW of 20 kD (described above ) is added to give a 20 - fold quencher and the byproducts thereof. molar reagent excess . After correction of the pH to 6 . 0 the 40 The retentate containing oxidized TNF -alpha is next mixture is incubated for 2 h in the dark at room temperature mixed with an aqueous m - toluidine solution (50 mm ) and under gentle stirring and quenched for 15 min at room incubated for 30 min at room temperature. Aminooxy -PEG temperature by the addition of a 1 M aqueous L - cysteine reagent with a MW of 20 kD is then added to give a 5 - fold solution to give a final concentration of 10 mM . molar reagent excess . This mixture is incubated for 2 . 5 h at The PEG - HGH conjugate is purified by means of ion 45 room temperature in the dark under gentle stirring . exchange chromatography (IEC ) . The conjugate containing Finally , the PEG - TNF -alpha conjugate is purified by fractions of the eluate are concentrated by UF /DF using a 10 ion -exchange chromatography . The conjugate containg frac kD membrane made of regenerated cellulose (88 cm2, tions of the eluate are collected and then subjected to UF/ DF cut -off 10 kD /Millipore ) . The final diafiltration step is per - using an appropriate MW cutoff membrane. The preparation formed against Hepes buffer (50 mM Hepes, 5 mM CaC12 , 50 is next analytically characterized by measuring total protein pH 7 . 5 ). ( Coomassie , Bradford ) and biological activity according to The preparation is analytically characterized by measur methods known in the art . ing total protein (Bradford and BCA procedure ) and bio Method 2 : logical activity according to known methods. TNF - alpha is PEGylated by use of a linear 20 kD PEGy 55 lation reagent containing an aminooxy group . An example of Example 38 this type of reagent is the Sunbright® CA series from NOF (NOF Corp ., Tokyo , Japan ) . TNF - alpha is transferred or PEGylation of TNF - Alpha Using an dissolved in reaction buffer ( e . g . 50 mM Hepes, 350 mM Aminooxy - PEG Reagent and m - Toluidine as a sodium chloride , 5 mM calcium chloride, pH 6 .0 ) to get a Nucleophilic Catalyst 60 final protein concentration of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is corrected to 6 . 0 by drop wise addition Method 1 : of a 0 . 5N aqueous HCl solution . Subsequently a 40 mm TNF - alpha is PEGylated by use of a linear 20 kD PEGY aqueous sodium periodate solution is added within 10 min lation reagent containing an aminooxy group . An example of utes to give a concentration of 200 uM . The oxidation this type of reagent is the Sunbright® CA series from NOF 65 reaction is carried out for 30 + / – 5 min at a temperature ( T ) (NOF Corp ., Tokyo , Japan ) . TNF- alpha is dissolved in 7 .0 of T = + 22 + / - 2° C . Then the reaction is stopped by addition ml histidine buffer , pH 6 . 0 ( 20 mM L -histidine , 150 mM of an aqueous L - cysteine solution ( 1 M ) within 15 minutes US 10 ,414 ,793 B2 147 148 at T = + 22 + / - 2° C . to give a final concentration of 10 mM in Method 4 : the reaction mixture and incubation for 60 + / - 5 min . TNF -alpha is PEGylated by use of a linear 20 kD PEGy The oxidized TNF - alpha is further purified by ion lation reagent containing an aminooxy group . An example of exchange chromatography. The oxidized TNF - alpha con - this type of reagent is the Sunbright® CA series from NOF taining fractions of the eluate are collected and used for the 5 (NOF Corp ., Tokyo , Japan ). An initial concentration or conjugation reaction . weight of TNF - alpha is transferred or dissolved in Hepes The aminooxy -PEG reagent with a MW of 20 kD reagent buffer (50 mM Hepes , 150 mM sodium chloride , 5 mM is added in a 50 - fold molar excess to the eluate containing calcium chloride, pH 6 . 0 ) to get a final protein concentration the purified oxidized TNF alpha within a maximum time of 2 mg TNF -alpha /ml . Subsequently , an 5 mM aqueous period ( t ) of 15 minutes under gentle stirring . Then an 10 sodium periodate solution is added within 15 minutes to give aqueous m - toluidine solution ( 50 mM ) is added within 15 a final concentration of 100 uM , followed by addition of an minutes to get a final concentration of 10 mM . The reaction 50 mM aqueous m - toluidine solution to get a final concen mixture is incubated for 120 + / – 10 min . in the dark at a tration of 10 mM within a time period of 30 minutes . Then temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking the aminooxy - PEG reagent with a MW of 20 kD (described The obtained PEG - TNF - alpha conjugate is further puri - 15 above ) is added to give a 20 - fold molar reagent excess . After fied by ion exchange chromatography. The PEG - TNF - alpha correction of the pH to 6 . 0 the mixture is incubated for 2 h conjugate containing fractions are collected and concen - in the dark at room temperature under gentle stirring and trated by ultra -/ diafiltration (UF /DF ) using a membrane quenched for 15 min at room temperature by the addition of made of regenerated cellulose with an appropriate molecular an 1 M aqueous L -cysteine solution to give a final concen weight cut off (Millipore ) . 20 tration of 10 mM . The conjugate prepared by use of this procedure are The PEG - TNF - alpha conjugate is purified by means of analytically characterized by measuring total protein and ion exchange chromatography ( IEC ) . The conjugate con biological activity according to methods known in the art. taining fractions of the eluate are concentrated by UF /DF Method 3 : using a 10 kD membrane made of regenerated cellulose (88 TNF - alpha is PEGylated by use of a linear 20 kD PEGY- 25 cm2, cut -off 10 kD /Millipore ). The final diafiltration step is lation reagent containing an aminooxy group . An example of performed against Hepes buffer (50 mM Hepes, 5 mM this type of reagent is the Sunbright® CA series from NOF CaC12 , pH 7 . 5 ) . ( NOF Corp. , Tokyo , Japan ) . TNF - alpha is dissolved in The preparation is analytically characterized by measur Hepes buffer ( 50 mM Hepes, 150 mM sodium chloride , 5 ing total protein ( Bradford and BCA procedure ) and bio mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous 30 logical activity according to known methods . sodium periodate solution ( 10 mM ) , and an aqueous m - toluidine solution ( 50 mM ) . Subsequently the aminooxy Example 39 reagent is added to give a 20 - fold molar reagent excess. The mixture is incubated for 2 h in the dark at room temperature PEGylation of Insulin Using an Aminooxy - PEG under gentle stirring and quenched for 15 min at room 35 Reagent and m - Toluidine as a Nucleophilic temperature by the addition of 8 ul of aqueous cysteine Catalyst solution ( 1 M ) . Finally , the PEG - TNF -alpha conjugate is purified by Method 1 : ion - exchange chromatography on Q - Sepharose FF . 1 . 5 mg As described herein , the amino acid sequence of insulin is protein /ml gel is loaded on the column pre equilibrated with 40 first modified to incorporate at least one glycosylation site . 50 mM Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The Following purification , insulin is glycosylated in vitro conjugate is eluted with 50 mM Hepes buffer containing 5 according to methods known in the art . Insulin is PEGylated mM CaCl2 and 500 mM sodium chloride, pH 7 . 4 and is then by use of a linear 20 kD PEGylation reagent containing an subjected to UF /DF using a membrane . The preparation is aminooxy group . An example of this type of reagent is the analytically characterized by measuring total protein (Brad - 45 Sunbright® CA series from NOF (NOF Corp . , Tokyo , ford ) and biological activity according to methods known in Japan ). Insulin is dissolved in 7 . 0 mlhistidine buffer, pH 6 . 0 the art . (20 mM L -histidine , 150 mM NaCl, 5 mM CaCl2 ) . An In an alternative embodiment, Method 3 is carried out as aqueous sodium periodate solution ( 5 mM ) is then added follows. TNF -alpha is PEGylated by use of a linear 20 kD and the reaction mixture is incubated for 1 h in the dark at PEGylation reagent containing an aminooxy group . An 50 4° C . under gentle stirring and quenched for 15 min at room example of this type of reagent is the Sunbright® CA series temperature by the addition of 7 . 5 ul of a 1 M aqueous from NOF (NOF Corp . , Tokyo , Japan ) . TNF - alpha is dis - cysteine solution . The mixture is subsequently subjected to solved in Hepes buffer (50 mM Hepes , 150 mM sodium UF /DF employing Vivaspin centrifugal filtrators to remove chloride , 5 mM calcium chloride , pH 6 . 0 ) and mixed with an excess periodate , quencher and the byproducts thereof. aqueous sodium periodate solution ( 10 mM ), and an aque - 55 The retentate containing oxidized insulin is next mixed ous m -toluidine solution (50 mm ) . Subsequently , the ami- with an aqueous m - toluidine solution ( 50 mM ) and incu nooxy reagent is added to give a 20 - fold molar reagent bated for 30 min at room temperature . Aminooxy - PEG excess. The mixture is incubated for 2 h in the dark at room reagent with a MW of 20 kD is then added to give a 5 - fold temperature under gentle stirring and quenched for 15 min molar reagent excess . This mixture is incubated for 2 . 5 h at at room temperature by the addition of 8 ul of aqueous 60 room temperature in the dark under gentle stirring . cysteine solution ( 1 M ) . Finally , the PEG - insulin conjugate is purified by ion Finally , the PEG - TNF - alpha conjugate is purified by exchange chromatography ( e . g ., on Q -Sepharose FF ) . For ion -exchange chromatography . The conjugate containing example , 1. 5 mg protein /ml gel is loaded on the column fractions are collected and then subjected to UF /DF . The equilibrated with 50 mM Hepes buffer, pH 7 . 4 containing 5 preparation is analytically characterized by measuring total 65 mM CaC12 . The conjugate is eluted with 50 mM Hepes protein (Bradford ) and biological activity according to meth - buffer containing 5 mM CaC12 and 500 mM sodium chlo ods known in the art . ride, pH 7 .4 and is then subjected to UF /DF using an US 10 , 414 , 793 B2 149 150 appropriate MW cutoff membrane . The preparation is next The obtained PEG - insulin conjugate is further purified by analytically characterized by measuring total protein (Coo ion exchange chromatography. The PEG - insulin conjugate massie , Bradford ) and biological activity according to meth containing fractions are collected and concentrated by ultra - / ods known in the art. diafiltration (UF / DF) using a membrane made of regener In an alternative embodiment, Method 1 is carried out as 5 ated cellulose with an appropriate molecular weight cut off follows . As described herein , the amino acid sequence of (Millipore ). insulin is first modified to incorporate at least one glycosy The conjugate prepared by use of this procedure are lation site. Following purification , insulin is glycosylated in analytically characterized by measuring total protein and vitro according to methods known in the art . Insulin is biological activity according to methods known in the art. PEGylated by use of a linear 20 kD PEGylation reagent 10 Method 3 : containing an aminooxy group . An example of this type of As described herein , the amino acid sequence of insulin is reagent is the Sunbright® CA series from NOF (NOF Corp ., first modified to incorporate at least one glycosylation site . Tokyo , Japan ) . Insulin is dissolved in 7 . 0 mlhistidine buffer, Following purification , insulin is glycosylated in vitro pH 6 . 0 ( 20 mM L - histidine , 150 mM NaCl, 5 mM CaC12 ) . according to methods known in the art . An aqueous sodium periodate solution (5 mM ) is then added 15 Insulin is PEGylated by use of a linear 20 kD PEGylation and the reaction mixture is incubated for 1 h in the dark at reagent containing an aminooxy group . An example of this 4° C . under gentle stirring and quenched for 15 min at room type of reagent is the Sunbright® CA series from NOF (NOF temperature by the addition of 7 . 5 al of a 1 M aqueous Corp ., Tokyo , Japan ). Insulin is dissolved in Hepes buffer cysteine solution . The mixture is subsequently subjected to (50 mM Hepes, 150 mM sodium chloride , 5 mM calcium UF /DF employing Vivaspin centrifugal filtrators to remove 20 chloride , pH 6 . 0 ) and mixed with an aqueous sodium excess periodate , quencher and the byproducts thereof. periodate solution ( 10 mM ) , and an aqueous m - toluidine The retentate containing oxidized insulin is next mixed solution (50 mM ). Subsequently , the aminooxy reagent is with an aqueous m -toluidine solution (50 mM ) and incu - added to give a 20 - fold molar reagent excess . The mixture bated for 30 min at room temperature . Aminooxy - PEG is incubated for 2 h in the dark at room temperature under reagent with a MW of 20 kD is then added to give a 5 - fold 25 gentle stirring and quenched for 15 min at room temperature molar reagent excess . This mixture is incubated for 2 . 5 h at by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . room temperature in the dark under gentle stirring . Finally , the PEG - insulin conjugate is purified by ion Finally , the PEG - insulin conjugate is purified by ion exchange chromatography on Sepharose FF . 1 . 5 mg exchange chromatography . The conjugate containg fractions protein /ml gel is loaded on the column pre equilibrated with of the eluate are collected and then subjected to UF /DF using 30 50 mM Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The an appropriate MW cutoff membrane . The preparation is conjugate is eluted with 50 mM Hepes buffer containing 5 next analytically characterized by measuring total protein mM CaC12 and 500 mM sodium chloride , pH 7 . 4 and is then ( Coomassie , Bradford ) and biological activity according to subjected to UF/ DF using a membrane . The preparation is methods known in the art . analytically characterized by measuring total protein (Brad Method 2 : 35 ford ) and biological activity according to methods known in As described herein , the amino acid sequence of insulin is the art . first modified to incorporate at least one glycosylation site . In an alternative embodiment, Method 3 is carried out as Following purification , insulin is glycosylated in vitro follows. As described herein , the amino acid sequence of according to methods known in the art . insulin is first modified to incorporate at least one glycosy Insulin is PEGylated by use of a linear 20 kD PEGylation 40 lation site . Following purification , insulin is glycosylated in reagent containing an aminooxy group . An example of this vitro according to methods known in the art. Insulin is type of reagent is the Sunbright® CA series from NOF (NOF PEGylated by use of a linear 20 kD PEGylation reagent Corp ., Tokyo , Japan ). Insulin is transferred or dissolved in containing an aminooxy group . An example of this type of reaction buffer ( e . g . 50 mM Hepes, 350 mM sodium chlo - reagent is the Sunbright® CA series from NOF (NOF Corp . , ride, 5 mM calcium chloride , pH 6 . 0 ) to get a final protein 45 Tokyo , Japan ) . Insulin is dissolved in Hepes buffer ( 50 mM concentration of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the Hepes, 150 mM sodium chloride , 5 mM calcium chloride , solution is corrected to 6 . 0 by drop wise addition of a 0 .5 N pH 6 . 0 ) and mixed with an aqueous sodium periodate aqueous HCl solution . Subsequently , a 40 mM aqueous solution ( 10 mM ), and an aqueous m - toluidine solution (50 sodium periodate solution is added within 10 minutes to give mM ) . Subsequently the aminooxy reagent is added to give a concentration of 200 uM . The oxidation reaction is carried 50 a 20 - fold molar reagent excess . The mixture is incubated for out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . 2 h in the dark at room temperature under gentle stirring and Then the reaction is stopped by addition of an aqueous quenched for 15 min at room temperature by the addition of L - cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° 8 ul of aqueous cysteine solution ( 1 M ) . C . to give a final concentration of 10 mM in the reaction Finally , the insulin - conjugate is purified by ion - exchange mixture and incubation for 60 + / - 5 min . 55 chromatography . The conjugate containing fractions are The oxidized insulin is further purified by ion exchange collected and then subjected to UF /DF . The preparation is chromatography . The oxidized insulin containing fractions analytically characterized by measuring total protein (Brad of the eluate are collected and used for the conjugation ford ) and biological activity according to methods known in reaction . the art. The aminooxy -PEG reagent with a MW of 20 kD reagent 60 Method 4 : is added in a 50 - fold molar excess to the eluate containing As described herein , the amino acid sequence of insulin is the purified oxidized insulin within a maximum time period first modified to incorporate at least one glycosylation site . ( t) of 15 minutes under gentle stirring . Then an aqueous Following purification , insulin is glycosylated in vitro m - toluidine solution (50 mM ) is added within 15 minutes to according to methods known in the art . get a final concentration of 10 mM . The reaction mixture is 65 Insulin is PEGylated by use of a linear 20 kD PEGylation incubated for 120 + / – 10 min . in the dark at a temperature ( T ) reagent containing an aminooxy group . An example of this of T = + 22 + / - 2° C . under gentle shaking . type of reagent is the Sunbright® CA series from NOF (NOF US 10 , 414 , 793 B2 151 152 Corp ., Tokyo , Japan ). An initial concentration or weight of from NOF (NOF Corp ., Tokyo , Japan ) . Interferon -alpha is insulin is transferred or dissolved in Hepes buffer (50 mM dissolved in 7. 0 ml histidine buffer , pH 6 .0 ( 20 mM L -his Hepes , 150 mM sodium chloride, 5 mM calcium chloride , tidine , 150 mM NaCl, 5 mM CaCl2 ). An aqueous sodium pH 6 . 0 ) to get a final protein concentration of 2 mg insulin periodate solution ( 5 mm ) is then added and the reaction ml. Subsequently an 5 mM aqueous sodium periodate solu - 5 mixture is incubated for 1 h in the dark at 4° C . under gentle tion is added within 15 minutes to give a final concentration stirring and quenched for 15 min at room temperature by the of 100 uM , followed by addition of an 50 mM aqueous addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The m - toluidine solution to get a final concentration of 10 mM mixture is subsequently subjected to UF /DF employing within a time period of 30 minutes . Then the aminooxy -PEG Vivaspin centrifugal filtrators to remove excess periodate , reagent with a MW of 20 kD (described above ) is added to 10 quencher and the byproducts thereof. give a 20 - fold molar reagent excess. After correction of the The retentate containing oxidized interferon - alpha is next pH to 6 . 0 the mixture is incubated for 2 h in the dark at room mixed with an aqueous m - toluidine solution ( 50 mM ) and temperature under gentle stirring and quenched for 15 min incubated for 30 min at room temperature . Aminooxy -PEG at room temperature by the addition of a 1 M aqueous reagent with a MW of 20 kD is then added to give a 5 - fold L - cysteine solution to give a final concentration of 10 mM . 15 molar reagent excess . This mixture is incubated for 2 . 5 h at The PEG - insulin conjugate is purified by means of ion room temperature in the dark under gentle stirring . exchange chromatography ( IEC ) . The conjugate containing Finally , the PEG - interferon -alpha conjugate is purified by fractions of the eluate are concentrated by UF/ DF using a 10 ion - exchange chromatography The conjugate containing kD membrane made of regenerated cellulose ( 88 cm2, freactions are collected and then subjected to UF /DF using cut -off 10 kD /Millipore ) . The final diafiltration step is per - 20 an appropriate MW cutoff membrane . The preparation is formed against Hepes buffer ( 50 mM Hepes , 5 mM CaCl2 , next analytically characterized by measuring total protein pH 7 . 5 ) . ( Coomassie , Bradford ) and biological activity according to The preparation is analytically characterized by measur methods known in the art . ing total protein (Bradford and BCA procedure ) and bio - Method 2 : logical activity according to known methods . 25 Interferon -alpha is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An Example 40 example of this type of reagent is the Sunbright® CA series from NOF (NOF Corp . , Tokyo , Japan ) . Interferon - alpha is PEGylation of Interferon - Alpha Using an transferred or dissolved in reaction buffer ( e . g . 50 mM Aminooxy - PEG Reagent and m - Toluidine as a 30 Hepes, 350 mM sodium chloride , 5 mM calcium chloride , Nucleophilic Catalyst pH 6 . 0 ) to get a final protein concentration of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is corrected to 6 . 0 by Method 1 : drop wise addition of a 0 . 5 N aqueous HCl solution . Sub Interferon -alpha is PEGylated by use of a linear 20 kD sequently , a 40 mM aqueous sodium periodate solution is PEGylation reagent containing an aminooxy group . An 35 added within 10 minutes to give a concentration of 200 uM . example of this type of reagent is the Sunbright® CA series The oxidation reaction is carried out for 30 + / - 5 min at a from NOF (NOF Corp . , Tokyo , Japan ) . Interferon - alpha is temperature ( T ) of T = + 22 + / - 2° C . Then the reaction is dissolved in 7 .0 ml histidine buffer, pH 6 .0 (20 mM L -his - stopped by addition of an aqueous L -cysteine solution (1 M ) tidine , 150 mM NaC1, 5 mM CaC12 ) . An aqueous sodium within 15 minutes at T = + 22 + / - 2° C . to give a final concen periodate solution ( 5 mm ) is then added and the reaction 40 tration of 10 mM in the reaction mixture and incubation for mixture is incubated for 1 h in the dark at 4° C . under gentle 60 + / - 5 min . stirring and quenched for 15 min at room temperature by the The oxidized interferon -alpha is further purified by ion addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The exchange chromatography . The oxidized interferon -alpha mixture is subsequently subjected to UF /DF employing containing fractions of the eluate are collected and used for Vivaspin centrifugal filtrators to remove excess periodate , 45 the conjugation reaction . quencher and the byproducts thereof. The aminooxy - PEG reagent with a MW of 20 kD reagent The retentate containing oxidized interferon - alpha is next is added in a 50 - fold molar excess to the eluate containing mixed with an aqueous m - toluidine solution ( 50 mm ) and the purified oxidized interferon - alpha within a maximum incubated for 30 min at room temperature . Aminooxy - PEG time period ( t ) of 15 minutes under gentle stirring . Then an reagent with a MW of 20 kD is then added to give a 5 - fold 50 aqueous m - toluidine solution ( 50 mM ) is added within 15 molar reagent excess. This mixture is incubated for 2 . 5 h at minutes to get a final concentration of 10 mM . The reaction room temperature in the dark under gentle stirring . mixture is incubated for 120 + / – 10 min . in the dark at a Finally , the PEG - interferon - alpha conjugate is purified by temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . ion -exchange chromatography (e . g. , on Q -Sepharose FF ). The obtained PEG - interferon -alpha conjugate is further For example , 1 . 5 mg protein /ml gel is loaded on the column 55 purified by ion exchange chromatography . The PEG - inter equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 ferferon alpha conjugate containing fractions are collected and mM CaC12 . The conjugate is eluted with 50 mM Hepes concentrated by ultra - / diafiltration (UF /DF ) using a mem buffer containing 5 mM CaCl2 and 500 mM sodium chlo - brane made of regenerated cellulose with an appropriate ride, pH 7 . 4 and is then subjected to UF /DF using an molecular weight cut off (Millipore ) . appropriate MW cutoff membrane . The preparation is next 60 The conjugate prepared by use of this procedure are analytically characterized by measuring total protein (Coo - analytically characterized by measuring total protein and massie , Bradford ) and biological activity according to meth - biological activity according to methods known in the art . ods known in the art. Method 3 : In an alternative embodiment, Method 1 is carried out as Interferon -alpha is PEGylated by use of a linear 20 kD follows. Interferon - alpha is PEGylated by use of a linear 20 65 PEGylation reagent containing an aminooxy group . An KD PEGylation reagent containing an aminooxy group . An example of this type of reagent is the Sunbright® CA series example of this type of reagent is the Sunbright® CA series from NOF (NOF Corp ., Tokyo , Japan ) . Interferon -alpha is US 10 ,414 ,793 B2 153 154 dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium The preparation is analytically characterized by measur chloride, 5 mM calcium chloride, pH 6 . 0 ) and mixed with an ing total protein (Bradford and BCA procedure ) and bio aqueous sodium periodate solution ( 10 mM ), and an aque - logical activity according to known methods. ous m - toluidine solution (50 mm ) . Subsequently the ami nooxy reagent is added to give a 20 - fold molar reagent5 Example 41 excess . The mixture is incubated for 2 h in the dark at room temperature under gentle stirring and quenched for 15 min PEGylation of Interferon -Gamma Using an at room temperature by the addition of 8 ul of aqueous Aminooxy -PEG Reagent and m - Toluidine as a cysteine solution ( 1 M ) . Nucleophilic Catalyst Finally , the PEG - interferon -alpha conjugate is purified by 10 Method 1 : ion -exchange chromatography on Q - Sepharose FF. 1. 5 mg Interferon - gamma is PEGylated by use of a linear 20 kD protein /ml gel is loaded on the column pre equilibrated with PEGylation reagent containing an aminooxy group . An 50 mM Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The example of this type of reagent is the Sunbright® CA series conjugate is eluted with 50 mM Hepes buffer containingning 5 15 from NOF (NOF Corp . , Tokyo , Japan ). 10 mg Interferon mM CaCl2 and 500 mM sodium chloride, pH 7 . 4 and is then gamma is dissolved in 5 mlhistidine buffer, pH 6 . 0 (20 mM subjected to UF/ DF using a membrane . The preparation is L -histidine , 150 mM NaCl) . 100 ul of an aqueous sodium analytically characterized by measuring total protein (Brad - periodate solution (5 mm ) is then added and the reaction ora ) and biological activity according to methods known in mixture is incubated for 1 h in the dark at 4° C . under gentle the art . 20 stirring and quenched for 15 min at room temperature by the In an alternative embodiment, Method 3 is carried out as addition of 50 ul of a 1 M aqueous cysteine solution . The follows. Interferon - alpha is PEGylated by use of a linear 20 mixture is subsequently subjected to UF/ DF employing kD PEGylation reagent containing an aminooxy group . An Vivaspin 15R 10 kD centrifugal filtrators to remove excess example of this type of reagent is the Sunbright® CA series periodate , quencher and the byproducts thereof. from NOF (NOF Corp . , Tokyo , Japan ) . Interferon - alpha is 25 The retentate ( approx . 7 ml) , containing oxidized inter dissolved in Hepes buffer ( 50 mM Hepes, 150 mM sodium feron - gamma, is mixed with 2 ml of an aqueous m - toluidine chloride, 5 mM calcium chloride, pH 6 . 0 ) and mixed with an solution (50 mM ) and incubated for 30 min at room tem aqueous sodium periodate solution ( 10 mM ) , and an aque - perature . Then aminooxy - PEG reagent with a MW of 20 kD ous m - toluidine solution (50 mM ) . Subsequently the ami- ( described above ) is added to give a 5 - fold molar reagent nooxy reagent is added to give a 20 - fold molar reagent 30 excess . This mixture is incubated for 2 . 5 h at RT in the dark excess . The mixture is incubated for 2 h in the dark at room under gentle stirring . temperature under gentle stirring and quenched for 15 min Finally , the PEG - interferon - gamma conjugate is purified at room temperature by the addition of 8 ul of aqueous by ion - exchange chromatography on SP Sepharose FF . The cysteine solution ( 1 M ) . reaction mixture is diluted with 20 ml Buffer A ( 50 mM Finally , the PEG - interferon - alpha conjugate is purified by 35 Hepes, pH 6 . 5 ) and loaded onto a 20 ml HiPrep SPFF 16 / 10 ion - exchange chromatography. The conjugate containg frac - column (GE Healthcare, Fairfield , Conn . ) pre - equilibrated tions are collected and then subjected to UF /DF using a with Buffer A . Then the column is eluted with Buffer B (50 membrane. The preparation is analytically characterized by mM Hepes , 1 M NaCl, pH 6 . 5 ) . Free interferon - gamma is measuring total protein (Bradford ) and biological activity eluted by washing the column with 25 % Buffer B and the according to methods known in the art . 40 conjugate at 50 % Buffer B . The conjugate containing frac Method 4 : tions are concentrated by UF /DF using a 10 kD membrane Interferon -alpha is PEGylated by use of a linear 20 kD made of regenerated cellulose ( 88 cm2 , cut -off 10 kD /Mil PEGylation reagent containing an aminooxy group . An lipore ) . The final diafiltration step is performed against example of this type of reagent is the Sunbright® CA series histidine buffer, pH 6 . 9 containing 150 mM NaCl. The from NOF (NOF Corp ., Tokyo , Japan ) . An initial concen - 45 preparation is analytically characterized by measuring total tration or weight of interferon - alpha is transferred or dis - protein ( Bradford ) and biological activity according to meth solved in Hepes buffer ( 50 mM Hepes , 150 mM sodium ods known in the art . For the PEG - interferon - gamma con chloride , 5 mM calcium chloride, pH 6 . 0 ) to get a final jugate a specific activity of > 50 % in comparison to native protein concentration of 2 mg interferon -alpha /ml . Subse - Interferon gamma is determined . The conjugate is addition quently , an 5 mM aqueous sodium periodate solution is 50 ally analytically characterized by Size Exclusion HPLC added within 15 minutes to give a final concentration of 100 using a Agilent 1200 HPLC system equipped with a Shodex uM , followed by addition of an 50 mM aqueous m - toluidine KW 803 column under conditions as previously described solution to get a final concentration of 10 mM within a time (Kolarich et al, Transfusion 2006 ; 46 : 1959 -77 ) . It is shown period of 30 minutes . Then the aminooxy -PEG reagent with that the preparation contains no free Interferon - gamma. a MW of 20 kD (described above ) is added to give a 20 - fold 55 Method 2 : molar reagent excess . After correction of the pH to 6 . 0 the Interferon - gamma is PEGylated by use of a linear 20 kD mixture is incubated for 2 h in the dark at room temperature PEGylation reagent containing an aminooxy group . An under gentle stirring and quenched for 15 min at room example of this type of reagent is the Sunbright® CA series temperature by the addition of an 1 M aqueous L - cysteine from NOF (NOF Corp . , Tokyo , Japan ) . Interferon - gamma is solution to give a final concentration of 10 mM . 60 transferred or dissolved in reaction buffer ( e . g . 50 mM The PEG - interferon -alpha conjugate is purified by means Hepes, 350 mM sodium chloride, 5 mM calcium chloride , of ion exchange chromatography (IEC ) . The conjugate con - pH 6 .0 ) to get a final protein concentration of 1 .0 + / - 0 .25 taining fractions of the eluate are concentrated by UF /DF mg/ ml . Then the pH of the solution is corrected to 6 . 0 by using a 10 kD membrane made of regenerated cellulose ( 88 drop wise addition of a 0 . 5 N aqueous HCl solution . Sub cm2, cutoff 10 kD /Millipore ) . The final diafiltration step is 65 sequently a 40 mM aqueous sodium periodate solution is performed against Hepes buffer ( 50 mM Hepes, 5 mM added within 10 minutes to give a concentration of 200 uM . CaC12 , pH 7 . 5 ) . The oxidation reaction is carried out for 30 + / - 5 min at a US 10 ,414 ,793 B2 155 156 temperature ( T ) of T = + 22 + / - 2° C . Then the reaction is example of this type of reagent is the Sunbright® CA series stopped by addition of an aqueous L -cysteine solution ( 1 M ) from NOF (NOF Corp ., Tokyo , Japan ). An initial concen within 15 minutes at T = + 22 + / - 2° C . to give a final concen tration or weight of interferon - gamma is transferred or tration of 10 mM in the reaction mixture and incubation for dissolved in Hepes buffer (50 mM Hepes , 150 mM sodium 60 + / - 5 min . 5 chloride, 5 mM calcium chloride, pH 6 . 0 ) to get a final The oxidized interferon - gamma is further purified by ion protein concentration of 2 mg interferon - gamma/ml . Subse exchange chromatography. The oxidized interferon - gamma quently an 5 mM aqueous sodium periodate solution is containing fractions of the eluate are collected and used for added within 15 minutes to give a final concentration of 100 the conjugation reaction . UM , followed by addition of an 50 mM aqueous m - toluidine The aminooxy -PEG reagent with a MW of 20 kD reagent 10 solution to get a final concentration of 10 mM within a time is added in a 50 - fold molar excess to the eluate containing period of 30 minutes. Then the aminooxy -PEG reagent with the purified oxidized interferon - gamma within a maximum a MW of 20 kD ( described above ) is added to give a 20 - fold time period ( t ) of 15 minutes under gentle stirring . Then an molar reagent excess . After correction of the pH to 6 . 0 the aqueous m - toluidine solution (50 mM ) is added within 15 mixture is incubated for 2 h in the dark at room temperature minutes to get a final concentration of 10 mM . The reaction 15 under gentle stirring and quenched for 15 min at room mixture is incubated for 120 + / - 10 min . in the dark at a temperature by the addition of an 1 M aqueous L - cysteine temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . solution to give a final concentration of 10 mM . The obtained PEG - interferon - gamma conjugate is further The PEG - interferon - gamma conjugate is purified by purified by ion exchange chromatography. The PEG - inter - means of ion exchange chromatography ( IEC ) . The conju feron - gamma conjugate containing fractions are collected 20 gate containing fractions of the eluate are concentrated by and concentrated by ultra - / diafiltration (UF /DF ) using a UF /DF using a 10 kD membrane made of regenerated membrane made of regenerated cellulose with an appropri - cellulose ( 88 cm2 , cutoff 10 kD /Millipore ) . The final dia ate molecular weight cut off (Millipore ) . filtration step is performed against Hepes buffer (50 mM The conjugate prepared by use of this procedure are Hepes, 5 mM CaC12 , pH 7 . 5 ) . analytically characterized by measuring total protein and 25 The preparation is analytically characterized by measur biological activity according to methods known in the art . ing total protein (Bradford and BCA procedure ) and bio Method 3 : logical activity according to known methods . Interferon - gamma is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An Example 42 example of this type of reagent is the Sunbright® CA series 30 from NOF (NOF Corp . , Tokyo , Japan ) . 10 mg interferon PEGylation of G -CSF Using an Aminooxy -PEG gamma is dissolved in - 8 ml histidine -buffer , pH 6 . 0 ( 20 Reagent and m - Toluidine as a Nucleophilic mM L -histidine , 150 mM NaCl) . 200 ul of an aqueous Catalyst sodium periodate solution ( 5 mM ) and 2 ml of an aqueous m - toluidine solution (50 mM ) are then added . Subsequently 35 Method 1 : the aminooxy - PEG reagent with a MW of 20 kD (described G - CSF is PEGylated by use of a linear 20 kD PEGylation above ) is added to give a 5- fold molar reagent excess . The reagent containing an aminooxy group . An example of this mixture is incubated for 2 h in the dark at room temperature type ofreagent is the Sunbright® CA series from NOF (NOF under gentle stirring and quenched for 15 min at room Corp ., Tokyo , Japan ). G - CSF is dissolved in 7 . 0 mlhistidine temperature by the addition of 100 ul of 1 M aqueous 40 buffer, pH 6 . 0 (20 mM L -histidine , 150 mM NaC1, 5 mM cysteine solution . CaC12 ) . An aqueous sodium periodate solution ( 5 mm ) is Finally the PEG - interferon - gamma conjugate is purified then added and the reaction mixture is incubated for 1 h in by ion - exchange chromatography on SP - Sepharose FF. The the dark at 4° C . under gentle stirring and quenched for 15 reaction mixture is diluted with 20 ml Buffer A (50 mM min at room temperature by the addition of 7 . 5 ul of a 1 M Hepes, pH 6 . 5 ) and loaded onto a 20 ml HiPrep SP FF 16 / 10 45 aqueous cysteine solution . The mixture is subsequently column (GE Healthcare , Fairfield , Conn . ) pre -equilibrated subjected to UF /DF employing Vivaspin centrifugal filtra with Buffer A . Then the column is eluted with Buffer B (50 tors to remove excess periodate , quencher and the byprod mM Hepes, 1 M NaCl, pH 6 . 5 ) . Free intergferon - gamma is ucts thereof. eluted by washing the column with 25 % Buffer B and the The retentate containing oxidized G - CSF is next mixed conjugate at 50 % Buffer B . The conjugate containing frac - 50 with an aqueous m - toluidine solution ( 50 mM ) and incu tions are concentrated by UF /DF using a 10 kD membrane bated for 30 min at room temperature . Aminooxy - PEG made of regenerated cellulose ( 88 cm2, cut -off 10 kD /Mil - reagent with a MW of 20 kD is then added to give a 5 - fold lipore ) . The final diafiltration step is performed against molar reagent excess . This mixture is incubated for 2 . 5 h at histidine buffer, pH 6 . 9 containing 150 mM NaCl. The room temperature in the dark under gentle stirring. preparation is analytically characterized by measuring total 55 Finally , the PEG -G -CSF conjugate is purified by ion protein (Bradford ) and biological activity according to meth - exchange chromatography ( e . g . , on Q -Sepharose FF ) . For ods known in the art . For the PEG - interferon - gamma con - example , 1. 5 mg protein /ml gel is loaded on the column jugate a specific activity of > 50 % in comparison to native equilibrated with 50 mM Hepes buffer , pH 7 .4 containing 5 interferon - gamma is determined . The conjugate is addition - mM CaC12 . The conjugate is eluted with 50 mM Hepes ally analytically characterized by Size Exclusion HPLC 60 buffer containing 5 mM CaC12 and 500 mM sodium chlo using a Agilent 1200 HPLC system equipped with a Shodex ride , pH 7. 4 and is then subjected to UF /DF using an KW 803 column under conditions as previously described appropriate MW cutoff membrane . The preparation is next (Kolarich et al, Transfusion 2006 ; 46 : 1959 - 77 ) . It is shown analytically characterized by measuring total protein (Coo that the preparation contains no free interferon - gamma. massie , Bradford ) and biological activity according to meth Method 4 : 65 ods known in the art. Interferon - gamma is PEGylated by use of a linear 20 kD In an alternative embodiment, Method 1 is carried out as PEGylation reagent containing an aminooxy group . An follows. G -CSF is PEGylated by use of a linear 20 kD US 10 ,414 ,793 B2 157 158 PEGylation reagent containing an aminooxy group . An periodate solution ( 10 mM ) , and an aqueous m - toluidine example of this type of reagent is the Sunbright® CA series solution (50 mm ). Subsequently , the aminooxy reagent is from NOF (NOF Corp ., Tokyo , Japan ) . G -CSF is dissolved added to give a 20 - fold molar reagent excess. The mixture in 7 . 0 mlhistidine buffer, pH 6 . 0 (20 mM L -histidine , 150 is incubated for 2 h in the dark at room temperature under mM NaC1, 5 mM CaC12 ) . An aqueous sodium periodate 5 gentle stirring and quenched for 15 min at room temperature solution ( 5 mM ) is then added and the reaction mixture is by the addition of 8 ul of aqueous cysteine solution ( 1 M ). incubated for 1 h in the dark at 4° C . under gentle stirring Finally , the PEG - G - CSF conjugate is purified by ion and quenched for 15 min at room temperature by the exchange chromatography on Q - Sepharose FF. 1 .5 mg pro addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The mixture is subsequently subjected to UF /DF employing 10 tein /ml gel is loaded on the column pre equilibrated with 50 Vivaspin centrifugal filtrators to remove excess periodate , mM Hepes buffer, pH 7. 4 containing 5 mM CaC12 . The quencher and the byproducts thereof. conjugate is eluted with 50 mM Hepes buffer containing 5 The retentate containing oxidized G - CSF is next mixed mM CaCl2 and 500 mM sodium chloride, pH 7 . 4 and is then with an aqueous m - toluidine solution (50 mM ) and incu subjected to UF /DF using a membrane . The preparation is bated for 30 min at room temperature . Aminooxy - PEG 15 analytically characterized by measuring total protein ( Brad reagent with a MW of 20 kD is then added to give a 5 -fold ford ) and biological activity according to methods known in molar reagent excess . This mixture is incubated for 2 . 5 h at the art . room temperature in the dark under gentle stirring . In an alternative embodiment , Method 3 is carried out as Finally, the PEG - G - CSF conjugate is purified by ion follows. G -CSF is PEGylated by use of a linear 20 kD exchange chromatography ( The conjugate containg frac - 20 PEGylation reagent containing an aminooxy group . An tions of the eluate are collected and then subjected to UF /DF example of this type of reagent is the Sunbright® CA series using an appropriate MW cutoff membrane . The preparation from NOF (NOF Corp . , Tokyo , Japan ). G - CSF is dissolved is next analytically characterized by measuring total protein in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , (Coomassie , Bradford ) and biological activity according to 5 mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous methods known in the art . 25 sodium periodate solution ( 10 mM ) , and an aqueous Method 2 : m - toluidine solution (50 mM ). Subsequently , the aminooxy G -CSF is PEGylated by use of a linear 20 kD PEGylation reagent is added to give a 20 - fold molar reagent excess . The reagent containing an aminooxy group . An example of this mixture is incubated for 2 h in the dark at room temperature type of reagent is the Sunbright® CA series from NOF (NOF under gentle stirring and quenched for 15 min at room Corp . , Tokyo , Japan ) . G - CSF is transferred or dissolved in 30 temperature by the addition of 8 ul of aqueous cysteine reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chlo - solution ( 1 M ) . ride, 5 mM calcium chloride , pH 6 . 0 ) to get a final protein Finally , the PEG - G -CSF conjugate is purified by ion concentration of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the exchange chromatography . The conjugate containing frac solution is corrected to 6 . 0 by drop wise addition of a 0 . 5N tions of the eluate are collected and then subjected to UF /DF aqueous HCl solution . Subsequently a 40 mM aqueous 35 using a membrane . The preparation is analytically charac sodium periodate solution is added within 10 minutes to give terized by measuring total protein ( Bradford ) and biological a concentration of 200 uM . The oxidation reaction is carried activity according to methods known in the art . out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . Method 4 : Then the reaction is stopped by addition of an aqueous G -CSF is PEGylated by use of a linear 20 kD PEGylation L - cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° 40 reagent containing an aminooxy group . An example of this C . to give a final concentration of 10 mM in the reaction type of reagent is the Sunbright® CA series from NOF (NOF mixture and incubation for 60 + / - 5 min . Corp ., Tokyo , Japan ) . An initial concentration or weight of The oxidized G -CSF is further purified by ion exchange G - CSF is transferred or dissolved in Hepes buffer (50 mM chromatography . The oxidized G - CSF containing fractions Hepes, 150 mM sodium chloride , 5 mM calcium chloride , of the eluate are collected and used for the conjugation 45 pH 6 . 0 ) to get a final protein concentration of 2 mg G -CSF / reaction . ml. Subsequently , an 5 mM aqueous sodium periodate The aminooxy -PEG reagent with a MW of 20 kD reagent solution is added within 15 minutes to give a final concen is added in a 50 - fold molar excess to the eluate containing tration of 100 UM , followed by addition of an 50 mM the purified oxidized G -CSF within a maximum time period aqueous m - toluidine solution to get a final concentration of ( t ) of 15 minutes under gentle stirring . Then an aqueous 50 10 mM within a time period of 30 minutes . Then the m - toluidine solution (50 mM ) is added within 15 minutes to aminooxy - PEG reagent with a MW of 20 kD (described get a final concentration of 10 mM . The reaction mixture is above ) is added to give a 20 -fold molar reagent excess . After incubated for 120 + / – 10 min . in the dark at a temperature ( T ) correction of the pH to 6 . 0 the mixture is incubated for 2 h of T = + 22 + / - 2° C . under gentle shaking . in the dark at room temperature under gentle stirring and The obtained PEG - G -CSF conjugate is further purified by 55 quenched for 15 min at room temperature by the addition of ion exchange chromatography . The PEG - G -CSF conjugate an 1 M aqueous L -cysteine solution to give a final concen containing fractions are collected and concentrated by ultra - / tration of 10 mM . diafiltration (UF /DF ) using a membrane made of regener - The G -CSF conjugate is purified by means of ion ated cellulose with an appropriate molecular weight cut off exchange chromatography (IEC ). The conjugate containing (Millipore ) . 60 fractions of the eluate are concentrated by UF /DF using a 10 Method 3 : kD membrane made of regenerated cellulose (88 cm2, G -CSF is PEGylated by use of a linear 20 kD PEGylation cutoff 10 kD /Millipore ). The final diafiltration step is per reagent containing an aminooxy group . An example of this formed against Hepes buffer ( 50 mM Hepes , 5 mM CaCl2 , type of reagent is the Sunbright® CA series from NOF (NOF PH 7 . 5 ) . Corp ., Tokyo , Japan ) . G -CSF is dissolved in Hepes buffer 65 The preparation is analytically characterized by measur (50 mM Hepes, 150 mM sodium chloride , 5 mM calcium ing total protein ( Bradford and BCA procedure ) and bio chloride , pH 6 . 0 ) and mixed with an aqueous sodium logical activity according to known methods. US 10 ,414 ,793 B2 159 160 Example 43 type of reagent is the Sunbright® CA series from NOF (NOF Corp . , Tokyo , Japan ). Humira is transferred or dissolved in PEGylation of Humira Using an Aminooxy -PEG reaction buffer (e . g . 50 mM Hepes, 350 mM sodium chlo Reagent and m - Toluidine as a Nucleophilic ride, 5 mM calcium chloride, pH 6 . 0 ) to get a final protein Catalyst concentration of 1. 0 + / - 0 .25 mg/ ml . Then the pH of the Method 1 : solution is corrected to 6 . 0 by drop wise addition of a 0 .5N Humira is PEGylated by use of a linear 20 kD PEGylation aqueous HCl solution . Subsequently a 40 mM aqueous reagent containing an aminooxy group . An example of this sodium periodate solution is added within 10 minutes to give type of reagent is the Sunbright® CA series from NOF (NOF 10 a concentration of 200 uM . The oxidation reaction is carried Corp ., Tokyo , Japan ). Humira is dissolved in 7 .0 mlhistidine out for 30 + / – 5 min at a temperature ( T ) of T = + 22 + / - 2° C . buffer, pH 6 . 0 ( 20 mM L -histidine , 150 mM NaC1, 5 mM Then the reaction is stopped by addition of an aqueous CaCl2 ) . An aqueous sodium periodate solution (5 mM ) is L - cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° then added and the reaction mixture is incubated for 1 h in C . to give a final concentration of 10 mM in the reaction the dark at 4° C . under gentle stirring and quenched for 15 . 15 minmixture and incubation for 60 + / - 5 min . min at room temperature by the addition of 7 . 5 ul of a 1 M The oxidized Humira is further purified by ion exchange aqueous cysteine solution . The mixture is subsequently chromatography. The oxidized Humira containing fractions subjected to UF /DF employing Vivaspin centrifugal filtra of the eluate are collected and used for the conjugation tors to remove excess periodate , quencher and the byprod reaction . ucts thereof . 20 The aminooxy -PEG reagent with a MW of 20 kD reagent The retentate containing oxidized Humira is next mixed is added in a 50 - fold molar excess to the eluate containing with an aqueous m - toluidine solution (50 mM ) and incu - the purified oxidized Humira within a maximum time period bated for 30 min at room temperature . Aminooxy - PEG ( t) of 15 minutes under gentle stirring. Then an aqueous reagent with a MW of 20 kD is then added to give a 5 - fold m - toluidine solution (50 mM ) is added within 15 minutes to molar reagent excess. This mixture is incubated for 2 . 5 h at 25 get a final concentration of 10 mM . The reaction mixture is room temperature in the dark under gentle stirring . incubated for 120 + / – 10 min . in the dark at a temperature ( T ) Finally , the PEG -Humira conjugate is purified by ion - of T = + 22 + / - 2° C . under gentle shaking . exchange chromatography (e . g ., on Q - Sepharose FF ) . For The obtained PEG -Humira conjugate is further purified example , 1 . 5 mg protein /ml gel is loaded on the column by ion exchange chromatography . The PEG - Humira conju equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 30 gate containing fractions are collected and concentrated by mM CaC12 . The conjugate is eluted with 50 mM Hepes ultra -/ diafiltration (UF /DF ) using a membrane made of buffer containing 5 mM CaCl2 and 500 mM sodium chlo regenerated cellulose with an appropriate molecular weight ride, pH 7 . 4 and is then subjected to UF /DF using an cut off (Millipore ) . appropriate MW cutoff membrane . The preparation is next Method 3 : analytically characterized by measuring total protein (Coo - 35 Humira is PEGylated by use of a linear 20 kD PEGylation massie , Bradford ) and biological activity according to meth - reagent containing an aminooxy group . An example of this ods known in the art . type of reagent is the Sunbright® CA series from NOF (NOF In an alternative embodiment, Method 1 is carried out as Corp ., Tokyo , Japan ) . Humira is dissolved in Hepes buffer follows. Humira is PEGylated by use of a linear 20 kD (50 mM Hepes , 150 mM sodium chloride , 5 mM calcium PEGylation reagent containing an aminooxy group . An 40 chloride , pH 6 . 0 ) and mixed with an aqueous sodium example of this type of reagent is the Sunbright® CA series periodate solution ( 10 mm ) , and an aqueous m - toluidine from NOF ( NOF Corp ., Tokyo , Japan ) . Humira is dissolved solution ( 50 mM ) . Subsequently , the aminooxy reagent is in 7 . 0 ml histidine buffer , pH 6 .0 ( 20 mM L -histidine , 150 added to give a 20 - fold molar reagent excess. The mixture mM NaC1, 5 mM CaC12 ) . An aqueous sodium periodate is incubated for 2 h in the dark at room temperature under solution ( 5 mM ) is then added and the reaction mixture is 45 gentle stirring and quenched for 15 min at room temperature incubated for 1 h in the dark at 4° C . under gentle stirring by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . and quenched for 15 min at room temperature by the Finally , the PEG -Humira conjugate is purified by ion addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The exchange chromatography on Q - Sepharose FF . 1 . 5 mg pro mixture is subsequently subjected to UF /DF employing tein /ml gel is loaded on the column pre equilibrated with 50 Vivaspin centrifugal filtrators to remove excess periodate , 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The quencher and the byproducts thereof. conjugate is eluted with 50 mM Hepes buffer containing 5 The retentate containing oxidized Humira is next mixed mM CaCl2 and 500 mM sodium chloride , pH 7 . 4 and is then with an aqueous m - toluidine solution (50 mM ) and incu - subjected to UF /DF using a membrane . The preparation is bated for 30 min at room temperature . Aminooxy - PEG analytically characterized by measuring total protein (Brad reagent with a MW of 20 kD is then added to give a 5 - fold 55 ford ) and biological activity according to methods known in molar reagent excess. This mixture is incubated for 2 . 5 h at the art. room temperature in the dark under gentle stirring . In an alternative embodiment, Method 3 is carried out as Finally , the PEG -Humira conjugate is purified by ion - follows. Humira is PEGylated by use of a linear 20 kD exchange chromatography . The conjugate containg fractions PEGylation reagent containing an aminooxy group . An of the eluate are collected and then subjected to UF /DF using 60 example of this type of reagent is the Sunbright® CA series an appropriate MW cutoff membrane. The preparation is from NOF (NOF Corp . , Tokyo , Japan ). Humira is dissolved next analytically characterized by measuring total protein in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , ( Coomassie , Bradford ) and biological activity according to 5 mM calcium chloride, pH 6 .0 ) and mixed with an aqueous methods known in the art . sodium periodate solution ( 10 mm ) , and an aqueous Method 2 : 65 m - toluidine solution (50 mM ). Subsequently the aminooxy Humira is PEGylated by use of a linear 20 kD PEGylation reagent is added to give a 20 - fold molar reagent excess . The reagent containing an aminooxy group . An example of this mixture is incubated for 2 h in the dark at room temperature US 10 , 414 , 793 B2 161 162 under gentle stirring and quenched for 15 min at room example , 1 . 5 mg protein /ml gel is loaded on the column temperature by the addition of 8 ul of aqueous cysteine equilibrated with 50 mM Hepes buffer, pH 7 . 4 containing 5 solution ( 1 M ). mM CaC12 . The conjugate is eluted with 50 mM Hepes Finally, the PEG -Humira conjugate is purified by ion - buffer containing 5 mM CaC12 and 500 mM sodium chlo exchange chromatography . The conjugate containing frac - 5 ride , pH 7 . 4 and is then subjected to UF /DF using an tions are collected and then subjected to UF /DF using a appropriate MW cutoff membrane . The preparation is next membrane. The preparation is analytically characterized by analytically characterized by measuring total protein (Coo measuring total protein ( Bradford ) and biological activity massie , Bradford ) and biological activity according to meth according to methods known in the art. ods known in the art . Method 4 : 10 In an alternative embodiment, Method 1 is carried out as Humira is PEGylated by use of a linear 20 kD PEGylation follows. Prolia is PEGylated by use of a linear 20 kD reagent containing an aminooxy group . An example of this PEGylation reagent containing an aminooxy group . An type of reagent is the Sunbright® CA series from NOF (NOF example of this type of reagent is the Sunbright® CA series Corp . , Tokyo , Japan ) . An initial concentration or weight of from NOF (NOF Corp ., Tokyo , Japan ) . 10 mg rFIX is HJumira is transferred or dissolved in Hepes buffer (50 mM 15 dissolved in 5 ml histidine -buffer , pH 6 . 0 ( 20 mM L -histi Hepes, 150 mM sodium chloride , 5 mM calcium chloride , dine , 150 mM NaCl) . 100 ul of an aqueous sodium periodate pH 6 . 0 ) to get a final protein concentration of 2 mg Humiral solution ( 5 mM ) is then added and the reaction mixture is ml. Subsequently an 5 mM aqueous sodium periodate solu - incubated for 1 h in the dark at 4° C . under gentle stirring tion is added within 15 minutes to give a final concentration and quenched for 15 min at room temperature by the of 100 uM , followed by addition of an 50 mM aqueous 20 addition of 50 ul of a 1 M aqueous cysteine solution . The m - toluidine solution to get a final concentration of 10 mM mixture is subsequently subjected to UF /DF employing within a time period of 30 minutes . Then the aminooxy - PEG Vivaspin 15R 10 kD centrifugal filtrators to remove excess reagent with a MW of 20 kD (described above ) is added to periodate , quencher and the byproducts thereof. give a 20 - fold molar reagent excess. After correction of the The retentate ( approx . 7 ml) , containing oxidized Prolia , pH to 6 .0 the mixture is incubated for 2 h in the dark at room 25 is mixed with 2 ml of an aqueous m - toluidine solution (50 temperature under gentle stirring and quenched for 15 min mM ) and incubated for 30 min at room temperature . Then at room temperature by the addition of a 1 M aqueous aminooxy - PEG reagent with a MW of 20 kD ( described L -cysteine solution to give a final concentration of 10 mM . above ) is added to give a 5 - fold molar reagent excess. This The Humira conjugate is purified by means of ion mixture is incubated for 2 . 5 h at RT in the dark under gentle exchange chromatography (IEC ) . The conjugate containing 30 stirring . fractions of the eluate are concentrated by UF /DF using a 10 Finally the PEG - Prolia conjugate is purified by ion kD membrane made of regenerated cellulose ( 88 cm2, exchange chromatography on SP Sepharose FF . The reaction cut -off 10 kD /Millipore ) . The final diafiltration step is per- mixture is diluted with 20 ml Buffer A (50 mM Hepes, pH formed against Hepes buffer (50 mM Hepes, 5 mM CaC12 , 6 . 5 ) and loaded onto a 20 ml HiPrep SP FF 16 / 10 column pH 7 . 5 ) . 35 (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with Buf The preparation is analytically characterized by measur- fer A . Then the column is eluted with Buffer B (50 mM ing total protein (Bradford and BCA procedure) and bio Hepes , 1 M NaC1, pH 6 . 5 ) . Free Prolia is eluted by washing logical activity according to known methods. the column with 25 % Buffer B and the conjugate at 50 % Buffer B . The conjugate containing fractions are concen Example 44 40 trated by UF /DF using a 10 kD membrane made of regen erated cellulose (88 cm2, cut -off 10 kD /Millipore ). The final PEGylation of Prolia Using an Aminooxy -PEG diafiltration step is performed against histidine buffer, pH Reagent and m - Toluidine as a Nucleophilic 6 . 9 containing 150 mM NaCl. The preparation is analyti Catalyst cally characterized by measuring total protein (Bradford ) 45 and biological activity according to methods known in the Method 1 : art . For the PEG - Prolia conjugate a specific activity of > 50 % Prolia is PEGylated by use of a linear 20 kD PEGylation in comparison to native Prolia is determined . The conjugate reagent containing an aminooxy group . An example of this is additionally analytically characterized by Size Exclusion type of reagent is the Sunbright® CA series from NOF (NOF HPLC using a Agilent 1200 HPLC system equipped with a Corp . , Tokyo , Japan ) . Prolia is dissolved in 7 . 0 ml histidine 50 Shodex KW 803 column under conditions as previously buffer , pH 6 . 0 (20 mM L -histidine , 150 mM NaCl, 5 mM described (Kolarich et al, Transfusion 2006 ; 46 : 1959 - 77 ) . It CaC12 ) . An aqueous sodium periodate solution (5 mM ) is is shown that the preparation contains no free Prolia . then added and the reaction mixture is incubated for 1 h in Method 2 : the dark at 4° C . under gentle stirring and quenched for 15 Prolia is PEGylated by use of a linear 20 KD PEGylation min at room temperature by the addition of 7 .5 ul of a 1 M 55 reagent containing an aminooxy group . An example of this aqueous cysteine solution . The mixture is subsequently type of reagent is the Sunbright® CA series from NOF (NOF subjected to UF /DF employing Vivaspin centrifugal filtra - Corp ., Tokyo , Japan ). Prolia is transferred or dissolved in tors to remove excess periodate , quencher and the byprod reaction buffer ( e . g . 50 mM Hepes, 350 mM sodium chlo ucts thereof. ride, 5 mM calcium chloride , pH 6 .0 ) to get a final protein The retentate containing oxidized Prolia is next mixed 60 concentration of 1 . 0 + / - 0 .25 mg/ml . Then the pH of the with an aqueous m - toluidine solution (50 mM ) and incu - solution is corrected to 6 . 0 by drop wise addition of a 0 .5N bated for 30 min at room temperature . Aminooxy -PEG aqueous HCl solution . Subsequently , a 40 mM aqueous reagent with a MW of 20 kD is then added to give a 5 - fold sodium periodate solution is added within 10 minutes to give molar reagent excess. This mixture is incubated for 2 . 5 h at a concentration of 200 uM . The oxidation reaction is carried room temperature in the dark under gentle stirring . 65 out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . Finally, the PEG - Prolia conjugate is purified by ion - Then the reaction is stopped by addition of an aqueous exchange chromatography (e .g ., on Q -Sepharose FF ) . For L -cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° US 10 ,414 ,793 B2 163 164 C . to give a final concentration of 10 mM in the reaction Finally the PEG -Prolia conjugate is purified by ion mixture and incubation for 60 + / - 5 min . exchange chromatography on SP -Sepharose FF . The reac The oxidized Prolia is further purified by ion exchange tion mixture is diluted with 20 ml Buffer A (50 mM Hepes , chromatography . The oxidized Humira containing fractions pH 6 .5 ) and loaded onto a 20 ml HiPrep SPFF 16 / 10 column in 5 (GE Healthcare, Fairfield , Conn .) pre - equilibrated with Buf of the eluate are collected and used for the conjugation 5 fer A . Then the column is eluted with Buffer B ( 50 mM reaction . Hepes, 1 M NaC1, pH 6 .5 ) . Free Prolia is eluted by washing The aminooxy -PEG reagent with a MW of 20 kD reagent the column with 25 % Buffer B and the conjugate at 50 % is added in a 50 - fold molar excess to the eluate containing Buffer B . The conjugate containing fractions are concen the purified oxidized Prolia within a maximum time period trated by UF /DF using a 10 kD membrane made of regen ( t ) of 15 minutes under gentle stirring . Then an aqueous erated cellulose (88 cm2, cutoff 10 kD /Millipore ). The final m - toluidine solution (50 mM ) is added within 15 minutes to diafiltration step is performed against histidine buffer, pH get a final concentration of 10 mM . The reaction mixture is 6 . 9 containing 150 mM NaCl. The preparation is analyti cally characterized by measuring total protein ( Bradford ) incubated for 120 + / – 10 min . in the dark at a temperature ( T ) and biological activity according to methods known in the of T = + 22 + / - 2° C . under gentle shaking . 15 art. For the PEG - Prolia conjugate a specific activity of > 50 % The obtained PEG -Prolia conjugate is further purified by in comparison to native Prolia is determined . The conjugate ion exchange chromatography . The PEG - Prolia conjugate is additionally analytically characterized by Size Exclusion containing fractions are collected and concentratedentre by ultra -/ HPLC using a Agilent 1200 HPLC system equipped with a diafiltration (UF /DF ) using a membrane made of regener Shodex KW 803 column under conditions as previously ated cellulose with an appropriate molecular weight cut off 20 described (Kolarich et al, Transfusion 2006 ; 46 : 1959 -77 ). It (Millipore ). is shown that the preparation contains no free Prolia . Method 3 : Method 4 : Prolia is PEGylated by use of a linear 20 kD PEGylation Prolia is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An example of this reagent containing an aminooxy group . An example of this type of reagent is the Sunbright® CA series from NOF (NOF 25 type of reagent is the Sunbright® CA series from NOF (NOF Corp . , Tokyo , Japan ). EPO is dissolved in Hepes buffer (50 Corp ., Tokyo , Japan ). An initial concentration or weight of mM Hepes , 150 mM sodium chloride , 5 mM calcium HJumira is transferred or dissolved in Hepes buffer ( 50 mM chloride , pH 6 . 0 ) and mixed with an aqueous sodium Hepes , 150 mM sodium chloride , 5 mM calcium chloride , periodate solution ( 10 mM ) , and an aqueous m - toluidine pH 6 . 0 ) to get a final protein concentration of 2 mg Prolia / solution (50 mM ) . Subsequently the aminooxy reagent is 30 ml. Subsequently an 5 mM aqueous sodium periodate solu added to give a 20 - fold molar reagent excess. The mixture tion is added within 15 minutes to give a final concentration is incubated for 2 h in the dark at room temperature under of 100 uM , followed by addition of an 50 mM aqueous gentle stirring and quenched for 15 min at room temperature m - toluidine solution to get a final concentration of 10 mM by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . within a time period of 30 minutes . Then the aminooxy - PEG Finally, the PEG -Prolia conjugate is purified by ion - 35 reagent with a MW of 20 kD ( described above ) is added to exchange chromatography on Q -Sepharose FF . 1 . 5 mg pro - give a 20 - fold molar reagent excess . After correction of the tein /ml gel is loaded on the column pre equilibrated with 50 pH to 6 . 0 the mixture is incubated for 2 h in the dark at room mM Hepes buffer, pH 7 .4 containing 5 mM CaC12 . The temperature under gentle stirring and quenched for 15 min conjugate is eluted with 50 mM Hepes buffer containing 5 at room temperature by the addition of an 1 M aqueous mM CaC12 and 500 mM sodium chloride, pH 7 . 4 and is then 40 L -cysteine solution to give a final concentration of 10 mM . subjected to UF/ DF using a membrane. The preparation is The Prolia conjugate is purified by means of ion exchange analytically characterized by measuring total protein (Brad - chromatography ( IEC ) . The conjugate containing fractions ford ) and biological activity according to methods known in of the eluate are concentrated by UF/ DF using a 10 kD the art . membrane made of regenerated cellulose (88 cm2, cutoff 10 In an alternative embodiment, Method 3 is carried out as 45 kD /Millipore ) . The final diafiltration step is performed follows. against Hepes buffer ( 50 mM Hepes , 5 mM CaCl2 , pH 7 . 5 ) . Prolia is PEGylated by use of a linear 20 kD PEGylation The preparation is analytically characterized by measur reagent containing an aminooxy group . An example of this ing total protein (Bradford and BCA procedure ) and bio type of reagent is the Sunbright® CA series from NOF (NOF logical activity according to known methods. Corp ., Tokyo , Japan ) . 10 mg Prolia is dissolved in ~ 8 ml 50 histidine buffer, pH 6 . 0 (20 mM L -histidine , 150 mM NaCl) . Example 45 200 ul of an aqueous sodium periodate solution ( 5 mM ) and 2 ml of an aqueous m - toluidine solution (50 mM ) are then PEGylation of a Therapeutic Protein Using added . Subsequently, the aminooxy- PEG reagent with a Branched PEG MW of 20 kD (described above) is added to give a 5 - fold 55 molar reagent excess . The mixture is incubated for 2 h in the PEGylation of a therapeutic protein of the invention may dark at room temperature under gentle stirring and quenched be extended to a branched or linear PEGylation reagent, for 15 min at room temperature by the addition of 100 ul of which is made of an aldehyde and a suitable linker contain 1 M aqueous cysteine solution . ing an active aminooxy group .

SEQUENCE LISTING

< 160 > NUMBER OF SEQ ID NOS : 1 < 210 > SEQ ID NO 1 US 10 ,414 ,793 B2 165 166 - continued < 211 > LENGTH : 422 < 212 > TYPE : PRT < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 1 Leu Asn Arg Pro Lys Arg Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val 15 Gin Gly Asn Leu Glu Arg Glu Cys Met Glu Glu Lys Cys Ser Phe Glu 20 Glu Pro Arg Glu Val Phe Glu Asn Thr Glu Lys Thr Thr Glu Phe Trp 35 40 Lys Gin Tyr Val Asp Gly Asp Gin Cys Glu Ser Asn Pro Cys Leu Asn 55 Gly Gly Ser Cys Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro 65 70 Phe Gly Phe Glu Gly Lys Asn cys Glu Leu Asp Val Thr Cys Asn Ile 95 Lys Asn Gly Arg Cys Glu Gin Phe cys Lys Asn Ser Ala Asp Asn Lys 100

Val Val Cys Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn Gin Lys 115 120 Ser Cys Glu Pro Ala Val Pro Phe Pro Cys Gly Arg Val Ser Val Ser 135 Gin Thr Ser Lys Leu Thr Arg Ala Glu Ala Val Phe Pro Asp Val Asp 145 150 Tyr Val Asn Pro Thr Glu Ala Glu Thr Ile Leu Asp Asn Ile Thr Gin 175 Gly Thr Gin Ser Phe Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp 180 Ala Lys Pro Gly Gin Phe Pro Trp Gin Val Val Leu Asn Gly Lys Val 195 200 Asp Ala Phe Cys Gly Gly Ser Ile Val Asn Glu Lys Trp Ile Val Thr 215 Ala Ala His Cys Val Glu Thr Gly Val Lys Ile Thr Val Val Ala Gly 225 230 Glu His Asn Ile Glu Glu Thr Glu His Thr Glu Gin Lys Arg Asn Val 255 Ile Arg Ala Ile Ile Pro His His Asn Tyr Asn Ala Ala Ile Asn Lys 260 Tyr Asn His Asp Ile Ala Leu Leu Glu Leu Asp Glu Pro Leu Val Leu 275 280 Asn Ser Tyr Val Thr Pro Ile Cys Ile Ala Asp Lys Glu Tyr Thr Asn 295 Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser Gly Trp Ala Arg Val 305 310 Phe His Lys Gly Arg Ser Ala Leu Val Leu Gin Tyr Leu Arg Val Pro 335 Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile Tyr 340 Asn Asn Met Phe Cys Ala Gly Phe His Glu Gly Gly Arg Asp Ser Cys 355 360 Gin Gly Asp Ser Gly Gly Pro His Val Thr Glu Val Glu Gly Thr Ser 375 Phe Leu Thr Gly Ile Ile Ser Trp Gly Glu Glu Cys Ala Met Lys Gly USUS 10410 ,414 , 793 B2 167 168 - continued 385 390 395 400 Lys Tyr Gly Ile Tyr Thr Lys Val Ser Arg Tyr Val Asn Trp Ile Lys 405 410 415 Glu Lys Thr Lys Leu Thr 420

10 What is claimed : minutes and 120 minutes; a temperature between about 2° C . 1 . A method of conjugating a water soluble polymer to an and about 37° C . ; in the presence or absence of light; and oxidized carbohydrate moiety of Factor VIII (FVIII ) com with or without stirring . prising contacting the oxidized carbohydrate moiety with an 11 . The method of claim 10 wherein the final concentra activated water soluble polymer under conditions that allow 15 tion of sodium periodate is about 400 and the conditions conjugation ; comprise a time period of about 10 minutes , a temperature said water soluble polymer containing an active aminooxy of about 22° C . , the absence of light and with stirring . group is selected from the group consisting of polyeth - 12 . The method of claim 1 wherein the conjugating the ylene glycol (PEG ) , branched PEG , PolyPEG® (War - water soluble polymer to the oxidized carbohydrate moiety wick Effect Polymers ; Coventry , UK ) , and polysialiczu of FVIII is stopped by the addition of a quenching agent acid (PSA ) ; and selected from the group consisting of L - cysteine, methio said carbohydrate moiety oxidized by incubation with a nine , glutathione, glycerol, sodium meta bisulfite buffer comprising sodium periodate (NalO2 ) ; (Na2S203 ) , tryptophane , tyrosine , histidine or derivatives wherein an oxime linkage is formed between the oxidized 25 thereof, kresol, imidazole , and combinations thereof ; carbohydrate moiety and the active aminooxy group on wherein the quenching agent is added in an amount to the water soluble polymer ; and result in a final concentration between about 1 mM and wherein said oxime linkage formation is catalyzed by about 100 mM quenching agent, under conditions m - toluidine . comprising a time period between about 5 minutes and 2 . The method according to claim 1 wherein a solution 30 about 120 minutes ; a temperature between about 2° C . comprising an initial concentration of FVIII between about and about 37° C .; in the presence or absence of light ; 0 .3 mg/ml and about 3 . 0 mg/ ml is adjusted to a pH value and with or without stirring . between about 5 . 0 and about 8 . 0 prior to contacting with the 13 . The method of claim 12 wherein the quenching agent activated water soluble polymer . is L - cysteine . 3 . Themethod of claim 2 wherein the initial concentration 35 14 . The method of claim 13 wherein the L - cysteine is of FVIII is about 1 . 0 mg/ ml and the pH is about 6 . 0 . added to result in a final concentration of about 10 mM and 4 . The method of claim 1 wherein FVIII is contacted by the conditions comprise a time period of about 60 minutes , a desired excess concentration of activated water soluble a temperature of about 22° C . , the absence of light and with polymer , wherein the excess concentration is between about stirring . 1 - fold molar and about 300 - fold molar excess . 40 5 . The method of claim 4 wherein the excess concentra 15 . The method of claim 1 comprising : tion is about 50 - fold molar excess . a ) a first step comprising adjusting the pH value of a 6 . The method of claim 4 wherein FVIII is incubated with solution comprising FVIII to a pH value between about the activated water soluble polymer under conditions com 5 . 0 and about 8 . 0 , wherein the concentration of FVIII prising a time period between about 0 . 5 hours and about 24 45 is between about 0 . 3 mg/ml and about 3 .0 mg/ ml ; hours ; a temperature between about 2° C . and about 37° C .; b ) a second step comprising oxidizing one or more in the presence or absence of light; and with or without carbohydrates on FVIII, wherein sodium periodate is stirring. added to the solution in the first step to result in a final 7 . The method according to claim 6 wherein the condi concentration between about 50 uM and about 1000 tions comprise a time period of about 120 minutes , a 50 under conditions comprising a time period between temperature of about 22° C ., the absence of light; and with about 0 . 1 minutes and about 120 minutes ; a tempera stirring . ture between about 2° C . and about 37° C . ; in the 8 . The method according to claim 1 wherein m - toluidine presence or absence of light, and with or without is added in an amount to result in a final concentration stirring; between about 1 . 0 mM and about 50 mM , under conditions 55 c ) a third step comprising contacting FVIII with a desired comprising a time period between about 0 . 1 minutes and excess concentration of activated water soluble poly about 30 minutes ; a temperature between about 2° C . and mer , wherein the excess concentration is between about about 37° C . ; in the presence or absence of light; and with 1 - fold molar excess and about 300 - fold molar excess , or without stirring . under conditions comprising a time period between 9 . The method of claim 8 wherein the final concentration 60 about 0 . 5 hours and about 24 hours , a temperature of m - toluidine is about 10 mM , and the conditions comprise between about 2° C . and about 37° C .; in the presence a time period of up to about 15 minutes, a temperature of or absence of light; and with or without stirring ; about 22° C ., the absence of light; and with stirring . d ) a fourth step comprising adding m - toluidine to the 10 . The method according to claim 1 wherein sodium solution of the third step , wherein m - toluidine is added periodate is added in an amount to result in a final concen - 65 to result in a final concentration between about 1 mM tration between about 50 uM and about 1000 UM , under and about 50 mM , under conditions comprising a time conditions comprising a time period between about 0 . 1 period between about 0 . 1 minutes and about 30 min US 10 ,414 ,793 B2 169 170 utes; a temperature between about 2° C . and about 37° ine is added to result in a final concentration of about C . ; in the presence or absence of light, and with or 10 mM , under conditions comprising a time period of without stirring ; about 60 minutes , a temperature of about 22° C . , the e ) a fifth step wherein FVIII is incubated with the acti absence of light and with stirring . vated water soluble polymer and m - toluidine under 5 17 . The method according to claim 1 wherein the water conditions that allow conjugation of the activated soluble polymer is PSA . water - soluble polymer to one or more oxidized carbo - 18 . The method according to claim 1 wherein the water hydrates on FVIII, said conditions comprising a time soluble polymer is PEG . period between about 0 . 5 hours and about 24 hours, a 19 . The method according to claim 17 wherein the PSA is temperature between about 2° C . and about 37° C . ; in 10 comprised of about 10 - 300 sialic acid units . the presence or absence of light, and with or without 20 . The method according to claim 1 wherein the oxidized stirring ; and carbohydrate moiety of FVIII is located in the activation f ) a sixth step wherein the conjugating the water soluble peptide of FVIII. polymer to the one or more oxidized carbohydrates of 21. The method according to claim 17 wherein the PSA is FVIII in the fifth step is stopped by the addition of a 15 prepared by reacting an activated aminooxy linker with quenching agent selected from the group consisting of L -cysteine , methionine , glutathione , glycerol, Na2S205 wherein the aminooxy linker is selected from the group ( sodium meta bisulfite ), tryptophane , tyrosine , histidine consisting of: or derivatives thereof, kresol, imidazole , and combina a ) a 3 -oxa -pentane - 1, 5 -dioxyamine linker of the formula : tions thereof; wherein the quenching agent is added to 20 result in a final concentration of about 1 mM and about 100 mm , under conditions comprising a time period H2N NH , between about 5 minutes and about 120 minutes; a temperature between about 2° C . and about 37° C . ; in the presence or absence of light , and with or without 25 b ) a 3 , 6 , 9 - trioxa - undecane- 1 , 11- dioxyamine linker of the stirring . formula: 16 . The method of claim 1 comprising : a ) a first step comprising adjusting the pH value of a solution comprising FVIII to a pH value of about 6 . 0 , H2N ^ , O - NH2 wherein the initial concentration of FVIII is about 1 30 vºvº NH3 mg/ ml ; b ) a second step comprising oxidizing one or more and carbohydrates on FVIII, wherein sodium periodate is c ) a 3 ,6 , 9 , 12 , 15 pentaoxa -heptadecane - 1 , 17 -dioxyamine added to the solution in the first step to result in a final linker of the formula : HN o °CNH,

concentration of about 400 under conditions compris 40 wherein the PSA is oxidized by incubation with sodium ing a time period of about 10 minutes , a temperature of periodate to form a terminal aldehyde group at the about 22° C . , the absence of light and with stirring ; non -reducing end of the PSA . c ) a third step comprising contacting FVIII with a desired 22. The method according to claim 21 wherein the ami excess concentration of activated water soluble poly - 45 nooxy linker is 3 - oxa -pentane - 1, 5 - dioxyamine. mer, wherein the excess concentration is about 50 -mo - 23 . The method according to claim 1 wherein m - toluidine lar excess ; is provided at a concentration between about 1 mM and under conditions comprising a time period of about 15 about 50 mM . minutes, a temperature of about 22° C . , the absence of 24 . The method according to claim 1 wherein the m -tolui light and with stirring ; 50 dine is present in the conjugation reaction at a concentration d ) a fourth step comprising adding m - toluidine to the of about 10 mM . solution of the third step , wherein m - toluidine is added 25 . The method according to claim 1 further comprising to result in a final concentration of about 10 mm , under the step of reducing an oxime linkage in the conjugated conditions comprising a time period of about 15 min - FVIII by incubating the conjugated FVIII in a buffer com utes, a temperature of about 22° C ., the absence of light 55 prising a reducing compound selected from the group con and with stirring; sisting of sodium cyanoborohydride (NaCNBHZ ) , ascorbic e ) a fifth step wherein FVIII is incubated with the acti - acid ( vitamin C ) and NaBHz. vated water soluble polymer and m -toluidine under 26 . The method according to claim 25 wherein the reduc conditions that allow conjugation of the activated ing compound is sodium cyanoborohydride (NaCNBH ). water - soluble polymer to one or more oxidized carbo - 6027 . The method according to claim 1 further comprising hydrates on FVIII, said conditions comprising a time the step of purifying the conjugated FVIII . period of about 2 hours ; a temperature of about 22° C . ; 28 . The method according to claim 27 wherein the con the absence of light; and with stirring ; and jugated FVIII is purified by a method selected from the f ) a sixth step wherein the conjugating the water soluble group consisting of chromatography, filtration and precipi polymer to the one or more oxidized carbohydrates of 65 tation . FVIII in the fifth step is stopped by the addition of a 29 . The method according to claim 28 wherein the chro quenching agent L - cysteine; and wherein the L - cyste - matography is selected from the group consisting ofHydro US 10 ,414 ,793 B2 171 172 phobic Interaction Chromatography (HIC ), Ion Exchange polyethylene glycol (PEG ) , branched PEG , PolyPEG® chromatography (IEC ), Size exclusion chromatography (Warwick Effect Polymers ; Coventry , UK ) , and poly ( SEC ) , Affinity chromatography, and Reversed - phase chro matography. sialic acid (PSA ) . 30 . The method of claim 29 wherein an anti - chaotropic 5 46 . The method according to claim 1 wherein the water salt is used in a chromatography loading step and in a soluble polymer containing an active aminooxy group is chromatography washing step . prepared by a method comprising : 31 . The method of claim 29 wherein the chromatography a ) incubating a solution comprising an oxidized water takes place in a column . soluble polymer with an activated aminooxy linker 32 . The method of claim 31 wherein the column com - 10 comprising an active aminooxy group under conditions prises a chromatography resin selected from the group that allow the formation of a stable oxime linkage consisting of Phenyl -Sepharose FF and Butyl -Sepharose FF . between the oxidized water -soluble polymer and the 33 . The method of claim 32 wherein the resin is present activated aminooxy linker, said conditions comprising in the column at a bed height of between about 5 cm and a time period between about 1 minute and about 24 about 20 cm . 15 hours ; a temperature between about 2° C . and about 37° 34 . The method according to claim 33 wherein the bed C .; in the presence or absence of light, and with or height is about 10 cm . without stirring ; thereby forming a water soluble poly 35 . The method of claim 31 comprising one or more washing steps wherein flow direction is set to up - flow and mer containing an active aminooxy group ; wherein the flow rate is between about 0 . 2 cm /min and about 20 b ) incubating a solution comprising the water soluble 6 . 7 cm /min . polymer containing an active aminooxy group of step 36 . The method according to claim 35 wherein the flow a ) with m - toluidine under conditions comprising a time rate is about 2 cm /min . period between 1 minute and 24 hours ; a temperature 37 . Themethod of claim 1 comprising one or more elution between 2° C . and 37° C . ; in the presence or absence steps wherein flow direction is set to down - flow and wherein 25 of light; and with or without stirring ; the flow rate is between about 0 . 1 cm /min and about 6 . 7 cm /min . c ) incubating a solution comprising the water soluble 38 . The method according to claim 37 wherein the flow polymer containing an active aminooxy group of step rate is about 1 cm /min . b ) with a reducing agent under conditions that allow the 39 . The method of claim 1 further comprising concen - 30 formation of a stable alkoxamine linkage between the trating the conjugated FVIII by ultrafiltration /diafiltration oxidized water- soluble polymer and the activated ami (UF / DF) . nooxy linker , said conditions comprising a time period 40 . Themethod of claim 1 wherein the final concentration of FVIII is between about 0 . 5 and about 3 mg/ ml . between about 1 minute and about 24 hours ; a tem 41 . The method according to claim 1 wherein FVIII 35 perature between about 2° C . and about 37° C . ; in the comprises between about 5 and about 11 water soluble presence or absence of light ; and with or without polymer moieties . stirring ; and 42 . The method of claim 1 wherein the conjugated FVIII d ) purifying the water soluble polymer containing an is purified using chromatography ; wherein an anti - chao active aminooxy group by a method selected from the tropic salt is used for a loading step and for a washing step ; 40 group consisting of chromatography, filtration and pre the method comprising one or more washing steps wherein cipitation ; flow direction is set to up - flow and wherein the flow rate is between about 0 . 2 cm /min and about 6 . 7 cm /min and one or wherein the water soluble polymer is PSA . more elution steps wherein flow direction is set to down 47 . The method according to claim 1 wherein said water flow and wherein the flow rate is between about 0 . 2 cm /min 45 soluble polymer is oxidized by incubation with sodium and about 6 . 7 cm /min ; further comprising concentrating the periodate to form a terminal aldehyde group at the non conjugated FVIII by UF /DF . reducing end of the water - soluble polymer . 43. The method of claim 42 wherein the chromatography is hydrophobic interaction chromatography (HIC ); wherein 48 . The method according to claim 46 wherein the ami the one or more washing steps flow rate is about 2 cm /min ; 50sonooxy 100 linker is selected from the group consisting of: and wherein the one or more elution steps flow rate is about a ) a 3 -oxa -pentane - 1 , 5 -dioxyamine linker of the formula : 1 cm /min . 44 . A modified FVIII produced by the method according to claim 1 . 45 . A method of forming an oxime linkage between an 55 | HANNA - NHA, oxidized carbohydrate moiety on FVIII and an activated water soluble polymer containing an active aminooxy group comprising the steps of: b ) a 3 , 6 , 9 - trioxa - undecane- 1 , 11- dioxyamine linker of the a ) oxidizing a carbohydrate moiety on FVIII by incubat formula: ing said protein with sodium periodate (NaI02 ) ; and 60 b ) forming an oxime linkage between the oxidized car- HAN , bohydrate moiety of FVIII and the activated water ° NH , soluble polymer containing an active aminooxy group in the presence of m - toluidine under conditions allow ing formation of said oxime linkage ; 65 and wherein said water soluble polymer containing an active c ) a 3, 6 ,9 ,12 , 15 -penatoxa -heptadecane -1 , 17 -dioxyamine aminooxy group is selected from the group consisting linker of the formula : US 10 ,414 ,793 B2 173 174

H?N NH2NH ,

49 . The method according to claim 46 wherein the reduc ing agent is selected from the group consisting of sodium cyanoborohydride (NaCNBHz ) , ascorbic acid ( vitamin C ) and NaBHz 50 . The method according to claim 49 wherein the reduc - 10 ing agent is sodium cyanoborohydride (NaCNBHz ) . 51 . The method according to claim 46 wherein m -tolui dine is added in an amount to result in a final concentration between about 1 . 0 mM and about 50 mM . 52 . The method according to claim 46 further comprising 15 concentrating the conjugated FVIII by UF /DF . * * * * *