That the Tout Untuk Mun Mauto Mo at Hindi Hinthi

Home , Nucleoside transporter

THATTHE TOUT UNTUK USMUN 20170240590A1 MAUTO MO ATHINDI HINTHI (19 ) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2017/ 0240590 A1 Haider et al. ( 43 ) Pub . Date: Aug. 24 , 2017 ( 54 ) NUCLEOPHILIC CATALYSTS FOR OXIME filed on May 21 , 2010 , provisional application No . LINKAGE 61 / 228 ,828 , filed on Jul. 27 , 2009 . ( 71 ) Applicants : BAXALTA INCORPORATED , Publication Classification Bannockburn , IL (US ) ; BAXALTA (51 ) Int. Ci. GMBH , Glattpark (Opifkon ) (CH ) CO7K 1/ 107 ( 2006 . 01 ) CO7K 1 / 34 (2006 .01 ) (72 ) Inventors : Stefan Haider, Prinzersdorf (AT ) ; CO7K 1 / 20 (2006 .01 ) Andreas Ivens, Zurich (CH ) ; COZK 14 / 755 ( 2006 .01 ) Hanspeter Rottensteiner , Vienna (AT ) ; (52 ) U .S . CI. Juergen Siekmann , Vienna ( AT ) ; Peter CPC ...... CO7K 1/ 1077 ( 2013 .01 ) ; C07K 14 / 755 Turecek , Klosterneuburg (AT ) (2013 .01 ) ; A61K 47/ 4823 (2013 .01 ) ; A61K 47 /48215 ( 2013 .01 ) ; CO7K 1 /34 ( 2013 .01 ); (21 ) Appl. No. : 15 / 281, 616 C07K 1 / 20 (2013 .01 ) (57 ) ABSTRACT (22 ) Filed : Sep . 30 , 2016 The invention relates to materials and methods of conjugat ing a water soluble polymer to an oxidized carbohydrate Related U . S . Application Data moiety of a therapeutic protein comprising contacting the oxidized carbohydrate moiety with an activated water (63 ) Continuation of application No . 14 / 136 ,233 , filed on soluble polymer under conditions that allow conjugation . Dec. 20 , 2013 , now Pat . No . 9 ,492 ,555 , which is a More specifically , the present invention relates to the afore continuation of application No . 13 / 194 , 038 , filed on mentioned materials and methods wherein the water soluble Jul. 29 , 2011 , now Pat. No . 8 ,642 ,737 , which is a polymer contains an active aminooxy group and wherein an continuation - in -part of application No . 12 / 843 , 542 , oxime or hydrazone linkage is formed between the oxidized filed on Jul. 26 , 2010 , now Pat . No. 8 ,637 , 640 . carbohydrate moiety and the active aminooxy group on the (60 ) Provisional application No . 61 / 369, 186 , filed on Jul. water soluble polymer , and wherein the conjugation is 30 , 2010 , provisional application No. 61 /347 , 136 , carried out in the presence of a nucleophilic catalyst . Patent Application Publication Aug . 24 , 2017 Sheet 1 of 6 US 2017 /0240590 A1

Figure 1

cewe EGE-like Domain ABOMO DOD8007200000000000000OODS BOONActivation Peptide p0000200028129900000000COVERS0000000029ogleetaJ090039 202008100009330 0920020022003200020202aCatalytic Domain *00000000000 000000 992003 9099920000000000000000000000000000230000 como el Con000000 co200041000000ChuC00000000 O -linked Glycan Gla Domain 00000000 ¥ Ninkexi Glycan < FXla cleavage site Patent Application Publication Aug . 24 , 2017 Sheet 2 of 6 US 2017 /0240590 A1

Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

80 .0 % 70 . 0 % 60 . 0 % 50 .0 % TFIX 40 . 0 % 30 . 0 % & di- PSAylated < FIX 20 . 0 % mono- PsAylated : FIX 10 . 096 free Fox 0. 0 % nocatalyst 10mManiline10mMm10MMO-toluidine - aminobenzioc 10mMm -aminobenziac 10mMpacid -aminobenzioc acid10mmo -aminobenzamideacid 10MM sulfanilic acid US 2017 /0240590 A1 Aug . 24 , 2017

NUCLEOPHILIC CATALYSTS FOR OXIME containing aminooxy groups which react with aldehydes to LINKAGE form oxime linkages (WO 96 / 40662 , WO2008 / 025856 ). [0005 ] Additional examples describing conjugation of a FIELD OF THE INVENTION water soluble polymer to a therapeutic protein are described in WO 06 / 071801 which teaches the oxidation of carbohy [0001 ] The present invention relates to materials and drate moieties in Von Willebrand factor and subsequent methods for conjugating a water soluble polymer to a coupling to PEG using hydrazide chemistry ; US Publication protein . No . 2009/ 0076237 which teaches the oxidation of rFVIII and subsequent coupling to PEG and other water soluble BACKGROUND OF THE INVENTION polymers ( e . g . PSA , HES , dextran ) using hydrazide chem istry ; WO 2008 / 025856 which teaches oxidation of different [0002 ] The preparation of conjugates by forming a cova coagulation factors , e . g . rFIX , FVIII and FVIIa and subse lent linkage between the water soluble polymer and the quent coupling to e . g . , PEG , using aminooxy chemistry by therapeutic protein can be carried out by a variety of forming an oxime linkage ; and U . S . Pat. No . 5 ,621 ,039 chemical methods. PEGylation of polypeptide drugs pro which teaches the oxidation of FIX and subsequent coupling tects them in circulation and improves their pharmacody to PEG using hydrazide chemistry . namic and pharmacokinetic profiles (Harris and Chess , Nat [0006 ] Recently , an improved method was described com Rev Drug Discov. 2003 ; 2 : 214 - 21 ). The PEGylation process prising mild periodate oxidation of sialic acids to generate attaches repeating units of ethylene glycol (polyethylene aldehydes followed by reaction with an aminooxy group glycol (PEG )) to a polypeptide drug . PEG molecules have a containing reagent in the presence of catalytic amounts of large hydrodynamic volume ( 5 - 10 times the size of globular aniline (Dirksen A . , and Dawson PE , Bioconjugate Chem . proteins ) , are highly water soluble and hydrated , non - toxic , 2008 ; 19 , 2543 - 8 ; and Zeng Y et al. , Nature Methods 2009 ; non - immunogenic and rapidly cleared from the body . PEGY 6 : 207 - 9 ) . The aniline catalysis dramatically accelerates the lation of molecules can lead to increased resistance of drugs oxime ligation , allowing the use of very low concentrations to enzymatic degradation , increased half - life in vivo , of the reagent. The use of nucelophilic catalysts are also reduced dosing frequency, decreased immunogenicity , described in Dirksen , A . , et al. , J Am Chem Soc ., 128 : increased physical and thermal stability, increased solubility, 15602 - 3 ( 2006 ) ; Dirksen , A ., et al. , Angew chem . Int Ed ., increased liquid stability , and reduced aggregation . The first 45 :7581 - 4 (2006 ); Kohler , J. J ., ChemBioChem ., 10 :2147 PEGylated drugs were approved by the FDA in the early 50 (2009 ) ; Giuseppone, N ., et al ., J Am Chem Soc . , 127 : 1990s. Since then , the FDA has approved several PEGylated 5528 - 39 ( 2005 ) ; and Thygesen , M . B ., et al. , J Org Chem . , drugs for oral, injectable , and topical administration . 75 : 1752 -5 ( 2010 ). [ 0003] Polysialic acid (PSA ), also referred to as colominic [ 0007 ] Although aniline catalysis can accelerate the oxime acid (CA ) , is a naturally occurring polysaccharide . It is a ligation allowing short reaction times and the use of low homopolymer of N - acetylneuraminic acid with a (2 8 ) concentrations of the aminooxy reagent, aniline has toxic ketosidic linkage and contains vicinal diol groups at its properties that must be considered when , for example , the non -reducing end . It is negatively charged and a natural conjugated therapeutic protein to form the basis of a phar constituent of the human body. It can easily be produced maceutical. For example , aniline has been shown to induce from bacteria in large quantities and with pre - determined methemoglobinemia (Harrison , J . H . , and Jollow , D . J . , physical characteristics ( U . S . Pat. No . 5 ,846 , 951 ) . Because Molecular Pharmacology , 32 (3 ) 423 -431 , 1987 ). Long - term the bacterially - produced PSA is chemically and immuno dietary treatment of rats has been shown to induce tumors in logically identical to PSA produced in the human body, the spleen (Goodman , D G . , et al. , J Natl Cancer Inst. , bacterial PSA is non - immunogenic , even when coupled to 73 ( 1 ): 265 -73 , 1984 ). In vitro studies have also shown that proteins . Unlike some polymers, PSA acid is biodegradable . aniline has the potential to induce chromosome mutations Covalent coupling of colominic acid to catalase and aspara and has the potentially genotoxic activity ( Bombhard E . M . ginase has been shown to increase enzyme stability in the et Herbold B , Critical Reviews in Toxicology 35 ,783 - 835 , presence of proteolytic enzymes or blood plasma. Compara 2005 ) . tive studies in vivo with polysialylated and unmodified [ 0008 ] Considering the potentially dangerous properties of asparaginase revealed that polysialylation increased the aniline and notwithstanding the methods available of con half- life of the enzyme ( Fernandes and Gregoriadis , Int J jugating water soluble polymers to therapeutic proteins , Pharm . 2001; 217: 215 - 24 ) . there remains a need to develop materials and methods for [0004 ] Coupling of PEG - derivatives to peptides or pro conjugating water soluble polymers to proteins that teins is reviewed by Roberts et al. (Adv Drug Deliv Rev improves the protein ' s pharmacodynamic and / or pharma 2002 ; 54 :459 - 76 ). One approach for coupling water soluble cokinetic properties while minimizing the costs associated polymers to therapeutic proteins is the conjugation of the with the various reagents and minimizing the health risks to polymers via the carbohydrate moieties of the protein . the patient recipient. Vicinal hydroxyl (OH ) groups of carbohydrates in proteins can be easily oxidized with sodium periodate (NalO4 ) to form active aldehyde groups (Rothfus et Smith , J Biol Chem SUMMARY OF THE INVENTION 1963; 238 : 1402 - 10 ; van Lenten et Ashwell, J Biol Chem [0009 ] The present invention provides materials and meth 1971; 246 : 1889 - 94 ) . Subsequently the polymer can be ods for conjugating polymers to proteins that improves the coupled to the aldehyde groups of the carbohydrate by use protein 's pharmacodynamic and / or pharmacokinetic prop of reagents containing , for example , an active hydrazide erties while minimizing the costs associated with the various group (Wilchek M and Bayer EA , Methods Enzymol 1987 ; reagents and the health risks to the patient recipients when 138 :429 -42 ). A more recent technology is the use of reagents the conjugation reaction is catalyzed by a nucleophilic US 2017 /0240590 A1 Aug . 24 , 2017 catalyst . In various embodiments of the invention , alterna morphogenic protein - 10 , bone morphogenic protein - 11, tive catalysts to substitute for aniline are provided . bone morphogenic protein - 12, bone morphogenic protein [0010 ] In one embodiment, a method of conjugating a 13, bone morphogenic protein - 14, bone morphogenic pro water soluble polymer to an oxidized carbohydrate moiety tein - 15, bone morphogenic protein receptor IA , bone mor of a therapeutic protein is provided comprising contacting Piphogenic protein receptor IB , bone morphogenic protein the oxidized carbohydrate moiety with an activated water receptor II , brain derived neurotrophic factor, cardiotrophin soluble polymer under conditions that allow conjugation ; 1 , ciliary neutrophic factor, ciliary neutrophic factor recep said water soluble polymer containing an active aminooxy tor , cripto , cryptic , cytokine- induced neutrophil chemotactic group and is selected from the group consisting of polyeth factor 1 , cytokine - induced neutrophil , chemotactic factor ylene glycol (PEG ) , branched PEG , PolyPEG® (Warwick 2a , cytokine - induced neutrophil chemotactic factor 2B , B Effect Polymers ; Coventry, UK ), polysialic acid (PSA ) , endothelial cell growth factor, endothelin 1, epidermal starch , hydroxyalkyl starch (HAS ) , hydroxylethyl starch growth factor , epigen , epiregulin , epithelial- derived neutro (HES ) , carbohydrate , polysaccharides, pullulane , chitosan , phil attractant, fibroblast growth factor 4 , fibroblast growth hyaluronic acid , chondroitin sulfate , dermatan sulfate , factor 5 , fibroblast growth factor 6 , fibroblast growth factor starch , dextran , carboxymethyl - dextran , polyalkylene oxide 7 , fibroblast growth factor 8 , fibroblast growth factor 8b , ( PAO ), polyalkylene glycol (PAG ), polypropylene glycol fibroblast growth factor 8c , fibroblast growth factor 9 , (PPG ) , polyoxazoline , polyacryloylmorpholine , polyvinyl fibroblast growth factor 10 , fibroblast growth factor 11 , alcohol (PVA ) , polycarboxylate, polyvinylpyrrolidone , fibroblast growth factor 12 , fibroblast growth factor 13 , polyphosphazene , polyoxazoline , polyethylene - co - maleic fibroblast growth factor 16 , fibroblast growth factor 17 , acid anhydride , polystyrene - co -maleic acid anhydride , poly fibroblast growth factor 19 , fibroblast growth factor 20 , ( 1- hydroxymethylethylene hydroxymethylformal) (PHF ), fibroblast growth factor 21 , fibroblast growth factor acidic , 2 -methacryloyloxy - 2 '- ethyltrimethylammoniumphosphate fibroblast growth factor basic , glial cell line -derived neutro (MPC ) ; and said carbohydrate moiety oxidized by incuba phic factor receptor al, glial cell line - derived neutrophic tion with a buffer comprising an oxidizing agent selected factor receptor a2, growth related protein , growth related from the group consisting of sodium periodate (NalO4 ) , lead protein a , growth related protein B , growth related protein y , tetraacetate (Pb (OAC ) 4 ) and potassium perruthenate heparin binding epidermal growth factor , hepatocyte growth (KRu04 ) ; wherein an oxime linkage is formed between the factor, hepatocyte growth factor receptor , hepatoma -derived oxidized carbohydrate moiety and the active aminooxy growth factor, insulin - like growth factor I , insulin - like group on the water soluble polymer ; and wherein said oxime growth factor receptor, insulin - like growth factor II , insulin linkage formation is catalyzed by a nucleophilic catalyst like growth factor binding protein , keratinocyte growth selected from the group consisting of o -amino benzoic acid , factor, leukemia inhibitory factor, leukemia inhibitory factor m - amino benzoic acid , p - amino benzoic acid , sulfanilic receptor a , nerve growth factor nerve growth factor recep acid , c - aminobenzamide , o - toluidine , m - toluidine , p - tolui tor, neuropoietin , neurotrophin - 3 , neurotrophin - 4 , oncosta dine , o -anisidine , m - anisidine , and p - anisidine . tin M (OSM ), placenta growth factor, placenta growth factor [0011 ] In another embodiment, a method of conjugating a 2 , plateletderived endothelial cell growth factor, platelet water soluble polymer to an oxidized carbohydrate moiety derived growth factor, platelet derived growth factor A of a therapeutic protein is provided comprising contacting chain , platelet derived growth factor AA , platelet derived the oxidized carbohydrate moiety with an activated water growth factor AB , platelet derived growth factor B chain , soluble polymer under conditions that allow conjugation ; platelet derived growth factor BB , platelet derived growth said therapeutic protein selected from the group consisting factor receptor a , platelet derived growth factor receptor ß , of Factor IX ( FIX ) , Factor VIII ( FVIII ) , Factor VIIa (FVIIa ) , pre - B cell growth stimulating factor, stem cell factor (SCF ) , Von Willebrand Factor (VWF ) , Factor FV ( FV ) , Factor X stem cell factor receptor, TNF, TNFO , TNF1, TNF2 , trans ( FX ) , Factor XI ( FXI) , Factor XII ( FXII) , thrombin ( FII ) , forming growth factor a , transforming growth factor B , protein C , protein S , DPA , PAI - 1 , tissue factor ( TF ) , transforming growth factor B1, transforming growth factor ADAMTS 13 protease, IL - 1 alpha , IL - 1 beta , IL - 2 , IL - 3 , B1 . 2 , transforming growth factor B2 , transforming growth IL - 4 , IL - 5 , IL - 6 , IL - 11, colony stimulating factor- 1 ( CSF - 1 ) , factor B3 , transforming growth factor B5 , latent transform M -CSF , SCF, GM -CSF , granulocyte colony stimulating fac ing growth factor B1 , transforming growth factor ß binding tor ( G - CSF ) , EPO , interferon - alpha ( IFN - alpha ), consensus protein I , transforming growth factor B binding protein II , interferon , IFN -beta , IFN - gamma, IFN -omega , IL - 7 , IL - 8 , transforming growth factor ß binding protein III , thymic IL - 9, IL - 10 , IL - 12 , IL - 13, IL - 14 , IL - 15 , IL - 16 , IL - 17 , IL - 18 , stromal lymphopoietin ( TSLP ) , tumor necrosis factor recep IL - 19 , IL - 20 , IL -21 , IL - 22 , IL - 23 , IL - 24 , IL -31 , IL - 32 alpha , tor type I , tumor necrosis factor receptor type II , urokinase IL -33 , thrombopoietin (TPO ), Ang - 1 , Ang -2 , Ang - 4 , Ang - Y, type plasminogen activator receptor, phospholipase - activat angiopoietin - like polypeptide 1 ( ANGPTL1 ) , angiopoietin ing protein ( PUP ) , insulin , lectin ricin , prolactin , chorionic like polypeptide 2 ( ANGPTL2 ), angiopoietin - like polypep gonadotropin , follicle - stimulating hormone , thyroid - stimu tide 3 (ANGPTL3 ) , angiopoietin - like polypeptide 4 ( AN lating hormone , tissue plasminogen activator, IgG , IgE , GPTL4 ), angiopoietin - like polypeptide 5 ( ANGPTL5 ) , IgM , IgA , and IgD , a - galactosidase , B - galactosidase , angiopoietin - like polypeptide 6 ( ANGPTL6 ), angiopoietin DNAse , fetuin , leutinizing hormone , estrogen , insulin , albu like polypeptide 7 (ANGPTL7 ) , vitronectin , vascular min , lipoproteins, fetoprotein , transferrin , thrombopoietin , endothelial growth factor (VEGF ) , angiogenin , activin A , urokinase , integrin , thrombin , leptin , Humira (adalimumab ) , activin B , activin C , bone morphogenic protein - 1 , bone Prolia (denosumab ) , Enbrel ( etanercept) , a protein in Table morphogenic protein - 2 , bone morphogenic protein - 3, bone 1 , or a biologically active fragment, derivative or variant morphogenic protein - 4 , bone morphogenic protein - 5 , bone thereof; said water soluble polymer containing an active morphogenicm protein - 6 , bone morphogenic protein - 7 , bone aminooxy group and is selected from the group consisting of morphogenic protein - 8 , bone morphogenic protein - 9, bone polyethylene glycol (PEG ), branched PEG , PolyPEG® US 2017 /0240590 A1 Aug . 24 , 2017

(Warwick Effect Polymers ; Coventry, UK ), polysialic acid [ 0017 ] In another embodiment, an aforementioned method (PSA ), starch , hydroxyalkyl starch (HAS ) , hydroxylethyl is provided wherein the nucleophilic catalyst is added in an starch (HES ) , carbohydrate , polysaccharides , pullulane , chi amount to result in a final concentration between about 1 . 0 tosan , hyaluronic acid , chondroitin sulfate , dermatan sulfate , mM and about 50 mM nucleophilic catalyst, under condi starch , dextran , carboxymethyl- dextran , polyalkylene oxide tions comprising a time period between about 0 . 1 minutes ( PAO ) , polyalkylene glycol ( PAG ) , polypropylene glycol and about 30 minutes ; a temperature between about 2° C . ( PPG ) , polyoxazoline , polyacryloylmorpholine , polyvinyl and about 37° C . ; in the presence or absence of light; and alcohol (PVA ) , polycarboxylate , polyvinylpyrrolidone , with or without stirring . In another embodiment, the final polyphosphazene, polyoxazoline, polyethylene - co -maleic concentration of the nucleophilic catalyst is about 10 mM , acid anhydride, polystyrene - co -maleic acid anhydride, poly and the conditions comprise a time period of up to about 15 ( 1 -hydroxymethylethylene hydroxymethylformal) (PHF ) , minutes , a temperature of about 22° C . , the absence of light ; 2 -methacryloyloxy - 2 '- ethyltrimethylammoniumphosphate and with stirring. (MPC ); and said carbohydrate moiety oxidized by incuba [0018 ] In still another embodiment, an aforementioned tion with a buffer comprising an oxidizing agent selected method is provided wherein the oxidizing agent is added in from the group consisting of sodium periodate (NalO4 ) , lead an amount to result in a final concentration between about 50 tetraacetate (Pb (OAc ) 4 ) and potassium perruthenate UM and about 1000 uM oxidizing agent, under conditions (KRu04 ) ; wherein an oxime linkage is formed between the comprising a time period between about 0 . 1 minutes and 120 oxidized carbohydrate moiety and the active aminooxy minutes ; a temperature between about 2° C . and about 37° group on the water soluble polymer ; and wherein in said C . , in the presence or absence of light; and with or without oxime linkage formation is catalyzed by a nucleophilic stirring . In another embodiment, the final concentration of catalyst selected from the group consisting of o - amino oxidizing agent is about 400 uM , and the conditions com benzoic acid , m - amino benzoic acid , p - amino benzoic acid , prise a time period of about 10 minutes , a temperature of sulfanilic acid , o -aminobenzamide , o - toluidine , m - toluidine , about 22° C . , the absence of light and with stirring . p -toluidine , o -anisidine , m - anisidine , and p -anisidine . [0019 ] In yet another embodiment, an aforementioned [0012 ] In still another embodiment, an aforementioned method is provided wherein the conjugating the water method is provided wherein a solution comprising an initial soluble polymer to the oxidized carbohydrate moiety of the concentration of the therapeutic protein between about 0 . 3 therapeutic protein is stopped by the addition of a quenching mg/ml and about 3 .0 mg/ml is adjusted to a pH value agent selected from the group consisting of L - cysteine , between about 5 . 0 and about 8 . 0 prior to contacting with the methionine , glutathione , glycerol, sodium meta bisulfite activated water soluble polymer. (Na2S205 ) , tryptophane , tyrosine , histidine or derivatives [ 0013 ]. As used herein , the term “ about” means a value thereof, kresol, imidazol, and combinations thereof; wherein above or below a stated value . In various embodiments , the the quenching agent is added in an amount to result in a final term “ about” includes the stated value plus or minus 0 . 1 , 0 . 2 , concentration between about 1 mM and about 100 mm 0 .3 , 0 . 4 , 0 .5 , 0 .6 , 0 .7 , 0 .8 , 0 . 9, 1, 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 or 10 % quenching agent, under conditions comprising a time period of the stated value . between about 5 minutes and about 120 minutes ; a tempera 0014 ] In yet another embodiment, an aforementioned ture between about 2° C . and about 37° C .; in the presence method is provided wherein the initial concentration of the or absence of light; and with or without stirring . In another therapeutic protein is about 1 . 0 mg/ ml and the pH is about embodiment, the quenching agent is L - cysteine . In still 6 . 0 . In a related embodiment, the initial concentration of the another embodiment, the L -cysteine is added to result in a therapeutic protein is about 0 . 75 mg/ ml and the pH is about final concentration of about 10 mM and the conditions 6 . 0 . In still another related embodiment, the initial concen comprise a time period of about 60 minutes , a temperature tration of the therapeutic protein is about 1 . 25 mg/ ml and the of about 22° C ., the absence of light and with stirring. [0020 ] In another embodiment, an aforementioned method pH is about 6 . 0 . is provided comprising : a ) a first step comprising adjusting [0015 ] In another embodiment, an aforementioned method the pH value of a solution comprising the therapeutic protein is provided wherein the therapeutic protein is contacted by to a pH value between about 5 . 0 and about 8 . 0 , wherein the a desired excess concentration of activated water soluble therapeutic protein concentration is between about 0 . 3 polymer , wherein the excess concentration is between about mg/ml and about 3 .0 mg/ml ; b ) a second step comprising 1 -molar and about 300 -molar excess . In another embodi oxidizing one or more carbohydrates on the therapeutic ment, the excess concentration is about 50 - fold molar protein , wherein the oxidizing agent is added to the solution excess . in the first step to result in a final concentration between [ 0016 ] In still another embodiment, an aforementioned about 50 uM and about 1000 uM , under conditions com method is provided wherein the therapeutic protein is incu - prising a time period between about 0 . 1 minutes and about bated with the activated water soluble polymer under con 120 minutes ; a temperature between about 2° C . and about ditions comprising a time period between about 0 . 5 hours 37° C . ; in the presence or absence of light, and with or and about 24 hours ; a temperature between about 2° C . and without stirring ; c ) a third step comprising contacting the about 37° C . ; in the presence or absence of light; and with therapeutic protein with a desired excess concentration of or without stirring. In another embodiment, the conditions activated water soluble polymer, wherein the excess con comprise a time period of about 120 minutes , a temperature centration is between about 1 -molar excess and about 300 of about 22° C . , the absence of light ; and with stirring . As molar excess , under conditions comprising a time period used herein , the term “ stirring ” is meant to include stirring between about 0 . 5 hours and about 24 hours , a temperature at various speeds and intensities ( e. g ., gentle stirring ) by between about 2° C . and about 37° C . ; in the presence or commonly used laboratory or manufacturing equipment and absence of light ; and with or without stirring ; d ) a fourth step products . comprising adding a nucleophilic catalyst to the solution of US 2017 /0240590 A1 Aug . 24 , 2017 the third step , wherein the nucleophilic catalyst is added to [ 0022 ] In still another embodiment , an aforementioned result in a final concentration between about 1 mM and method is provided wherein the therapeutic protein is FIX . about 50 mM , under conditions comprising a time period In another embodiment, the therapeutic protein is FVIIa . In between about 0 . 1 minutes and about 30 minutes ; a tem another embodiment, the therapeutic protein is FVIII. perature between about 2° C . and about 37° C . ; in the [0023 ] In yet another embodiment, an aforementioned presence or absence of light, and with or without stirring ; e ) method is provided wherein the oxidizing agent is sodium a fifth step wherein the therapeutic protein is incubated with periodate (NalO4 ) . the activated water soluble polymer and nucleophilic cata lyst under conditions that allow conjugation of the activated [0024 ]. In another embodiment, an aforementioned method water- soluble polymer to one or more oxidized carbohy is provided wherein the oxidized carbohydrate moiety of the drates on the therapeutic protein , said conditions comprising therapeutic protein is located in the activation peptide of the a time period between about 0 . 5 hours and about 24 hours , blood coagulation protein . a temperature between about 2° C . and about 37° C . ; in the [0025 ] In one embodiment, an aforementioned method is presence or absence of light, and with or without stirring ; provided wherein PSA is prepared by reacting an activated and f ) a sixth step wherein the conjugating the water soluble aminooxy linker with oxidized PSA ; wherein the aminooxy polymer to the one or more oxidized carbohydrates of the linker is selected from the group consisting of: therapeutic protein in the fifth step is stopped by the addition [0026 ] a ) a 3- oxa - pentane - 1, 5 -dioxyamine linker of the of a quenching agent selected from the group consisting of formula : L - cysteine , methionine , glutathione, glycerol, Na2S205 ( so dium meta bisulfite ) , tryptophane , tyrosine, histidine or derivatives thereof, kresol, imidazol, and combinations H2N thereof; wherein the quenching agent is added to result in a final concentration of about 1 mM and about 100 mM , under conditions comprising a time period between about 5 min utes and about 120 minutes ; a temperature between about 2° [0027 ] b ) a 3 , 6 , 9 - trioxa - undecane- 1 , 11 - dioxyamine C . and about 37° C . ; in the presence or absence of light , and linker of the formula : with or without stirring. In another embodiment, the initial concentration of the therapeutic protein in the first step is HN NH2 about 1 mg/ ml and the pH is about 6 . 0 ; wherein the final o vom NH2 concentration of oxidizing agent in the second step is about 400 uM , and the conditions in the fifth step comprise a time period of about 10 minutes , a temperature of about 22° C ., and the absence of light and with stirring ; wherein the excess [ 0028 ] c ) a 3 ,6 ,9 ,12 , 15 -penatoxa -heptadecane - 1, 17 -di concentration in the third step is about 50 molar excess ; oxyamine linker of the formula :

H2N nonoº-NH2 NH , wherein the conditions in the third step comprise a time [0029 ] wherein the PSA is oxidized by incubation with a period of about 15 minutes , a temperature of about 22° C ., oxidizing agent to form a terminal aldehyde group at the the absence of light and with stirring ; wherein the final non -reducing end of the PSA . In a related embodiment, the concentration of the nucleophilic catalyst in the fourth step aminooxy linker is 3 -oxa -pentane - 1 , 5 - dioxyamine . is about 10 mm , and the conditions in the fourth step [0030 ] In still another embodiment, an aforementioned comprise a time period of about 15 minutes , a temperature method is provided wherein the oxidizing agent is NaI04. of about 22° C . , the absence of light and with stirring ; 0031 ) In another embodiment, an aforementioned method wherein the conditions of incubating the therapeutic protein is provided wherein the nucleophilic catalyst is provided at with the activated water soluble polymer and nucleophilic a concentration between about 1 mM and about 50 mM . catalyst in the fifth step comprise a time period of about 2 [0032 ] In one embodiment, the nucleophilic catalyst is hours ; a temperature of about 22° C .; the absence of light; m - toluidine. In still another embodiment, the m - toluidine is and with stirring ; and wherein the quenching agent in the present in the conjugation reaction at a concentration of sixth step is L - cysteine ; and wherein the L - cysteine is added about 10 mM . to result in a final concentration of about 10 mM and the [0033 ] In yet another embodiment, an aforementioned conditions in the sixth step comprise a time period of about method is provided further comprising the step of reducing 60 minutes , a temperature of about 22° C ., the absence of an oxime linkage in the conjugated therapeutic protein by light and with stirring . incubating the conjugated therapeutic protein in a buffer [0021 ] In another embodiment, an aforementioned method comprising a reducing compound selected from the group is provided wherein the water soluble polymer is PSA . In consisting of sodium cyanoborohydride (NaCNBH3 ) , ascor another embodiment the PSA is comprised of about 10 - 300 bic acid (vitamin C ) and NaBH3. In one embodiment , the sialic acid units . In another embodiment, the water soluble reducing compound is sodium cyanoborohydride polymer is PEG . In another embodiment, the water soluble (NaCNBH3 ) . polymer is HES . In still another embodiment, the water [0034 ] In still another embodiment , an aforementioned soluble polymer is HAS . method is provided further comprising the step of purifying US 2017 /0240590 A1 Aug . 24 , 2017

the conjugated therapeutic protein . In another embodiment, [0041 ] In still another embodiment , a method of forming the conjugated therapeutic protein is purified by a method an oxime linkage between an oxidized carbohydrate moiety selected from the group consisting of chromatography, fil on a therapeutic protein and an activated water soluble tration and precipitation . In another embodiment, the chro polymer containing an active aminooxy group is provided matography is selected from the group consisting of Hydro comprising the steps of: a ) oxidizing a carbohydrate moiety phobic Interaction Chromatography (HIC ) , Ion Exchange on a therapeutic protein by incubating said protein with an chromatography (IEC ), Size exclusion chromatography oxidizing agent selected from the group consisting of ( SEC ) , Affinity chromatography, and Reversed - phase chro sodium periodate (NalO4 ) , lead tetraacetate ( Pb (OAc ) 4 ) and matography. In still another embodiment, an anti- chaotropic potassium perruthenate (KRu04 ); and b ) forming an oxime salt is used in a chromotagraphy loading step and in a linkage between the oxidized carbohydrate moiety of the chromatography washing step . In yet another embodiment, therapeutic protein and the activated water soluble polymer the chromatography takes place in a column. In another containing an active aminooxy group in the presence of a embodiment, the column comprises a chromatography resin nuclephilic catalyst under conditions allowing formation of selected from the group consisting of Phenyl- Sepharose FF said oxime linkage ; wherein said water soluble polymer and Butyl -Sepharose FF . In another embodiment , the resin is containing an active aminooxy group is selected from the present in the column at a bed height of between about 5 cm group consisting polyethylene glycol (PEG ) , branched PEG , and about 20 cm . In one embodiment, thebed height is about PolyPEG® (Warwick Effect Polymers; Coventry , UK ), 10 cm . polysialic acid (PSA ), starch , hydroxyalkyl starch ( HAS) , [ 0035 ] In another embodiment, an aforementioned method hydroxylethyl starch (HES ) , carbohydrate , polysaccharides , is provided comprising one or more washing steps wherein pullulane , chitosan , hyaluronic acid , chondroitin sulfate , flow direction is set to up - flow and wherein the flow rate is dermatan sulfate , starch , dextran , carboxymethyl -dextran , between about 0 . 2 cm / min and about 6 . 7 cm /min . As used polyalkylene oxide (PAO ), polyalkylene glycol (PAG ), herein , the term “ down - flow ” refers to a flow direction from polypropylene glycol (PPG ), polyoxazoline , polyacryloyl the top of the chromatographic column to the bottom of the morpholine , polyvinyl alcohol (PVA ), polycarboxylate , chromatographic column (normal flow direction / standard polyvinylpyrrolidone , polyphosphazene , polyoxazoline , mode ) . As used herein , the term “ up - flow ” refers to a flow polyethylene - co -maleic acid anhydride , polystyrene - co -ma direction from the bottom to the top of the column ( reversed leic acid anhydride, poly ( 1 -hydroxymethylethylene flow direction ). In one embodiment, the flow rate is about 2 hydroxymethylformal ) (PHF ) , 2 -methacryloyloxy - 2 '- ethylt cm /min . rimethylammoniumphosphate (MPC ) ; wherein the nucleo [ 0036 ] In another embodiment, an aforementioned method philic catalyst is selected from the group consisting of is provided comprising one or more elution steps wherein 0 - amino benzoic acid , m -amino benzoic acid , p - amino ben flow direction is set to down - flow and wherein the flow rate zoic acid , sulfanilic acid , c -aminobenzamide , o - toluidine , is between about 0 . 1 cm /min and about 6 . 7 cm /min . In a m - toluidine , p - toluidine, o - anisidine , m -anisidine , and related embodiment, the flow rate is about 1 cm /min . p -anisidine . [0037 ] In still another embodiment, an aforementioned [0042 ] In yet another embodiment, a method of forming method is provided comprising concentrating the conjugated an oxime linkage between an oxidized carbohydrate moiety therapeutic protein by ultra - / diafiltration (UF /DF ) . In on a therapeutic protein and an activated water soluble another embodiment, the final concentration of therapeutic polymer containing an active aminooxy group is provided protein is between about 0 . 5 and about 3 mg/ ml. comprising the steps of: a ) oxidizing a carbohydrate moiety [ 0038 ] In another embodiment, an aforementioned method on a therapeutic protein by incubating said protein with an is provided wherein the therapeutic protein comprises oxidinzing agent selected from the group consisting of sodium periodate (NalO4 ) , lead tetraacetate ( Pb (OAc ) 4 ) and between about 5 and about 11 water - soluble polymer moi potassium perruthenate (KRu04 ) ; and b ) forming an oxime eties . In another embodiment, the therapeutic protein com linkage between the oxidized carbohydrate moiety of the prises between about 1 and about 3 water - soluble polymers . therapeutic protein and the activated water soluble polymer [0039 ] In still another embodiment, an aforementioned containing an active aminooxy group in the presence of a method is provided wherein the conjugated therapeutic nuclephilic catalyst under conditions allowing formation of protein is purified using chromatography ; wherein an anti said oxime linkage ; wherein the therapeutic protein is chaotropic salt is used for a loading step and for a washing selected from the group consisting of Factor IX (FIX ) , step ; the method comprising one or more washing steps Factor VIII (FVIII ), Factor VIIa ( FVIIa ), Von Willebrand wherein flow direction is set to up - flow and wherein the flow Factor (VWF ) , Factor FV ( FV ) , Factor X ( FX ), Factor XI rate is between about 0 .2 cm /min and about 6 .7 cm /min and (FXI ) , Factor XII ( FXII) , thrombin (FII ), protein C , protein one or more elution steps wherein flow direction is set to S , DPA , PAI- 1 , tissue factor ( TF ) , ADAMTS 13 protease , down - flow and wherein the flow rate is between about 0 . 2 IL - 1 alpha , IL - 1 beta , IL - 2 , IL - 3 , IL - 4 , IL - 5 , IL - 6 , IL - 11, cm /min and about 6 . 7 cm /min ; further comprising concen colony stimulating factor -1 (CSF - 1 ), M -CSF , SCF, GM trating the conjugated therapeutic protein by ultra -/ diafiltra CSF , granulocyte colony stimulating factor ( G - CSF ), EPO , tion (UF /DF ) . In another embodiment, the chromatography interferon - alpha (IFN -alpha ), consensus interferon , IFN is hydrophobic interaction chromatography (HIC ) ; wherein beta , IFN - gamma, IFN - omega , IL - 7 , IL - 8 , IL - 9 , IL - 10 , the one or more washing steps flow rate is about 2 cm /min ; IL - 12 , IL - 13, IL - 14 , IL - 15 , IL - 16 , IL - 17 , IL - 18 , IL - 19 , and wherein the one or more elution steps flow rate is about IL - 20 , IL - 21 , IL -22 , IL -23 , IL - 24 , IL - 31, IL - 32 alpha , IL - 33 , 1 cm /min . thrombopoietin (TPO ) , Ang - 1 , Ang - 2 , Ang - 4 , Ang - Y , [0040 ] In another embodiment, a modified therapeutic angiopoietin - like polypeptide 1 ( ANGPTL1) , angiopoietin protein produced by any of the aforementioned methods is like polypeptide 2 ( ANGPTL2 ) , angiopoietin - like polypep provided . tide 3 ( ANGPTL3) , angiopoietin - like polypeptide 4 ( AN US 2017 /0240590 A1 Aug . 24 , 2017

GPTL4 ), angiopoietin - like polypeptide 5 ( ANGPTL5 ), IgM , IgA , and IgD , a - galactosidase, B - galactosidase , angiopoietin - like polypeptide 6 ( ANGPTL6 ) , angiopoietin DNAse , fetuin , leutinizing hormone, estrogen , insulin , albu like polypeptide 7 (ANGPTL7 ) , vitronectin , vascular min , lipoproteins , fetoprotein , transferrin , thrombopoietin , endothelial growth factor (VEGF ) , angiogenin , activin A , urokinase, integrin , thrombin , leptin , Humira (adalimumab ), activin B , activin C , bone morphogenic protein - 1 , bone Prolia (denosumab ) , Enbrel ( etanercept ) , a protein from morphogenic protein - 2 , bone morphogenic protein - 3 , bone Table 1 , or a biologically active fragment, derivative or morphogenic protein - 4 , bone morphogenic protein - 5 , bone variant thereof; wherein said water soluble polymer con taining an active aminooxy group is selected from the group morphogenic protein - 6 , bone morphogenic protein - 7 , bone consisting of polyethylene glycol (PEG ), branched PEG , morphogenic protein - 8 , bone morphogenic protein - 9 , bone PolyPEG® ( Warwick Effect Polymers ; Coventry , UK ) , morphogenic protein - 10 , bone morphogenic protein - 11, polysialic acid (PSA ), starch , hydroxyalkyl starch ( HAS) , bone morphogenic protein - 12 , bone morphogenic protein hydroxylethyl starch (HES ) , carbohydrate, polysaccharides , 13, bone morphogenic protein - 14 , bone morphogenic pro pullulane , chitosan , hyaluronic acid , chondroitin sulfate , tein - 15, bone morphogenic protein receptor IA , bone mor dermatan sulfate , starch , dextran , carboxymethyl -dextran , phogenic protein receptor IB , bone morphogenic protein polyalkylene oxide (PAO ), polyalkylene glycol (PAG ), receptor II, brain derived neurotrophic factor, cardiotrophin polypropylene glycol (PPG ) , polyoxazoline , polyacryloyl 1 , ciliary neutrophic factor, ciliary neutrophic factor recep morpholine , polyvinyl alcohol (PVA ) , polycarboxylate , tor, cripto , cryptic , cytokine -induced neutrophil chemotactic polyvinylpyrrolidone , polyphosphazene , polyoxazoline, factor 1 , cytokine - induced neutrophil , chemotactic factor polyethylene - co -maleic acid anhydride , polystyrene - co -ma 2a , cytokine - induced neutrophil chemotactic factor 23 , B endothelial cell growth factor, endothelin 1 , epidermal leic acid anhydride , poly ( 1- hydroxymethylethylene growth factor, epigen , epiregulin , epithelial -derived neutro hydroxymethylformal) (PHF ) , 2 -methacryloyloxy - 2 ' - ethylt phil attractant, fibroblast growth factor 4 , fibroblast growth rimethylammoniumphosphate (MPC ) ; wherein the nucleo factor 5 , fibroblast growth factor 6 , fibroblast growth factor philic catalyst is selected from the group consisting of 7 , fibroblast growth factor 8 , fibroblast growth factor 8b , 0 -amino benzoic acid , m - amino benzoic acid , p - amino ben fibroblast growth factor 8c , fibroblast growth factor 9 , zoic acid , sulfanilic acid , c -aminobenzamide , 0 - toluidine , fibroblast growth factor 10 , fibroblast growth factor 11 , m -toluidine , p - toluidine , o - anisidine, m - anisidine , and fibroblast growth factor 12 , fibroblast growth factor 13 , p - anisidine . fibroblast growth factor 16 , fibroblast growth factor 17 , [0043 ] In yet another embodiment , a method of forming a fibroblast growth factor 19 , fibroblast growth factor 20 , hydrazone linkage between an oxidized carbohydrate moi fibroblast growth factor 21, fibroblast growth factor acidic , ety on a therapeutic protein and an activated water soluble fibroblast growth factor basic , glial cell line- derived neutro polymer containing an active hydrazide group is provided phic factor receptor al, glial cell line - derived neutrophic comprising the steps of: a ) oxidizing a carbohydrate moiety factor receptor a2 , growth related protein , growth related on a therapeutic protein by incubating said protein with an protein a , growth related protein B , growth related protein y , oxidinzing agent selected from the group consisting of heparin binding epidermal growth factor, hepatocyte growth sodium periodate (NalO4 ) , lead tetraacetate (Pb (OAC ) 4 ) and factor, hepatocyte growth factor receptor , hepatoma- derived potassium perruthenate (KRU04 ) ; and b ) forming a hydra growth factor, insulin - like growth factor 1, insulin - like zone linkage between the oxidized carbohydrate moiety of growth factor receptor, insulin -like growth factor II , insulin the therapeutic protein and the activated water soluble like growth factor binding protein , keratinocyte growth polymer containing an active hydrazide group in the pres factor, leukemia inhibitory factor, leukemia inhibitory factor ence of a nuclephilic catalyst under conditions allowing receptor a , nerve growth factor nerve growth factor recep formation of said hydrazone linkage ; wherein said water tor, neuropoietin , neurotrophin - 3 , neurotrophin - 4 , oncosta soluble polymer containing an active hydrazide group is tin M (OSM ) , placenta growth factor, placenta growth factor selected from the group consisting of polyethylene glycol 2 , platelet - derived endothelial cell growth factor, platelet (PEG ) , branched PEG , PolyPEG (Warwick Effect Poly derived growth factor, platelet derived growth factor A mers ; Coventry , UK ), polysialic acid (PSA ), starch , chain , platelet derived growth factor AA , platelet derived hydroxyalkyl starch (HAS ), hydroxylethyl starch (HES ), growth factor AB , platelet derived growth factor B chain , carbohydrate , polysaccharides, pullulane , chitosan , platelet derived growth factor BB , platelet derived growth hyaluronic acid , chondroitin sulfate , dermatan sulfate , factor receptor a , platelet derived growth factor receptor ß , starch , dextran , carboxymethyl- dextran , polyalkylene oxide pre - B cell growth stimulating factor, stem cell factor ( SCF ) , (PAO ) , polyalkylene glycol (PAG ) , polypropylene glycol stem cell factor receptor, TNF, TNFO , TNF1, TNF2, trans (PPG ), polyoxazoline, polyacryloylmorpholine , polyvinyl forming growth factor a , transforming growth factor B , alcohol (PVA ) , polycarboxylate , polyvinylpyrrolidone , transforming growth factor B1, transforming growth factor polyphosphazene , polyoxazoline , polyethylene- co -maleic B1 . 2 , transforming growth factor B2 , transforming growth acid anhydride , polystyrene - co -maleic acid anhydride, poly factor B3 , transforming growth factor B5 , latent transform ( 1- hydroxymethylethylene hydroxymethylformal ) (PHF ), ing growth factor B1 , transforming growth factor ß binding 2 -methacryloyloxy - 2 '- ethyltrimethylammoniumphosphate protein I, transforming growth factor ß binding protein II , (MPC ) ; wherein the nucleophilic catalyst is selected from transforming growth factor B binding protein III , thymic the group consisting of o - amino benzoic acid , m - amino stromal lymphopoietin ( TSLP ) , tumor necrosis factor recep benzoic acid , p - amino benzoic acid , sulfanilic acid , o - amin tor type I , tumor necrosis factor receptor type II , urokinase obenzamide, o - toluidine, m - toluidine , p - toluidine , o - ani type plasminogen activator receptor , phospholipase - activat sidine , m - anisidine , and p - anisidine. ing protein (PUP ) , insulin , lectin ricin , prolactin , chorionic [0044 ] In another embodiment, a method of forming a gonadotropin , follicle - stimulating hormone, thyroid - stimu - hydrazone linkage between an oxidized carbohydrate moi lating hormone , tissue plasminogen activator , IgG , IgE , ety on a therapeutic protein and an activated water soluble US 2017 /0240590 A1 Aug . 24 , 2017

polymer containing an active hydrazide group comprising growth factor receptor, insulin - like growth factor II, insulin the steps of: a ) oxidizing a carbohydrate moiety on a like growth factor binding protein , keratinocyte growth therapeutic protein by incubating said protein with an oxid factor, leukemia inhibitory factor, leukemia inhibitory factor inzing agent selected from the group consisting of sodium receptor a , nerve growth factor nerve growth factor recep periodate (NalO4 ) , lead tetraacetate (Pb ( OAC) 4 ) and potas tor, neuropoietin , neurotrophin - 3 , neurotrophin - 4 , oncosta sium perruthenate (KRu04 ) ; and b ) forming a hydrazone tin M (OSM ) , placenta growth factor, placenta growth factor linkage between the oxidized carbohydrate moiety of the 2 , platelet - derived endothelial cell growth factor, platelet therapeutic protein and the activated water soluble polymer derived growth factor, platelet derived growth factor A containing an active hydrazide group in the presence of a chain , platelet derived growth factor AA , platelet derived nuclephilic catalyst under conditions allowing formation of growth factor AB , platelet derived growth factor B chain , said hydrazone linkage ; wherein the therapeutic protein is platelet derived growth factor BB , platelet derived growth selected from the group consisting of Factor IX (FIX ), factor receptor a , platelet derived growth factor receptor ß , Factor VIII ( FVIII ) , Factor VIIa (FVIIa ), Von Willebrand pre - B cell growth stimulating factor, stem cell factor (SCF ) , Factor ( VWF ), Factor FV (FV ) , Factor X ( FX ) , Factor XI stem cell factor receptor , TNF , TNFO , TNF1, TNF2, trans (FXI ), Factor XII (FXII ), thrombin (FII ), protein C , protein forming growth factor a , transforming growth factor B , S , tPA , PAI- 1 , tissue factor ( TF ) , ADAMTS 13 protease , transforming growth factor B1, transforming growth factor IL - 1 alpha , IL - 1 beta , IL - 2 , IL - 3 , IL - 4 , IL - 5 , IL - 6 , IL - 11 , 31. 2 , transforming growth factor B2 , transforming growth colony stimulating factor - 1 (CSF - 1) , M -CSF , SCF , GM factor B3 , transforming growth factor B5 , latent transform CSF , granulocyte colony stimulating factor ( G - CSF ), EPO , ing growth factor B1, transforming growth factor ß binding interferon - alpha ( IFN - alpha ) , consensus interferon , IFN protein I , transforming growth factor ß binding protein II , beta , IFN - gamma, IFN -omega , IL - 7 , IL - 8 , IL - 9 , IL - 10 , transforming growth factor ß binding protein III, thymic IL - 12 , IL - 13 , IL - 14 , IL - 15 , IL - 16 , IL - 17 , IL - 18 , IL - 19 , stromal lymphopoietin ( TSLP ) , tumor necrosis factor recep IL - 20 , IL -21 , IL -22 , IL - 23, IL -24 , IL -31 , IL -32 alpha, IL - 33, tor type I , tumor necrosis factor receptor type II , urokinase thrombopoietin ( TPO ) , Ang - 1 , Ang - 2 , Ang- 4 , Ang - Y , type plasminogen activator receptor, phospholipase -activat angiopoietin - like polypeptide 1 ( ANGPTL1 ) , angiopoietin ing protein (PUP ), insulin , lectin ricin , prolactin , chorionic like polypeptide 2 (ANGPTL2 ), angiopoietin - like polypep gonadotropin , follicle - stimulating hormone , thyroid - stimu tide 3 (ANGPTL3 ) , angiopoietin - like polypeptide 4 (AN lating hormone , tissue plasminogen activator, IgG , IgE , GPTL4 ) , angiopoietin - like polypeptide 5 ( ANGPTL5 ) , IgM , IgA , and IgD , A - galactosidase, B - galactosidase , angiopoietin - like polypeptide 6 ( ANGPTL6 ), angiopoietin DNAse , fetuin , leutinizing hormone , estrogen , insulin , albu like polypeptide 7 (ANGPTL7 ) , vitronectin , vascular min , lipoproteins, fetoprotein , transferrin , thrombopoietin , endothelial growth factor (VEGF ) , angiogenin , activin A , urokinase , integrin , thrombin , leptin , Humira (adalimumab ) , activin B , activin C , bone morphogenic protein - 1 , bone Prolia ( denosumab ) , Enbrel ( etanercept) , a protein from morphogenic protein -2 , bone morphogenic protein -3 , bone Table 1 , or a biologically active fragment, derivative or morphogenic protein - 4 , bone morphogenic protein - 5 , bone variant thereof; wherein said water soluble polymer con morphogenic protein - 6 , bone morphogenic protein - 7 , bone taining an active hydrazide group is selected from the group morphogenic protein - 8 , bone morphogenic protein - 9 , bone consisting of polyethylene glycol (PEG ) , branched PEG , morphogenic protein - 10 , bone morphogenic protein - 11 , PolyPEG® (Warwick Effect Polymers; Coventry , UK ), bone morphogenic protein - 12 , bone morphogenic protein polysialic acid (PSA ), starch , hydroxyalkyl starch ( HAS) , 13 , bone morphogenic protein - 14 , bone morphogenic pro hydroxylethyl starch (HES ), carbohydrate , polysaccharides , tein - 15 , bone morphogenic protein receptor IA , bone mor pullulane, chitosan , hyaluronic acid , chondroitin sulfate , phogenic protein receptor IB , bone morphogenic protein dermatan sulfate , starch , dextran , carboxymethyl -dextran , receptor II , brain derived neurotrophic factor, cardiotrophin polyalkylene oxide (PAO ), polyalkylene glycol (PAG ), 1 , ciliary neutrophic factor, ciliary neutrophic factor recep polypropylene glycol (PPG ) , polyoxazoline , polyacryloyl tor, cripto , cryptic , cytokine - induced neutrophil chemotactic morpholine , polyvinyl alcohol (PVA ) , polycarboxylate , factor 1 , cytokine - induced neutrophil, chemotactic factor polyvinylpyrrolidone, polyphosphazene , polyoxazoline , 2a , cytokine - induced neutrophil chemotactic factor 2B , B polyethylene -co -maleic acid anhydride , polystyrene - co -ma endothelial cell growth factor, endothelin 1 , epidermal leic acid anhydride , poly ( 1 - hydroxymethylethylene growth factor, epigen , epiregulin , epithelial -derived neutro hydroxymethylformal ) (PHF ) , 2 -methacryloyloxy - 2' - ethylt phil attractant, fibroblast growth factor 4 , fibroblast growth rimethylammoniumphosphate (MPC ) ; wherein the nucleo factor 5 , fibroblast growth factor 6 , fibroblast growth factor philic catalyst is selected from the group consisting of 7 , fibroblast growth factor 8 , fibroblast growth factor 8b , 0 -amino benzoic acid , m -amino benzoic acid , p -amino ben fibroblast growth factor 8c , fibroblast growth factor 9 , zoic acid , sulfanilic acid , o - aminobenzamide , o - toluidine , fibroblast growth factor 10 , fibroblast growth factor 11 , m - toluidine , p -toluidine , o -anisidine , m -anisidine , and fibroblast growth factor 12 , fibroblast growth factor 13 , p -anisidine . fibroblast growth factor 16 , fibroblast growth factor 17 , [0045 ] In another embodiment, an aforementioned method fibroblast growth factor 19 , fibroblast growth factor 20 , is provided wherein the water soluble polymer containing an fibroblast growth factor 21 , fibroblast growth factor acidic , active aminooxy group is prepared by a method comprising : fibroblast growth factor basic , glial cell line -derived neutro incubating a solution comprising an oxidized water- soluble phic factor receptor al, glial cell line - derived neutrophic polymer with an activated aminooxy linker comprising an factor receptor a2 , growth related protein , growth related active aminooxy group under conditions that allow the protein a , growth related protein ß , growth related protein y , formation of a stable oxime linkage between the oxidized heparin binding epidermal growth factor, hepatocyte growth water- soluble polymer and the activated aminooxy linker , factor, hepatocyte growth factor receptor, hepatoma - derived said conditions comprising a time period between about 1 growth factor, insulin - like growth factor 1 , insulin - like minute and about 24 hours; a temperature between about 2° US 2017 /0240590 A1 Aug . 24 , 2017

C . and about 37° C . , in the presence or absence of light, and vated aminooxy linker, said conditions comprising a time with or without stirring ; thereby forming a water soluble period between about 1 minute and about 24 hours; a polymer containing an active aminooxy group ; and b ) puri temperature between about 2° C . and about 37° C .; in the fying the water soluble polymer containing an active ami presence or absence of light, and with or without stirring ; nooxy group by a method selected from the group consisting thereby forming a water soluble polymer containing an of chromatography, filtration and precipitation . The term active aminooxy group ; b ) incubating a solution comprising " activated water- soluble polymer” referes, in one embodi the water soluble polymer containing an active aminooxy ment, to a water -soluble polyer containing an aldehyde group of step a ) with a nucleophilic catalyst under condi group . tions comprising a time period between 1 minute and 24 [0046 ] In yet another embodiment, an aforementioned hours; a temperature between 2° C . and 37° C . ; in the method is provided wherein the water soluble polymer presence or absence of light; and with or without stirring ; c ) containing an active aminooxy group is prepared by a incubating a solution comprising the water soluble polymer method comprising : a ) incubating a solution comprising an containing an active aminooxy group of step b ) with a oxidized water- soluble polymer with an activated aminooxy reducing agent under conditions that allow the formation of linker comprising an active aminooxy group under condi a stable alkoxamine linkage between the oxidized water tions that allow the formation of a stable oxime linkage soluble polymer and the activated aminooxy linker, said between the oxidized water -soluble polymer and the acti conditions comprising a time period between about 1 minute vated aminooxy linker, said conditions comprising a time and about 24 hours; a temperature between about 2° C . and period between about 1 minute and about 24 hours ; a about 37° C . , in the presence or absence of light; and with temperature between about 2° C . and about 37° C .; in the or without stirring ; and d ) purifying the water soluble presence or absence of light, and with or without stirring ; polymer containing an active aminooxy group by a method thereby forming a water soluble polymer containing an selected from the group consisting of chromatography, fil active aminooxy group ; b ) incubating a solution comprising tration and precipitation . the water soluble polymer containing an active aminooxy [0049 ] In another embodiment, an aforementioned method group of step a ) with a reducing agent under conditions that is provided wherein the oxidized water soluble polymer is allow the formation of a stable alkoxamine linkage between selected from the group consisting of polyethylene glycol the oxidized water -soluble polymer and the activated ami (PEG ) , branched PEG , PolyPEG® (Warwick Effect Poly nooxy linker, said conditions comprising a time period mers ; Coventry , UK ), polysialic acid (PSA ), starch , between about 1 minute and about 24 hours ; a temperature hydroxyalkyl starch (HAS ), hydroxylethyl starch (HES ) , between about 2° C . and about 37° C .; in the presence or carbohydrate, polysaccharides , pullulane , chitosan , absence of light; and with or without stirring ; and c ) puri hyaluronic acid , chondroitin sulfate , dermatan sulfate , fying the water soluble polymer containing an active ami starch , dextran , carboxymethyl- dextran , polyalkylene oxide nooxy group by a method selected from the group consisting (PAO ) , polyalkylene glycol ( PAG ) , polypropylene glycol of chromatography, filtration and precipitation . (PPG ) , polyoxazoline, polyacryloylmorpholine , polyvinyl [0047 ] In still another embodiment , an aforementioned alcohol (PVA ) , polycarboxylate , polyvinylpyrrolidone , method is provided wherein the water soluble polymer polyphosphazene, polyoxazoline, polyethylene -co -maleic containing an active aminooxy group is prepared by a acid anhydride , polystyrene - co -maleic acid anhydride , poly method comprising : a ) incubating a solution comprising an ( 1 -hydroxymethylethylene hydroxymethylformal ) (PHF ) , oxidized water- soluble polymer with an activated aminooxy 2 -methacryloyloxy - 2 ' - ethyltrimethylammoniumphosphate linker comprising an active aminooxy group under condi (MPC ) , and wherein said water -soluble polymer is oxidized tions that allow the formation of a stable oxime linkage by incubation with a oxidizing agent to form a terminal between the oxidized water- soluble polymer and the acti aldehyde group at the non - reducing end of the water - soluble vated aminooxy linker, said conditions comprising a time polymer. In one embodiment, the water - soluble polymer is period between about 1 minute and about 24 hours ; a PSA . temperature between about 2° C . and about 37° C . ; in the [0050 ] In another embodiment, an aforementioned method presence or absence of light, and with or without stirring ; is provided wherein the oxidizing agent is NalO4 . thereby forming a water soluble polymer containing an [0051 ] In still another embodiment, an aforementioned active aminooxy group; b ) incubating a solution comprising method is provided wherein the aminooxy linker is selected the water soluble polymer containing an active aminooxy from the group consisting of: group of step a ) with a nucleophilic catalyst under condi [ 0052 ] a ) a 3 -oxa -pentane - 1 , 5 -dioxyamine linker of the tions comprising a time period between 1 minute and 24 formula : hours ; a temperature 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 HNH2N 0 a NH . active aminooxy group by a method selected from the group consisting of chromatography, filtration and precipitation . [0048 ] In yet another embodiment, an aforementioned [0053 ] b ) a 3 ,6 , 9 -trioxa -undecane -1 ,11 -dioxyamine method is provided wherein the water soluble polymer linker of the formula : containing an active aminooxy group is prepared by a method comprising : a ) incubating a solution comprising an oxidized water- soluble polymer with an activated aminooxy H N . NH, linker comprising an active aminooxy group under condi tions that allow the formation of a stable oxime linkage between the oxidized water- soluble polymer and the acti US 2017 /0240590 A1 Aug . 24 , 2017 and [0054 ] c ) a 3 ,6 ,9 ,12 , 15 -penatoxa -heptadecane - 1, 17 - di oxyamine linker of the formula :

HN NH2

[ 0055 ] In yet another embodiment, an aforementioned FIGURES method is provided wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride [0062 ] FIG . 1 shows the primary structure of coagulation (NaCNBH3 ) , ascorbic acid ( vitamin C ) and NaBH3. In one Factor IX ( SEQ ID NO : 1 ) . embodiment, the reducing agent is sodium cyanoborohy [ 0063 ] FIG . 2 shows the coupling of oxidized rFIX to dride (NaCNBH3 ) . aminooxy - PSA . [0056 ] In another embodiment, an aforementioned method [0064 ] FIG . 3 shows the synthesis of the water soluble is provided wherein the nucleophilic catalyst is selected di - aminoxy linkers 3 -oxa - pentane - 1 , 5 - dioxyamine and 3 , 6 , from the group consisting of o - amino benzoic acid , m - amino 9 - trioxa -undecane - 1 , 11- dioxyamine . benzoic acid , p - amino benzoic acid , sulfanilic acid , o -amin [0065 ] FIG . 4 shows the preparation of aminooxy - PSA . obenzamide, o -toluidine , m - toluidine, p - toluidine, o - ani [0066 ] FIG . 5 shows the visualization of PSA - FIX conju sidine , m -anisidine , and p -anisidine . In one embodiment, the gates prepared in the presence of different catalysts by SDS nucleophilic catalyst is m - toluidine . In another embodiment, PAGE . a ) Comparison of aniline with m - toluidine using the nucleophilic catalyst is added in an amount to result in different concentrations ; b ) Comparison of aniline with a final concentration between about 1 . 0 mM and about 50 0 -aminobenzoic acid , m - aminobenzoic acid , p -aminoben mM nucleophilic catalyst . zoic acid , p - aminobenzamide and sulfanilic acid ; c ) Com [0057 ] In another embodiment, an aforementioned method parison of aniline and m - toluidine with 0 - anisidine and is provided further comprising concentrating the conjugated m - anisidine . therapeutic protein by ultra - / diafiltration (UF / DF ) . [0067 ] FIG . 6 shows percent of polysialylation with vari [ 0058 ] In another embodiment, a method of conjugating a ous nucleophilic catalysts . water soluble polymer to an oxidized carbohydrate moiety of a blood coagulation protein is provided comprising con DETAILED DESCRIPTION OF THE tacting the oxidized carbohydrate moiety with an activated INVENTION water soluble polymer under conditions that allow conjuga [0068 ] The pharmacological and immunological proper tion ; ties of therapeutic proteins can be improved by chemical [ 0059 ] said blood coagulation protein selected from the modification and conjugation with polymeric compounds group consisting of Factor IX ( FIX ) , Factor VIII (FVIII ) , such as polyethylene glycol (PEG ) , branched PEG , poly Factor VIIa (FVIIa ) , Von Willebrand Factor ( VWF ) , Factor sialic acid ( PSA ) , hydroxyalkyl starch (HAS ) , hydroxylethyl FV (FV ) , Factor X ( FX ) , Factor XI (FXI ) , Factor XII (FXII ) , starch (HES ) , carbohydrate , polysaccharides , pullulane , chi thrombin ( FII ) , protein C , protein S , PA , PAI- 1, tissue factor tosan , hyaluronic acid , chondroitin sulfate , dermatan sulfate , ( TF ) and ADAMTS 13 protease or a biologically active starch , dextran , carboxymethyl- dextran , polyalkylene oxide ( PAO ), polyalkylene glycol (PAG ) , polypropylene glycol fragment, derivative or variant thereof; (PPG ) , polyoxazoline , polyacryloylmorpholine , polyvinyl [0060 ] said water soluble polymer containing an active alcohol (PVA ) , polycarboxylate , polyvinylpyrrolidone , aminooxy group and is selected from the group consisting of polyphosphazene , polyoxazoline , polyethylene - co -maleic polyethylene glycol (PEG ) , branched PEG , polysialic acid acid anhydride , polystyrene -co -maleic acid anhydride, poly (PSA ), carbohydrate , polysaccharides , pullulane, chitosan , ( 1 -hydroxymethylethylene hydroxymethylformal ) ( PHF ) , hyaluronic acid , chondroitin sulfate , dermatan sulfate , 2 -methacryloyloxy - 2 ' - ethyltrimethylammoniumphosphate starch , dextran , carboxymethyl- dextran , polyalkylene oxide (MPC ) . The properties of the resulting conjugates generally (PAO ) , polyalkylene glycol (PAG ) , polypropylene glycol strongly depend on the structure and the size of the polymer . ( PPG ) , polyoxazoline , polyacryloylmorpholine , polyvinyl Thus , polymers with a defined and narrow size distribution alcohol (PVA ) , polycarboxylate , polyvinylpyrrolidone , are usually preferred in the art . Synthetic polymers like PEG polyphosphazene, polyoxazoline, polyethylene - co -maleic can be manufactured easily with a narrow size distribution , acid anhydride, polystyrene - co -maleic acid anhydride, poly while PSA can be purified in such a manner that results in ( 1- hydroxymethylethylene hydroxymethylformal) (PHF ), a final PSA preparation with a narrow size distribution . In 2 -methacryloyloxy - 2 ' - ethyltrimethylammoniumphosphate addition PEGylation reagents with defined polymer chains (MPC ) ; and and narrow size distribution are on the market and commer [0061 ] said carbohydrate moiety oxidized by incubation cially available for a reasonable price . with a buffer comprising an oxidizing agent selected from [0069 ] The addition of a soluble polymer , such as through the group consisting of sodium periodate (NalO4 ) , lead polysialylation , is one approach to improve the properties of tetraacetate (Pb (OAc ) 4 ) and potassium perruthenate therapeutic proteins such as the blood coagulation protein (KRu04 ); wherein an oxime linkage is formed between the FIX , as well as other coagulation proteins (e .g . , VWF, FVIIa oxidized carbohydrate moiety and the active aminooxy ( see , e . g . , US 2008 /0221032A1 , incorporated herein by group on the water soluble polymer. reference ) and FVIII ). US 2017 /0240590 A1 Aug . 24 , 2017 10

Therapeutic Proteins receptor al, glial cell line - derived neutrophic factor receptor a2 , growth related protein , growth related protein a , growth [0070 ] In certain embodiments of the invention , the afore related protein ß , growth related protein y , heparin binding mentioned polypeptides and polynucleotides are exempli epidermal growth factor, hepatocyte growth factor, hepato fied by the following therapeutic proteins: enzymes, anti cyte growth factor receptor, hepatoma- derived growth fac gens, antibodies , receptors , blood coagulation proteins , tor, insulin - like growth factor I, insulin - like growth factor growth factors , hormones , and ligands . In certain embodi receptor, insulin - like growth factor II , insulin - like growth ments, the therapeutic protein is a blood coagulation protein factor binding protein , keratinocyte growth factor, leukemia such as Factor IX (FIX ) , Factor VIII (FVIII ) , Factor VIIa inhibitory factor, leukemia inhibitory factor receptor a , ( FVIIa ), Von Willebrand Factor (VWF ) , Factor FV ( FV ) , nerve growth factor nerve growth factor receptor, neuropoi Factor X (FX ) , Factor XI ( FXI) , Factor XII (FXII ) , thrombin etin , neurotrophin - 3 , neurotrophin - 4 , oncostatin M ( OSM ) , ( FII) , protein C , protein S , DPA , PAI- 1 , tissue factor ( TF ) or placenta growth factor, placenta growth factor 2 , platelet ADAMTS 13 protease . In one embodiment, a therapeutic derived endothelial cell growth factor, platelet derived protein according to the invention is a glycoprotein or, in growth factor, platelet derived growth factor A chain , plate various embodiments, a protein that is not naturally glyco let derived growth factor AA , platelet derived growth factor sylated in vivo ( i . e . , a protein that does not contain a natural AB , platelet derived growth factor B chain , platelet derived glycosylation site or a protein that is not glycosylated in a growth factor BB , platelet derived growth factor receptor a , host cell prior to purification ) . platelet derived growth factor receptor B , pre - B cell growth 10071] In certain embodiments, the therapeutic protein is stimulating factor , stem cell factor ( SCF ) , stem cell factor immunoglobulins , cytokines such IL - 1 alpha , IL - 1 beta , receptor, TNF, including TNFO , TNF1 , TNF2 , transforming IL - 2 , IL - 3 , IL - 4 , IL - 5 , IL -6 , IL - 11, colony stimulating growth factor a , transforming growth factor ß , transforming factor- 1 (CSF - 1 ) , M -CSF , SCF, GM - CSF , granulocyte growth factor B1 , transforming growth factor $ 1 . 2 , trans colony stimulating factor ( G - CSF ) , EPO , interferon -alpha forming growth factor B2 , transforming growth factor B3 , ( IFN - alpha ) , consensus interferon , IFN -beta , IFN - gamma, transforming growth factor B5, latent transforming growth IFN -omega , IL - 7 , IL - 8 , IL - 9, IL - 10 , IL - 12 , IL - 13 , IL - 14 , factor B1 , transforming growth factor ß binding protein I , IL - 15 , IL - 16 , IL - 17 , IL - 18 , IL - 19 , IL - 20 , IL - 21 , IL - 22 , transforming growth factor ß binding protein II , transform IL - 23 , IL - 24 , IL -31 , IL -32 alpha , IL - 33 , thrombopoietin ing growth factor ß binding protein III , thymic stromal ( TPO ) , angiopoietins , for example Ang - 1 , Ang - 2 , Ang - 4 , lymphopoietin ( TSLP ), tumor necrosis factor receptor type Ang - Y , the human angiopoietin - like polypeptides I , tumor necrosis factor receptor type II , urokinase - type ANGPTL1 through 7 , vitronectin , vascular endothelial plasminogen activator receptor, vascular endothelial growth growth factor (VEGF ) , angiogenin , activin A , activin B , factor, and chimeric proteins and biologically or immuno activin C , bone morphogenic protein - 1 , bone morphogenic logically active fragments thereof. protein - 2 , bone morphogenic protein - 3 , bone morphogenic [0072 ] In certain embodiments , the therapeutic protin is protein - 4 , bone morphogenic protein - 5 , bone morphogenic alpha - , beta - , and gamma - interferons, colony stimulating protein - 6 , bone morphogenic protein - 7 , bone morphogenic factors including granulocyte colony stimulating factors , protein - 8 , bone morphogenic protein - 9 , bone morphogenic fibroblast growth factors , platelet derived growth factors , protein - 10 , bone morphogenic protein - 11 , bone morpho phospholipase - activating protein ( PUP ) , insulin , plant pro genic protein - 12 , bone morphogenic protein - 13 , bone mor teins such as lectins and ricins , tumor necrosis factors and phogenic protein - 14 , bone morphogenic protein - 15 , bone related alleles, soluble forms of tumor necrosis factor recep morphogenic protein receptor IA , bonemorphogenic protein tors , interleukin receptors and soluble forms of interleukin receptor IB , bone morphogenic protein receptor II, brain receptors , growth factors such as tissue growth factors , such derived neurotrophic factor, cardiotrophin - 1 , ciliary neutro as TGFas or TGFBs and epidermal growth factors , hor phic factor, ciliary neutrophic factor receptor, cripto , cryptic , mones, somatomedins, pigmentary hormones, hypothalamic cytokine - induced neutrophil chemotactic factor 1, cytokine releasing factors , antidiuretic hormones , prolactin , chorionic induced neutrophil , chemotactic factor 2a , cytokine - in gonadotropin , follicle - stimulating hormone , thyroid - stimu duced neutrophil chemotactic factor 2ß , ß endothelial cell lating hormone , tissue plasminogen activator , and immuno growth factor, endothelin 1 , epidermal growth factor, epi globulins such as IgG , IgE , IgM , IgA , and IgD , a galacto gen , epiregulin , epithelial - derived neutrophil attractant, sidase, a - galactosidase , B - galactosidase , DNAse , fetuin , fibroblast growth factor 4 , fibroblast growth factor 5 , fibro leutinizing hormone , estrogen , corticosteroids , insulin , albu blast growth factor 6 , fibroblast growth factor 7 , fibroblast min , lipoproteins, fetoprotein , transferrin , thrombopoietin , growth factor 8 , fibroblast growth factor 8b , fibroblast urokinase , DNase , integrins , thrombin , hematopoietic growth factor 8c , fibroblast growth factor 9 , fibroblast growth actors , leptin , glycosidases, Humira ( adalimumab ) , growth factor 10 , fibroblast growth factor 11, fibroblast Prolia (denosumab ), Enbrel ( etanercept) , and fragments growth factor 12 , fibroblast growth factor 13 , fibroblast thereof, or any fusion proteins comprising any of the above growth factor 16 , fibroblast growth factor 17 , fibroblast mentioned proteins or fragments thereof. In addition to the growth factor 19, fibroblast growth factor 20 , fibroblast aforementioned proteins, the following Table 1 provides growth factor 21, fibroblast growth factor acidic , fibroblast therapeutic proteins contemplated by the present invention : growth factor basic , glial cell line - derived neutrophic factor US 2017 /0240590 A1 Aug . 24 , 2017

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 2017 /0240590 A1 Aug . 24 , 2017

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 Ribonuclease8 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 Group10secretoryphospholipaseA2GroupIIDsecretoryphospholipaseA2 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 Secretogranin-2 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 Glycoproteinhormonesalphachain Hepatocytegrowthfactor-likeprotein lymphocytegrowthfactor;cellStem Fibroblastgrowthfactor10 epitheliumnasalLongpalatelungand, carcinoma-associatedprotein1 Interferonalpha-1/13 Interferon-inducedhelicaseCdomain CMP-Nacetylneuraminatebeta Dipeptidylpeptidase4 Ephrintype-Areceptor3 Ephrintype-Breceptor6 CoagulationfactorX CoagulationfactorVIII Fibrinogenalphachain containingprotein1Interferonalpha-2 Insulin-likegrowthfactorIB secretedC-typelectin Endothelin-1 relatedprotein7 Alpha-2HSglycoprotein Fibrinogenbetachain N-acetylglucosamine1 IndianhedgehogproteinNeuralcelladhesionmolecule Interleukin-13 Interleukin-2 B1Ephrin- EMILIN-1 protein1 Fibrillin-2 GranzymeA Interferonbeta Interferongamma ProteinCYR61 protein Dentinsialophosphoprotein protein Endoplasmin Gastrin member10D L1-likeprotein

Membranemetallo-endopeptidaselike1 Carcinoembryonicantigen-relatedcell Pulmonarysurfactant-associatedproteinA1 Gammaaminobutyric-acidtypeBreceptor Glycoproteinhormonealpha-2 Epithelialdiscoidindomain-containing VascularendothelialgrowthfactorA kexinsubtilisinconvertase/Proprotein stimulatingfactorreceptorsubunitalpha Granulocytecolony-stimulatingfactor Cholinetransporter-likeprotein4 CMP-Nacetylneuraminatebeta1,4 galactosidealpha-2,3sialyltransferase Tumornecrosisfactorreceptorsuperfamily Pro-neuregulin1,membranebound chainXI)-2(alphaCollagenGranulocytemacrophage -colony Interleukin-15receptorsubunitalpha Peptidyl-glycinealphaamidating Tumornecrosisfactorreceptorsuperfamily C-typelectindomainfamily4memberM C-typelectindomainfamily7memberA AmyloidbetaA4protein C-motifchemokine4like Apolipoprotein-IA Brain-derivedneurotrophicfactor Insulin-likegrowthfactorII CMRF35-likemolecule1 likeAreceptor-Fc Integrinalpha-7 Proteoglycan4 Prostate-associatedmicroseminoprotein Alpha-amylase1 adhesionmolecule1 activatorplasminogenTissue-type member6 subunit1 receptor1 Mucin-1 Fibulin-1 Prolactinreceptor CD209antigen Mucin-4 member25 Spermineoxidase isoform type6 Elastin Midkine monooxygenase Attractin receptor Kallikrein US 2017 /0240590 A1 Aug . 24 , 2017

Histidinetriadnucleotide-bindingprotein2 Abhydrolasedomain-containingprotein15 Nuclearporecomplex-interactingprotein Proactivatorpolypeptide-like1 Methyltransferase-likeprotein7B AlkylatedDNArepairproteinalkB Insulingrowthfactor-likefamilymember3Nuclearporecomplex-interactingprotein PutativeuncharacterizedproteinC10orf31 PutativeuncharacterizedproteinCllorf45 Hepatocellularcarcinoma-associated specificprotein18endocrineRegulated-ComplementClqtumornecrosisfactor Proteinspinsterhomolog2vonWillebrandfactorCdomain containingprotein2-like UPF0514membraneproteinFAM159A Transmembraneemp24domain domain-containingprotein7 Collagenalpha-5(VI)chain WAPfour-disulfidecoredomainprotein UncharacterizedproteinC12orf28 UncharacterizedproteinC17orf67 Placenta-specificprotein9 Bonemorphogeneticprotein8A Ribonuclease-likeprotein9 UncharacterizedproteinC2orf66C17orf99protein Uncharacterized Tetraspanin-18 containingprotein6 PutativeV-setandimmunoglobulin Secretedphosphoprotein1 Bmelanomaantigen5 UPF0369proteinC6orf57 Ig-likedomaincontainingprotein Urotensin-2B ProteinTEX261 homolog7 XK-relatedprotein5 Beta-defensin121 Beta-defensin130 TD26protein relatedprotein8 ProteinWnt-8a like2 like1 Apelin ProteinWFDC13 ProteinFAM150A Latherin 10A Persephin ENSP00000270642

Endonucleasedomain-containing1 Calcium-bindingandcoiledcoildomain UDP-GlcNAc:betaGalbeta1,3N Oncoprotein-inducedtranscript3protein FCHanddoubleSH3domainsprotein1 ComplementClqtumornecrosisfactor Poliovirusreceptor-relatedprotein4 acetylglucosaminyltransferase4 Trypsin-X3(EC3.421) NCU-G1proteinLysosomal Solutecarrierfamily22member15 Serumparaoxonase/lactonase3 Long-chainfattyacidCoAligase EMIdomaincontaining-protein1 UncharacterizedproteinCorf15 Proline-richacidicprotein1 Transmembraneprotein43 Tolloid-likeprotein2 RINGfingerandSPRYdomain containingprotein1 containingprotein1 diphosphohydrolase8 15Peptidaseinhibitor Interleukin-12subunitalpha inhibitor16Peptidase Semaphorin-3B member4-like2 ProteinWnt-2 ProteinWnt-8b Collectin-10 HHIP-likeprotein2 ProteinWnt-11 ProteinWnt-7a UPF0577proteinKIAA1324 relatedprotein9 GPIinositol-deacylase protein Somatostatin Trypsin-2 ACSBG2 Urocortin Fractalkine protein2 Mucin-17 SDCCAG10 TABLE1-continued familyDehydrogenasereductaseSDR/Tumornecrosisfactorreceptorsuperfamily TumornecrosisfactorligandsuperfamilyHepatoma-derivedgrowthfactorrelated ProcollagenC-endopeptidaseenhancer1Prolyl4-hydroxylasesubunitalpha3Transmembraneandubiquitin-likedomainPeptidylprolylcistransisomerase

Inter-alphatrypsininhibitorheavychainH1Transcobalamin1 Inter-alphatrypsininhibitorheavychainH2 Trefoilfactor2chainH3trypsininhibitorheavy-alpha Inter Testican-1 metalloproteinasewithdisintegrinandAEctonucleosidetriphosphate Lymphocyteantigen6complexlocus Platelet-derivedgrowthfactorsubunitA Plasminogenactivatorinhibitor1 Plasminogenactivatorinhibitor2 Chymotrypsin-likeelastasefamily Pancreaticsecretorytrypsininhibitor Calcium-activatedchloridechannel Bactericidal/permeability-increasing thrombospondinmotifs4 Microfibrillar-associatedprotein1 Mesencephalicastrocyte-derived Pigmentepithelium-derivedfactor InhibinbetaAchain Prostate-specificantigen Hepatictriacylglycerollipase Lutropinsubunitbeta 72kDatypeIVcollagenase Stromelysin-1 Oxytocin-neurophysin1 Neurotrophin-3 containingprotein2Proteindisulfide-isomerase Kallikrein-4 regulator4 protein-like1 Eosinophillysophospholipase neurotrophicfactorMatrixGlaprotein Neutrophilcollagenase Beta-nervegrowthfactor Sonichedgehogprotein member2A member21 Plasmakallikrein Leptin proteinG6c Mucin-5AC Mucin-6 member18 Phosphopantothenoylcysteine decarboxylase PepsinA Mesothelin Norrin Gastricsin

HLAclassIhistocompatibilityantigen,A-2 factorstimulating1colonyMacrophage- Interleukin-22receptorsubunitalpha2 Deformedepidermalautoregulatoryfactor1 Autophagy-relatedprotein161 Low-densitylipoproteinreceptorrelated GPItransamidasecomponentPIG-T interactingmultifunctionalprotein1 PeroxisomalN(1)-acetyl Cholesterylestertransferprotein Collagenalpha-1(II)chain Stromalcell-derivedfactor1 Disintegrinandmetalloproteinasedomain Adisintegrinandmetalloproteinasewiththrombospondinmotifs13 T-cellsurfaceglycoproteinCD8alphachain Breastcanceranti-estrogenresistance Neuralcelladhesionmolecule1 Tumornecrosisfactorligandsuperfamily EGF-likedomaincontainingprotein8AminoacyltRNAsynthetasecomplex Prostaglandin-H2Disomerase spermine/spermidineoxidaseProbablepalmitoyltransferaseZDHHC4 containingprotein12 EGFR-coamplifiedandoverexpressed E3ubiquitin-proteinligaseLRSAM1 Neuroligin-4,Xlinked SLAMfamilymember7 ADAMTS-likeprotein4 CoagulationfactorXI ComplementfactorB Pro-interleukin16 Folatereceptoralpha Seizure6-likeprotein Tumornecrosisfactor Acyl-CoAbindingprotein Leptinreceptor protein3 Cadherin-23 protein8 Alpha-1antitrypsin Alpha-1antichymotrypsin alphachain Decorin Tenascin protein Netrin-G1 Kitligand Uromodulin member13 ProteinCREG1 homolog US 2017 /0240590 A1 Aug . 24 , 2017 14

11E2serineTransmembraneprotease, Submaxillaryglandandrogen-regulated Putativeneurofibromin1-likeprotein4/6 Histidine-richcarboxylterminusprotein1 C-typelectindomainfamily2memberALeucine-richrepeatcontainingprotein70 BTB/POZdomain-containingprotein17 C-typelectindomainfamily9memberA ComplementC1q-likeprotein4 Abhydrolasedomain-containingprotein Putativemacrophage-stimulatingprotein SerineproteaseinhibitorKazal-type Placenta-specific1likeprotein UncharacterizedproteinC18orf20 UncharacterizedproteinC12orf53 CMRF35-likemolecule4 Transmembraneprotein14E Transmembraneprotein207TOMM20-likeprotein1 UncharacterizedproteinC3orf41 Inactivecarboxylesterase4 Calcium-activatedchloridechannel regulatorfamilymember3 UncharacterizedproteinC4orf26 UncharacterizedproteinC4orf40C5orf55proteinUncharacterized UncharacterizedproteinC15orf61 domain-containingprotein6 Neuritin-likeprotein FAM108A2/A3 Bmelanomaantigen1 Four-jointedboxprotein1 Kielin/chordin-likeprotein UPF0624proteinC6orf186 Peroxidasin-likeprotein Putativeuncharacterizedprotein Probableserineprotease ChymotrypsinogenB2 PutativeV-setandimmunoglobulin Beta-defensin110 SerpinA13 3Aprotein ProteinHSN2 SCO-spondin UNQ9165/PRO28630 UNQ9391/PRO34284 Beta-defensin108A Beta-defensin111 ProteinFAM151B Osteocrin Humanin MSTP9 5-like3

Carboxypeptidase-likeproteinX2 Dehydrogenase/reductaseSDRfamily protein-betasecretoryE3Epididymal Fibroblastgrowthfactor-bindingprotein2 SerineproteaseinhibitorKazal-type6 Vitellinemembraneouterlayerprotein1 Secretoglobinfamily3Amember2 protein2Angiopoietinrelated-Angiopoietin-relatedprotein6 Brain-specificserineprotease4DBH -likemonooxygenaseprotein1 discoidin-likeIrepeatandEGFlike Fibroblastgrowthfactor17 Fibroblastgrowthfactor22 Growth/differentiationfactor3 Hyaluronanandproteoglycanlink Choriogonadotropinsubunitbeta Matrixmetalloproteinase-28 Olfactomedin-likeprotein1Olfactomedin-likeprotein2A Adisintegrinandmetalloproteinasewith thrombospondinmotifs2 UncharacterizedproteinClorf56 Coorf126proteinColipaselike-UncharacterizedproteinCilorf83 UncharacterizedproteinC16orf89 Cytokine-likeprotein1 Dickkopf-relatedprotein2 Dickkopf-likeprotein1 domain-containingprotein3 GLIPR1-likeprotein1 Lysozyme-likeprotein1 WAPfour-disulfidecoredomain Disintegrinandmetalloproteinase domain-containingprotein28 ArylsulfataseK Cerebellin-3 Cerebellin-4 Cystatin-9like Interleukin-17B Interleukin-170 Interleukin-17D 27proteaseSerine Augurin member13 Beta-defensin123 Beta-defensin132 ProteinFAM55D protein3 homolog variant1 Nephronectin protein12 TABLE1-continued

PeptidoglycanrecognitionproteinI-alpha Extracellularsuperoxidedismutase[Cu-Zn] Pulmonarysurfactant-associatedproteinC Transforminggrowthfactorbeta-1 Submaxillaryglandandrogen-regulated C-typelectindomainfamily11,memberA arylesterase1Serumparaoxonase/Alkalinephosphatase,placentaltype Peptidyl-prolylcistransisomeraseB Basicsalivaryproline-richprotein1 Basementmembrane-specificheparan Lymphocyteantigen6complexlocus SerumamyloidP-component sulfateproteoglycancoreprotein Structuralmaintenanceofchromosomes Metalloproteinaseinhibitor1Metalloproteinaseinhibitor2 inhibitor3Metalloproteinase Urokinase-typeplasminogenactivator Tumornecrosisfactorreceptorsuperfamily Vascularendothelialgrowthfactor UncharacterizedproteinC14orf93 Majorfacilitatorsuperfamilydomain Prolactin-inducibleprotein Secretogranin-1 VitaminD-bindingprotein Zinc-alpha2glycoprotein Alpha-1,3mannosyltransferaseALG2 isoformCRA_b containingprotein7 Plateletfactor4 Bonemarrowproteoglycan hormoneParathyroid Spondin-1 Syntaxin-1A Trypsin-1 vonWillebrandfactor Biglycan Plasminogen Antileukoproteinase Stabilin-1 Somatotropin SerpinB5 protein3 Tetranectin Thyroglobulin Lactotransferrin 3Bprotein member1A receptor1 Vitronectin proteinG5c Retinoschisin

CUBandsushidomain-containingprotein1 Transmembraneprotease,serine6 Choriogonadotropinsubunitbeta Parathyroidhormone-relatedprotein Prolyl4-hydroxylasesubunitalpha2 Cysteine-richwithEGFlikedomain Leucine-richrepeatcontainingprotein8A Urokinaseplasminogenactivatorsurface Dehydrogenase/reductaseSDRfamily Advancedglycosylationendproduct Epidermalgrowthfactorreceptor Ecto-NOXdisulfidethiolexchanger2 Interleukin-1receptorantagonistprotein Interleukin-6receptorsubunitbeta Interleukin-1receptorlike JunctionaladhesionmoleculeA SLITandNTRK-likeprotein2 Sulfatase-modifyingfactor1 Sulfatase-modifyingfactor2 Tumornecrosisfactorreceptorsuperfamily V-setdomaincontainingTcellactivation amidasealanineacetylmuramoyl-LN Interleukin-18bindingprotein Myeloid-associateddifferentiationmarker Hyaluronidase-1 Fcreceptor-likeprotein5 Lymphotoxin-alpha Sulfhydryloxidase1 KinofIRRE-likeprotein2 1-acylsnglycerol3phosphate Versicancoreprotein Multipleinositolpolyphosphatephosphatase1 Neuropilin-1 ProteinRIC-3 acyltransferasegamma CD276antigen 1protein Ficolin-2 Plexin-A4 Plexin-B1 member10B inhibitor1 member4 specificreceptor Insulin Glycodelin Nurim ProteinGPR89 Periostin receptor Glucagon Chordin US 2017 /0240590 A1 Aug . 24 , 2017

SerineproteaseinhibitorKazal-type9 Putativescavengerreceptorcysteine-rich KazalinhibitorproteasePutativeserine Dehydrogenase/reductaseSDRfamily relatedprotein3lipasePancreatic- Testis,prostateandplacenta-expressed Neuronalpentraxin-likeproteinC16orf38 Iron/zincpurpleacidphosphatase-like Plasminogen-relatedproteinA Ribonuclease-likeprotein10 Ribonuclease-likeprotein12 Ribonuclease-likeprotein13 Putativetestisserineprotease2 Secretedfrizzled-relatedprotein4 ComplementClq-likeprotein2 PutativeuncharacterizedproteinC17orf69 PeptidaseS1domain-containingprotein Growthdifferentiationfactor7 Putativelipocalin1-likeprotein PutativepeptideYY-2 PutativepeptideYY-3 Kunitz-typeproteaseinhibitor4 FLJ37543Uncharacterizedprotein Putativecystatin-13 Putativeserineprotease29 domain-containingproteinLOC619207 LipasememberN Neuromedin-S Ovostatinhomolog1 Polyserase-3 Meteorin-likeprotein IgA-inducingproteinhomolog like25-type Beta-defensin131 Beta-defensin134 Beta-defensin136 Beta-defensin116 Beta-defensin115 Beta-defensin114 NeuropeptideS Beta-defensin112 Beta-defensin109 Beta-defensin113 Beta-defensin135 7Cmember ProteinFAM132A ProteinFAM132B protein Otolin-1 protein SerpinA11 ProteinFAM24A LOC136242

SerineproteaseinhibitorKazal-type7 Zymogengranuleprotein16homologB SerineproteaseinhibitorKazal-type4 Transmembraneprotease,serine11D ComplementClqtumornecrosisfactor Keratinocyte-associatedprotein3 DDRGKdomain-containingprotein1 Liver-expressedantimicrobialpeptide2 Bactericidal/permeabilityincreasing- Scrapie-responsiveprotein1PutativeannexinA2-likeprotein protein10morphogeneticBone UncharacterizedproteinClorf54 UncharacterizedproteinC7orf34 Collagenalpha-1(VIII)chainUncharacterizedproteinC18orf54 Fibroblastgrowthfactor5ProbableserineproteaseHTRA3 Interleukin-1familymember8 Lysyloxidasehomolog1 Lysyloxidasehomolog2 Longpalate,lungandnasalepithelium carcinoma-associatedprotein4 Lysozymeg-likeprotein2 phosphodiesterase3b Neurexophilin-4ProteinWnt-9b Semaphorin-3D Secretogranin3- CarboxypeptidaseA6 NeuropeptideBKinesin-likeproteinKIF7 like2protein- relatedprotein4 C-motifchemokine25Chymotrypsin-likeelastasefamily Glycerophosphodiesterphosphodiesterase member2B ProteinCEI ProteinFAM55C ProteinFAM19A2 ProteinFAMSB Otospiralin Endomucin likereceptor2 TABLE1-continued Collagenalpha-1(XXVI)chainLy6/PLAURdomaincontainingprotein NADHdehydrogenase?ubiquinone]1betaAcidsphingomyelinase-like PotassiumchannelsubfamilyKmember17UncharacterizedproteinC9orf47 C2domain-containingprotein2Leucinerichrepeatcontainingprotein8D ChondroitinsulfateApolipoproteinL4glucuronyltransferase Leucine-richrepeatLGIfamilymember3Cmotifchemokine19 UncharacterizedproteinCoorfiProbableG-proteincoupledreceptor114 Cystatin-like1Leucinerichrepeatcontainingprotein8B Leukocyte-associatedimmunoglobulinLeucinerichrepeatsandimmunoglobulin ComplementfactorH-relatedprotein4 Pregnancy-specificbeta1glycoprotein2 Carbonicanhydrase-relatedprotein10 Chitinasedomain-containingprotein1 Dehydrogenase/reductaseSDRfamily ProbableG-proteincoupledreceptor113 Sialicacid-bindingIglikelectin6 Ly6/PLAURdomain-containingprotein5 Macrophagemigrationinhibitoryfactor Mitochondrialcarrierhomolog1 Long-chainfattyacidtransportprotein3 subcomplexsubunit11,mitochondrial UPF0546membraneproteinClorf91 UncharacterizedproteinCoorf89 TransmembraneproteinC9orf7 Crumbsproteinhomolog3 Glycerol-3phosphateacyltransferase4 KDELmotif-containingprotein2 MLN64N-terminaldomainhomolog 2-acylglycerolOacyltransferase3 Protocadherinalpha-6 Protocadheringamma-A12 Voltage-gatedhydrogenchannel1 Leucine-richrepeattransmembrane protein1 neuronal CMRF35-likemolecule9 CytochromeP450251 domain-containingprotein5 ApolipoproteinL6 All-transretinol13,14reductase Regulatorofmicrotubuledynamics Vesicle-traffickingproteinSEC22c likedomainsprotein3 Cholecystokinin Codanin-1 member7 ENEDProtein Gliomedin Gremlin-1 Layilin protein2 R-spondin4 Claudin-1

Tubulointerstitialnephritisantigen-like ComplementClqsubcomponentsubunitA ComplementClqsubcomponentsubunitC BifunctionalUDP-Nacetylglucosamine2 NLRfamilyCARDdomain-containing Inter-alphatrypsininhibitorheavychainH5 Sialicacid-bindingIglikelectin10 Long-chainfattyacidCoAligase5 Leucine-richrepeatcontainingprotein20 Lymphocyteantigen6complexlocus Solublecalcium-activatednucleotidase1 Fibroblastgrowthfactorreceptor3 Fibroblastgrowthfactorreceptor4 Pro-neuregulin2,membranebound antigen11ASpermassociated- Oocyte-secretedprotein1homolog Interleukin-7receptorsubunitalpha Platelet-derivedgrowthfactorD Tumornecrosisfactorligandsuperfamily Interleukin-1familymember7 Anthraxtoxinreceptor2 ApolipoproteinIIIC- Collagenalpha-6(IV)chainCollagenalpha-2(VI)chain Collagenalpha-1(XI)chain Lowaffinityimmunoglobulinepsilon Growtharrest-specificprotein6 epimerase/N-acetylmannosaminekinase inhibitorproteaseC1Plasma ProteinS100-A7 Amelogenin,Xisoform Crumbshomolog1 Neutrophildefensin1 ApolipoproteinL1 C-motifchemokine15 CD97antigen( Contactin-4ComplementC2 Cystatin-C Endothelin-3 Growthhormonereceptor protein4 isoform Serumalbumin Cochlin member13B Claudin-14 proteinG5b Acetylcholinesterase Angiogenin AnnexinA2 Calcitonin Fcreceptor US 2017 /0240590 A1 Aug . 24 , 2017 16

Insulingrowthfactor-likefamilymember2 Putativezinc-alpha2glycoproteinlike1 Insulingrowthfactor-likefamilymember4 SerineproteaseinhibitorKazal-type8 Putativezinc-alpha2glycoproteinlike Replicationinitiation-likeprotein Prostateandtestisexpressedprotein3 Secretedfrizzled-relatedprotein2peptideIB-1 Basicprolinerich UDP-GlcNAc:betaGalbeta1,3N Putativeabhydrolasedomain-containing cDNAFLJ55667,highlysimilarto Secretedproteinacidicandrichin Putativestereocilin-likeprotein UncharacterizedproteinC2orf72 PutativeuncharacterizedproteinClorf191 UncharacterizedproteinFLJ90687 Fibroblastgrowthfactor16 Plateletbasicprotein-like2 Stressinducedsecretedprotein1 PutativeuncharacterizedproteinClorf134 acetylglucosaminyltransferase9 UncharacterizedproteinCilorf44 UncharacterizedproteinC12orf73like 2cystatin-9Putative Putativetestis-specificprionprotein PutativeuncharacterizedproteinFP248 Secretoglobin-likeprotein Beta-defensin108Blike UncharacterizedproteinKIAA0495 Secretedphosphoprotein1 ProteinWnt(Fragment) PutativeserineproteaseLOC138652 Putativeuncharacterizedprotein Probablefolatereceptordelta Proline-richprotein1 Bmelanomaantigen4 NPIP-likeproteinENSP00000346774 UPF0670proteinC8orf55 Otopetrin-1 LipasememberK KIR2DL4 SerpinE3 CR1receptor ProteinWnt TOM1 FLJ46089 FAM108A5protein Beta-defensin133 Fibrosin-1 RPE-spondin SPARCprotein cysteine

Adipocyteenhancer-bindingprotein1 Interphotoreceptormatrixproteoglycan1 Proapoptoticcaspaseadapterprotein WAPfour-disulfidecoredomainprotein8 SH3domain-bindingprotein5like ApolipoproteinA-Ibindingprotein GRAMdomain-containingprotein1B NMDAreceptor-regulatedprotein2 Parkinsondisease7domain-containing Sialicacid-bindingIglikelectin12 Coiled-coildomaincontainingprotein80 TRACH2015180,highlysimilarto Calcium-dependentphospholipaseA2 Zonapellucida-bindingprotein2 Collagenalpha-1(XXVIII)chain Phosphatidylinositolglycananchor Pro-neuregulin4,membranebound Leucine-richrepeatneuronalprotein3 NADH-cytochromeb5reductase1 C-typelectindomainfamily12memberB Solutecarrierfamily35memberF5 WDrepeat-containingprotein82 Ecto-NOXdisulfidethiolexchanger1 Neuronalgrowthregulator1 Secretedfrizzled-relatedprotein2 Integrinbeta-likeprotein1 inhibitor3proteasetypeKunitz- UncharacterizedproteinC12orf59 Inactivecaspase-12 UncharacterizedproteinC7orf58 Dentinmatrixprotein4 UncharacterizedproteinC16orf48 biosynthesisclassUproteinInterleukin-27subunitalpha FK506-bindingprotein11 ADAMTS-likeprotein3 cDNAFLJ36603fis,clone Tolloid-likeprotein1 ProteinWnt-5b ProteinWnt-7b Adipocyteadhesionmolecule Carboxylesterase3 GPN-loopGTPase3 LipasememberH ProteinTMEM155 Prosalusin Proteinamnionless ProteinWFDC10B 17Claudin- FAM20BProtein isoform protein1 ProteinFAM19A3 TABLE1-continued

Platelet-derivedgrowthfactorsubunitB Tetratricopeptiderepeatprotein14 Zonapellucidasperm-bindingprotein3Leucine-richrepeatcontainingprotein39 Lipopolysaccharide-bindingprotein pyrophosphatase/phosphodiesterasefamily Serine/threonine-proteinkinase32B XTP3-transactivatedgeneBprotein Transmembraneprotein139 Carbohydratesulfotransferase9 Cysteine-richmotorneuron1protein Connectivetissuegrowthfactor Fibroblastgrowthfactor19 Follistatin-relatedprotein3 FXYDdomain-containingiontransport EGF-containingfibulinlikeextracellular Group3secretoryphospholipaseA2 Tumornecrosisfactorligandsuperfamily Kunitz-typeproteaseinhibitor1 SLAMfamilymember9 Tryptasealpha-1 PalmitoyltransferaseZDHHC15 Pancreatictriacylglycerollipase Leukemiainhibitoryfactor Proteineyesshuthomolog Hedgehog-interactingprotein Interleukin-17receptorB GroupXVphospholipaseA2 InactiveserineproteasePAMR1 Xylosyltransferase1 CD5antigen-like Mucin-likeprotein1 Prokineticin-1 Galectin-1C-motifchemokine21 regulator5 Endotheliallipase matrixprotein2 Ribonuclease7 Spondin-2 Testican-2 Vasohibin-1 Oncostatin-M Transthyretin Noggin Otoraplin member14Plexin-A2 Papilin Torsin-2A Vasorin Ectonucleotide member6

Immunoglobulinsuperfamilymember8 Pulmonarysurfactant-associatedproteinB Multipleepidermalgrowthfactor-like BaculoviralIAPrepeat-containingprotein7 Mannan-bindinglectinserineprotease1Mannan-bindinglectinserineprotease2 Neutrophilgelatinase-associatedlipocalin Poliovirusreceptor-relatedprotein1 Signalinglymphocyticactivationmolecule EpididymalsecretoryproteinEl acetylgalactosaminyltransferase1 galactosamide-alpha2,3sialyltransferase WAPfour-disulfidecoredomainprotein2 subunit-1AsecretaseAPHGamma Coxsackievirusandadenovirusreceptor familyphosphodiesterasepyrophosphatase/ EGF-likedomaincontainingprotein7 UDP-GalNAc:beta1,3N SH2domain-containingprotein3C Interleukin-4receptoralphachain Lamininsubunitbeta-3 Fibroblastgrowthfactor23 Interleukin-23subunitalpha ADAMTS-likeprotein1 Transmembraneprotein25 Interleukin-15(IL) Mucinandcadherin-likeprotein CMP-Nacetylneuraminatebeta Transmembraneprotein9 Chorionicsomatomammotropinhormone Leucyl-cystinylaminopeptidase NeuropeptideY Aggrecancoreprotein Semenogelin-1 Tissuefactorpathwayinhibitor Chemokine-likefactor AdenosineA3receptor ComplementfactorH ERO1-likeproteinalpha Kallikrein-14 Kallikrein-6 Ribonucleasepancreatic Tectonic-1 Ribonuclease4 Alpha-S1casein Cyclin-L1 Renin Usherin domains Basigin Calumenin Ectonucleotide member2 US 2017 /0240590 A1 Aug . 24 , 2017

FibroblastgrowthfactorreceptorFGFR-1 Prostateandtestisexpressedprotein4 domainreceptorcysteine-richScavenger COBW-likeplacentalprotein(Fragment) Glycosyltransferase54domain-containing Putativesolubleinterleukin18receptor1 C-typelectindomainfamily18member Collagenalpha-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 Putativeuncharacterizedprotein ComplementC3 Uncharacterizedprotein Serpin-likeproteinHMSD Putativeuncharacterizedprotein Putativeuncharacterizedprotein Tachykinin-3 LOC284297proteincontaining Tryptasebeta-1 Hyaluronidase-3 Chordin-likeprotein1 Putativeuncharacterizedprotein NetrinreceptorUNC5B Uncharacterizedprotein UNQ6125/PRO20090 UNQ6126/PRO20091 Cystatin-SR-spondin1 UNQ6975/PRO21958 Tryptasedelta protein9 Beta-defensin103 Beta-defensin106 UNQ9370/PRO34162 C&orf2 Opiorphin Sclerostin protein ENSP00000244321 ECE2 EPA6

Psoriasissusceptibility1candidategene Signalpeptidasecomplexsubunit3 Spermequatorialsegmentprotein1 Acyl-CoAsynthetasefamilymember2, Zonapellucidasperm-bindingprotein4Endoplasmicreticulumresidentprotein DnajhomologsubfamilyBmember9 Integralmembraneprotein2ASFT2Bprotein transportVesicle vonWillebrandfactorAdomain CD164sialomucin-like2protein TransmembraneproteinC3orf1 UncharacterizedproteinC6orf72 UncharacterizedproteinC11orf24 ProbableUDP-sugartransporterprotein C-typelectindomainfamily1memberAC-typelectindomainfamily3memberA memberEfamilydomain4typelectinC- C-typelectindomainfamily4memberG TransmembraneproteinC16orf54 Thioredoxindomain-containing Adenosinemonophosphate-protein Prenylcysteineoxidase-like Phytanoyl-CoAhydroxylaseinteracting FXYDdomain-containingiontransport Growth/differentiationfactor11 Cerebraldopamineneurotrophicfactor Mucin-19(MUC) containingprotein3AProteinshisa-2homolog Probablecationtransporting- CytochromeP4504F12 CytochromeP4504X1421 P450Cytochrome Cerebellin-2 UPF0480proteinC15orf24 Dipeptidase3FAM174A proteinMembrane GPN-loopGTPase2 Cadherin-16 Cadherin-19 ATPase13A4 ProteinCREG2 15protein ProteinFAM19A4 transferaseFICD likeprotein- regulator4 2protein mitochondrial SLC35A5 ERp27 TABLE1-continued

Hyaluronanandproteoglycanlinkprotein1 Transmembraneprotease,serine11E Leukocyteimmunoglobulin-likereceptor SerineproteaseinhibitorKazal-type5 Pregnancy-specificbeta1glycoprotein4 Thrombospondintype-1domaincontaining AngiogenicfactorwithGpatchandFHA HERV-FRD_6p24.1provirusancestralEnv HLAclassIhistocompatibilityantigen, Adisintegrinandmetalloproteinasewiththrombospondinmotifs6 Interleukin-1receptoraccessoryprotein Matrixmetalloproteinase-16Pituitaryadenylatecyclase-activating Latent-transforminggrowthfactorbeta Wntinhibitoryfactor1 Deoxyribonucleasegamma acetylgalactosaminyltransferase1N- FRAS1-relatedextracellularmatrix subfamilyAmember3 Interferonalpha-14 TGF-betareceptortypeIII ThyrotropinsubunitbetaUncharacterizedproteinC19orf36 Angiopoietin-2 CarboxypeptidaseA5 C—Xmotifchemokine2C—Xmotifchemokine5 bindingprotein3 Cw-16alphachain C-typenatriureticpeptide C-motifchemokine14 Interleukin-5 Interleukin-10 Interleukin-17F Kallikrein-15 Collagenase3 Prokineticin-2 Derlin-1 polyprotein Prostasin Polypeptide Fibulin-2 Ficolin-1 SLcytokine Follistatin protein1 Enamelin polypeptide Somatoliberin protein1 domains1

ProteinO-linkedmannosebeta1,2N Sushi,vonWillebrandfactortypeAEGFandpentraxindomain-containingprotein1 Transmembraneandcoiled-coildomain Transmembraneprotease,serine3 LowaffinityimmunoglobulingammaFc Leucine-richrepeatcontainingGprotein Latent-transforminggrowthfactorbeta Myelinproteinzero-likeprotein1 acetylglucosaminyltransferase1 Sarcolemmalmembrane-associatedprotein Tumornecrosisfactorreceptorsuperfamily receptorsuperfamilyfactorTumornecrosis regionreceptorIII-B Leucine-richrepeattransmembraneprotein TransmembraneglycoproteinNMB Interleukin-31receptorAJunctionaladhesionmoleculeB Neurobeachin-likeprotein2 CoagulationfactorIX protein2receptor-likeFc ADP-ribosepyrophosphatase, SerumamyloidAproteinSexhormone-bindingglobulin SLAMfamilymember6 globulinbindingThyroxine- containingprotein1 Tryptasebeta-2 IgmuchainCregion Interleukin-1alpha Lipocalin-1 coupledreceptor6 bindingprotein1 Protocadherin-15 Placentagrowthfactor ProbablehydrolasePNKD Reticulon-4receptor Ficolin-3 Matrilin-3 Poliovirusreceptor FLRT3 Gelsolin Granulysin Granulins Heparanase Nicastrin mitochondrial Pleiotrophin member10C member11B Serotransferrin ProteinYIPF5 US 2017 /0240590 A1 Aug . 24 , 2017

Putativeabhydrolasedomain-containing Methylthioribose-1phosphateisomerase Methyltransferase-likeprotein7A WNT1inducedsecretedprotein1splice Chemokine-likefactorsuperfamily2 domain-containinglikeprotein Bcellmaturationantigentranscriptvariant superfamilymember17) 17-betahydroxysteroiddehydrogenase13 Interleukin-family1member10 protein3Olfactomedinlike- Extracellularglycoproteinlacritin Keratinocytesassociatedtransmembrane RTFV9368(SLE-dependent Leucine-richrepeatandimmunoglobulinlikedomain-containingnogoreceptor FAM108A6protein PutativeV-setandimmunoglobulin 4(Tumornecrosisfactorreceptor UPF0672proteinC3orf58 AminopeptidaseB variantx(Fragment) Proteoglycan3Insulin-likepeptideINSL5 Retinoldehydrogenase13Neutrophildefensin3 Clq/TNFrelated-protein8 KIR2DL4(Fragment) transcriptvariant2 upregulation1) interactingprotein4 N-acetyltransferase15 Ephrin-A4 ProteinPlunc Kallikrein-11 protein1 POM121-like ENSP00000303034 Dermcidin Meteorin NL3 PLA2G2D GLGQ5807 TUFT1 DRLV8200 IDLW5808 UBAP2 GKGM353 MATL2963 NINP6167 KCNQ2 ELCV5929 KVVM3106

Polycystickidneydiseaseprotein1-like Transmembrane4L6familymember20 WAP,kazalimmunoglobulinkunitzand Leucine-richrepeatcontainingprotein33MANSCdomain-containingprotein1 Mesodermdevelopmentcandidate2 FibronectintypeIIIdomain-containing Solutecarrierfamily35memberE3 Abhydrolasedomain-containingprotein associatedprotein2-like NTRdomain-containingprotein1 KDELmotif-containingprotein1 Collagenalpha-6(VI)chain PDZdomain-containingprotein2 Matrixmetalloproteinase-26 Ankyrinrepeatdomain-containing Brain-specificangiogenesisinhibitor1 Transmembraneprotein107 Transmembraneprotein143 Growthhormone-inducible Chondromodulin-1 Dickkopf-relatedprotein1 Olfactoryreceptor1061 Seizure6-likeprotein2 transmembraneprotein Lactase-likeprotein Lipocalin-15 ArylsulfataseI ProteinPARM-1 Semaphorin-3ASemaphorin-4C Proteinshisa-4 Neuromedin-U Synaptogyrin-2 protein2 protein1 Noelin-2 36protein Nodalhomolog 104protein Glycerophosphodiester Adipophilin Podocan Neurotrimin Proepiregulin WLPL514 14A TABLE1-continued pyrophosphatase/phosphodiesterasefamilydomaincontainingphosphodiesterase- RELT-likeprotein2ProcollagenCendopeptidaseenhancer Leucine-richrepeatandimmunoglobulinCoiledcoildomaincontaining Leucine-richrepeatLGIfamilymember4ZinctransporterZIP9 acetylgalactosaminyltransferase-like Carbonicanhydrase-relatedprotein11 Secretoglobinfamily3Amember1 proteinrelated2factorComplementH- tumornecrosisComplementClq Angiopoietin-relatedprotein3 Angiopoietin-relatedprotein7 Cysteine-richsecretoryproteinLCCL Spermacrosomemembrane-associated Immunoglobulinsuperfamilycontaining likedomain-containingnogoreceptor Hematopoieticcellsignaltransducer ProbablecarboxypeptidaseX1 Fibroblastgrowthfactor21 Plasmaalpha-Lfucosidase Glutathioneperoxidase7 Apolipoprotein-IC Left-rightdeterminationfactor1 BRCA1-AcomplexsubunitAbraxasLeucinezipperprotein 2 Kazal-typeserineproteaseinhibitordomain-containingprotein1 KinofIRRE-likeprotein3 factor-relatedprotein2 Slithomolog1protein Ecto-ADPribosyltransferase5 Probableribonuclease11 C—Xmotifchemokine14 domain-containing2 Neurexophilin-3 Claudin-2(SP82) leucine-richrepeatprotein interactingprotein1 Follitropinsubunitbeta PolypeptideN Growthhormonevariant Beta-defensin127 Beta-defensin129 Gastrokine-1 Gastrokine-2 HHIP-likeprotein1Interferonkappa Ectonucleotide member5 protein2 ProteinFAM3D Osteomodulin protein3 Tsukushin

cDNA,FLJ96669highlysimilartoHomosapienssecretedprotein,acidiccysteine cDNAFLJ77519,highlysimilartoHomo Programmedcelldeath1ligand2 cDNAFLJ75759,highlysimilartoHomo rich(osteonectin)SPARC,mRNA sapienssecretedfrizzledrelatedprotein Tumornecrosisfactorreceptorsuperfamily UPF0414transmembraneproteinC20orf30 C-typelectindomainfamily4member UPF0317proteinC14orf159,mitochondrial Sushidomaincontaining-protein4 T-cellsurfaceglycoproteinCD8betachain ComplementClqtumornecrosisfactor EGF-likemodulecontainingmucin sapiensfollistatin-like3(secretedglycoprotein)(FSTL3,mRNA Angiotensin-convertingenzyme2 Angiopoietin-relatedprotein4 proteinB/Cassociated Fibrinogen-likeprotein1 C-motifchemokine3like1 Fibroblastgrowthfactor8Sialomucincoreprotein24 hormonereceptor-like3 WNT1-induciblesignalingpathway Vesicle-associatedmembraneprotein T-celldifferentiationantigenCD6 Sperm-associatedantigen11BCoagulationfactorXII Tomoregulin2- Chordin-likeprotein2 C4b-bindingproteinbetachain Secretedandtransmembrane1 Odorant-bindingprotein2b Interleukin-32 MetalloreductaseSTEAP2 ApolipoproteinM relatedprotein6 Urotensin-2 Matrilin-4 member6B Netrin-G2 ProteinYIF1B Noelin-3 protein3 mRNA Pikachurin Hepcidin Klotho Serglycin Vitrin Adiponectin US 2017 /0240590 A1 Aug . 24 , 2017

S100calciumbindingproteinA7-like3 CR1C3b/C4breceptorSCR9(or16)C GTWW5826LP5085(protein) carcinoma-associatedprotein3(Ligand term.exonSCR=shortconsensusrepeat KTIS8219(HCG2020043)Hyaluronanandproteoglycan link Longpalate,lungandnasalepithelium bindingproteinRYA3) ISPF6484 LKHP9428 VNFT9373 ACAH3104 RVLA1944 Wpep3002 ZDHHC11 AGLW2560 TSSP3028 RFVG5814 SHSS3124 MMP19 GSQS6193 VGPW2523 LMNE6487 ALLA2487 GAL11870 FRSS1829 MRSS6228 GRPR5811 AVLL5809 PIKR2786 protein4 Micronovel SAMK3000 VFLL3057 CVWG5837 VGSA5840 GHPS3125 GRTR3118 PAMP6501 LTLL9335 VCEW9374 AHPA9419 MDHV1887 HSAL5836 LHLC1946 LPPA601 PINK1 SERH2790 FLFF9364

Mitochondrialuncouplingprotein4 Zonapellucidasperm-bindingprotein1 Zonapellucidasperm-bindingprotein2 Heparansulfateglucosamine3-0 protein6Spermatogenesisassociated- Twistedgastrulationproteinhomolog1 ConservedoligomericGolgicomplex Adiponectinreceptorprotein2 PhospholipaseAlmemberABasicsalivaryproline-richprotein2 Sushirepeat-containingproteinSRPX2 C-typelectindomainfamily18memberB Transmembraneprotein178 Transmembraneprotein205 Transmembraneprotein41A Transmembraneprotein50A Transmembraneprotein50B Neuronalpentraxin-2 Transmembraneprotein92 Transmembraneprotein95 Transmembraneprotein9B ProbablecarboxypeptidasePM20D1 UPF0513transmembraneprotein ProbablepalmitoyltransferaseZDHHC24 Leptinreceptoroverlappingtranscript ProteinHEGhomolog1FibrinogenCdomain-containing Lysosomal-associatedtransmembrane Majorfacilitatorsuperfamilydomaincontainingprotein5 Interleukin-28B Tetraspanin-12 Tetraspanin-1315Tetraspanin- Polyserase-2 InhibinbetaCchain Semaphorin-3C sulfotransferase2 SPARC-likeprotein1 ProteinWnt-5a Acrosin-bindingprotein Semaphorin-3E Angiopoietin-1 subunit7 Fibulin-7 protein1 Torsin-1B 4Aprotein Collectrin Brorin like1 Ameloblastin TABLE1-continued

Melanomainhibitoryactivityprotein3 Endothelin-convertingenzymelike1 Signalpeptide,CUBandEGF-likedomain ComplementfactorH-relatedprotein3 Proheparin-bindingEGFlikegrowthfactor Leucine-richrepeatcontainingprotein4 proteinneuronal1repeatLeucine-rich 3-hydroxybutyratedehydrogenasetype2 CarboxypeptidaseNcatalyticchain Short-chaindehydrogenase/reductase3 Insulin-likegrowthfactorbindingprotein5 acetylgalactosaminyltransferase1 Insulin-likegrowthfactorbindingprotein7 FibronectintypeIIIdomain-containing Chondroitinsulfatesynthase1 Chondroitinsulfatesynthase2 DeltaandNotch-likeepidermalgrowth Globosidealpha-1,3N Glycerol-3phosphateacyltransferase Hyaluronan-bindingprotein2 Interleukin-20receptorbetachain SerumamyloidA-4protein CIGALT1-specificchaperone1 TransmembraneproteinC2orf18 CMRF35-likemolecule7 Proteincanopyhomolog3 Integralmembraneprotein2B Endothelialcell-selectiveadhesion Follistatin-relatedprotein1 Carbohydratesulfotransferase14 pyrophosphatase/phosphodiesterase Leukemiainhibitoryfactorreceptor Zinctransporter5 Apicalendosomalglycoprotein Beta-1,4galactosyltransferase7 Delta-likeprotein4. containingprotein1 Gamma-glutamylhydrolase G-proteincoupledreceptor56 Histidine-richglycoprotein familymember3 Kappa-casein factor-relatedreceptor Dolicholkinase ProrelaxinH1 Cadherin-24 3Bprotein Probetacellulin Beta-casein CD320 molecule Ectonucleotide Kallistatin

Allograftinflammatoryfactor1-like Glycosyltransferase8domain-containing Pulmonarysurfactant-associatedproteinA2Splicingfactor,arginine/serine-rich16 alpha-2,6 acetylgalactosaminideAlphaN Bactericidalpermeability-increasingprotein Cartilageintermediatelayerprotein1 DnajhomologsubfamilymemberC10 CoagulationfactorXIIIAchain Leucine-richgliomainactivatedprotein1 Proproteinconvertasesubtilisin/kexin Armadillorepeat-containingXlinked acetylgalactosaminyltransferase1 Claudindomain-containingprotein1 ProbableG-proteincoupledreceptor125 GPIethanolaminephosphatetransferase2 GPIethanolaminephosphatetransferase3 proteinSCAMC-2(Smallcalciumbinding mitochondrialcarrierprotein2) Apolipoprotein-VA CUBdomain-containingprotein1 Collagenalpha-1(V)chain Collagenalpha-1(XXV)chainEstradiol17-betadehydrogenase11 EGF-likedomaincontainingprotein6 proteinPeptidoglycanrecognition Interferon-induced17kDaprotein Interleukin-20receptoralphachain Calcium-bindingmitochondrialcarrier Glucose-6phosphateisomerase Appetite-regulatinghormone Interleukin-12subunitbeta Lymphocyteantigen96 sulfateNChondroitin Chitotriosidase-1 Golgimembraneprotein1 Prolyl3-hydroxylase1 SingleIgIL-1relatedreceptor Beta-AlaHisdipeptidase Interleukin-22 Intelectin-1 ProteinWnt-4 NKG2Dligand4 sialyltransferase6 Mucin-20 protein3 protein1 Galectin-7 L-aminoacidoxidase Asporin Matrilysin type9 Erlin-2 US 2017 /0240590 A1 Aug . 24 , 2017

Stromalcell-derivedfactor2likeprotein1 cDNAFLJ53955,highlysimilarto Secretedfrizzled-relatedprotein4 Deoxyribonuclease-1like2 Endothelialcell-specificmolecule1 Butyrophilinsubfamily1memberAl ImmunoglobulinalphaFcreceptor Interleukin-27subunitbeta Keratinocyte-associatedtransmembrane Ephrintype-Areceptor10 Follistatin-relatedprotein4 Follistatin-relatedprotein5 Transmembraneprotein66Growthdifferentiationfactor2 Stanniocalcin-2 Carboxylesterase7 ProteinNOVhomolog UPF0528proteinFAM172A Exostosin-like2 Beta-defensin108B Beta-defensin118 Beta-defensin124 Beta-defensin125 Beta-defensin126 ProteinF???? protein2 EMILIN-2 APELIN GLSH6409 SFVP2550 RRLF9220 PTML5838 VLGN1945 AVPC1948 AWQG2491 PSVL6168 LCI13035 PPRR6495 RLSC6348 CSRP2BP GLLV3061 GWS16489 PPIF VSSW1971 KLIA6249 ALLW1950 GVEI466 ESFI5812 GNNC2999 AAGG6488 HHSL751

EpididymalsecretoryproteinE3-alpha proteinbinding3growthfactorFibroblast- Multipleepidermalgrowthfactor-like WAPfour-disulfidecoredomainprotein1 Myelinproteinzero-like2 Cartilageintermediatelayerprotein2 Phosphatidylethanolamine-binding Proto-oncogeneproteinWnt1 Serineprotease1-likeprotein Coiled-coildomaincontainingprotein70 CUBdomain-containingprotein2 Cardiotrophin-1(CT) Acidsphingomyelinase-like ADAMTS-likeprotein5 Bonemorphogeneticprotein3b Bonemorphogeneticprotein5 Bonemorphogeneticprotein8B (EpithelialV-likeantigen1) UncharacterizedproteinC4orf29 UncharacterizedproteinCorf58 UncharacterizedproteinC10orf25 Fibroblastgrowthfactor20 Transmembraneprotein204 Angiopoietin-4 phosphodiesterase3a Putativetrypsin-6 Carboxypeptidaseo Trem-liketranscript4protein ChymotrypsinogenB C—Xmotifchemokine9 C—Xmotifchemokine13 CoagulationfactorV CoagulationfactorVII Cerebellin-1 C-motifchemokine28 Cystatin-8 Folatereceptorgamma Galanin-likepeptide Hemicentin-1 Interleukin-6 domains9 ProteinFAM26D C1q-relatedfactor Isthmin-1 EMILIN-3 ProteinFAM5C protein4 Mucin-7 Spexin Chondroadherin Secretagogin Epiphycan Pro-MCH TABLE1-continued

Transmembranechannel-likeprotein5 Neuropilinandtolloid-likeprotein2 Peptidyl-prolylcistransisomeraselike1 Transmembraneprotease,serine4 Thioredoxindomain-containingprotein12 Neuropilinandtolloid-likeprotein1glycoprotein11specific-beta1Pregnancy UDP-GlcNAc:betaGalbeta1,3N factorcytokine1Cardiotrophin-like member1Immunoglobulinsuperfamily Ly6/PLAURdomain-containingprotein3C-typelectindomainfamily14memberA Sialicacid-bindingIglikelectin9 Metastasis-suppressorKiSS1 VascularendothelialgrowthfactorB VascularendothelialgrowthfactorC acetylglucosaminyltransferase3 Calcitoningene-relatedpeptide2 Dentinmatrixacidicphosphoprotein1 Disintegrinandmetalloproteinasedomain Chitobiosyldiphosphodolicholbeta SLITandNTRK-likeprotein1 Collagenalpha-2(VIII)chain Downsyndromecelladhesionmolecule Thioredoxin-relatedtransmembrane containingprotein32 FK506-bindingprotein14 Protocadherinbeta-13 Prenylcysteineoxidase1 Prostatestemcellantigen Proteinpatchedhomolog2 Proteinsel-1homolog Trem-liketranscript2protein Proteincornichonhomolog Isletamyloidpolypeptide Reticulocalbin-3 ADP-dependentglucokinaseAlpha-amylase2B CarboxypeptidaseE Crumbshomolog2 Lin-7homologB protein1 ProteinFAM151A Fibrillin-1 ProteinFAMJA ProteinG7c B4Serpin ADAMDEC1 Peflin mannosyltransferase ProSAAS Statherin Testisin

Scavengerreceptorcysteine-richtype1 Complementreceptortype1 Insulin-likegrowthfactorbindingprotein3 Tumornecrosisfactorligandsuperfamily Tumornecrosisfactorreceptorsuperfamily ProteinZ-dependentproteaseinhibitor Bonemorphogeneticprotein4 Collagenalpha-2(I)chainCollagenalpha-1(III)chain Collagenalpha-1(IV)chain Collagenalpha-3(IV)chain Collagenalpha-5(IV)chain Collagenalpha-3(VI)chain ComplementcomponentC6 Collagenalpha-1(IX)chain Collagenalpha-1(X)chain Collagenalpha-1(XVII)chain Collagenalpha-1(XXI)chain Deoxyribonuclease-1Extracellularmatrixprotein1 FcregionreceptorIII-A Heparin-bindinggrowthfactor2 Growth/differentiationfactor5 Glialcellline-derivedneurotrophicfactor PalmitoyltransferaseZDHHC9 Pancreaticalpha-amylaseNatriureticpeptidesB CarboxypeptidaseB2 CarboxypeptidaseZ ComplementfactorI Coatomersubunitalpha Lowaffinityimmunoglobulingamma Fibrinogengammachain Insulin-likegrowthfactorIA Iggamma-lchainCregion 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 2017 /0240590 A1 Aug . 24 , 2017 12

member21Immunoglobulinsuperfamily Pregnancy-specificbetaglycoprotein15 Triggeringreceptorexpressedonmyeloid GDNFfamilyreceptoralpha-4 GRAMdomain-containingprotein10 protein2Apolipoproteinlike(a)- Alpha-1,3mannosylglycoprotein4beta ComplementClqtumornecrosisfactor Cytokinereceptor-likefactor1 VitaminK-dependentproteinS Butyrophilin-likeprotein8 Lamininsubunitbeta-4 Lymphaticvesselendothelialhyaluronic Transmembraneprotein59 Tubulointerstitialnephritisantigen Transmembraneemp24domain GroupIIEsecretoryphospholipaseA2 Stromalcell-derivedfactor2 Iggamma-4chainCregion Lymphocyteantigen86 Interferonalpha-10 Interferonalpha-16 Interferonalpha-6 1-acylsnglycerol3phosphate Lysozyme-likeprotein2 Lysozyme-likeprotein4 Retinol-bindingprotein4 containingprotein5 Podocan-likeprotein1 Left-rightdeterminationfactor2 Lysozyme-likeprotein6 InhibinbetaEchain Kelch-likeprotein11ProteinWnt-16 acyltransferasedelta Carbonicanhydrase14 NeuropeptideW N-acetylglucosaminyltransferaseB relatedprotein3 NKG2Dligand2 Kallikrein-13 Kallikrein-9 acidreceptor1Cystatin-SA Macrophagemetalloelastase SerpinA9 Agrin Prolactin Properdin Reelin Keratocan cells1 Secretin

Embryonicgrowth/differentiationfactor1 Pregnancy-specificbeta1glycoprotein Melanomainhibitoryactivityprotein2 Pregnancy-specificbeta1glycoprotein8 ProbableG-proteincoupledreceptor171 Microfibril-associatedglycoprotein4 Spermacrosomemembrane-associated MAMdomain-containingprotein2 Microfibrillar-associatedprotein2 Matrixmetalloproteinase-19 containingprotein3 Lamininsubunitgamma-3 Neurotensin/neuromedinN. Matrixmetalloproteinase-24Matrixmetalloproteinase-25 Alpha-Nacetylgalactosaminidealpha Alpha-Nacetylgalactosaminidealpha FMRFamide-relatedpeptides containingprotein1 Stromelysin-2 Pappalysin-2 Neuromedin-B Insulin-likepeptideINSL6 ProteinWnt-2b Neurexophilin-2 Plateletfactor4variant Prolactin-releasingpeptide Interleukin-8 Gremlin-2 Interleukin-11 Interleukin-17A Interleukin-18 Interleukin-26 Interleukin-29 2,6-sialyltransferase1 2,6-sialyltransferase3 Otoconin-90 V-setandtransmembranedomain Serineprotease33 Mimecan protein4 Netrin-1 Netrin-3 protein Nociceptin Retbindin TABLE1-continued Thyrotropin-releasinghormonedegradingMelanomaderivedgrowthregulatory Leucine-richrepeatcontainingprotein31Lysyloxidasehomolog3 Immunoglobulinlambda-likepolypeptide1Neurexophilin Proline-richprotein4LeucinerichrepeatLGIfamilymember2 Pregnancy-specificbeta1glycoprotein10Interleukin28A Leucine-richrepeattransmembraneproteinTransmembraneemp24domain glucosamine3-0sulfateHeparan Pancreaticlipase-relatedprotein1 Matrix-remodelingassociatedprotein5 Basicsalivaryproline-richprotein3 Collagenandcalcium-bindingEGF Germcell-specificgene1likeprotein subunit-1BsecretaseAPHGamma Caspaserecruitmentdomain-containing Cholinetransporter-likeprotein3 17-betahydroxysteroiddehydrogenase14Neurturin DnajhomologsubfamilyBmember14 Methionine-RsulfoxidereductaseB3, ProbableG-proteincoupledreceptor78 Interleukin-6receptorsubunitalpha Leucine-richalpha2glycoprotein Hepatocytegrowthfactorreceptor domain-containingprotein1 Neutrophildefensin4Amphoterin-inducedprotein3 ApolipoproteinIVC- VesicletransportproteinGOT1B IntegralmembraneproteinGPR177 HEPACAMfamilymember2 Interleukin-4 Interleukin-24 memberILipase C-motifchemokine22 Dipeptidase2 Apolipoprotein0 ArylsulfataseG Glia-activatingfactor sulfotransferase3A1 F-boxonlyprotein8 Ladinin-1 Netrin-4 Nyctalopin Osteocalcin FLRT2 R-spondin3Sialoadhesin Trypsin-3 Dystroglycan 18protein ectoenzyme Guanylin Fibroleukin mitochondrial

Transforminggrowthfactorbeta-2 Fibroblastgrowthfactor-bindingprotein1 Vasopressin-neurophysin2copeptin Phosphatidylinositol-glycanspecific Protransforminggrowthfactoralpha Inter-alphatrypsininhibitorheavychain Lamininsubunitalpha-2 Lamininsubunitalpha-4 Lamininsubunitbeta-1 VitaminK-dependentproteinZ Salivaryacidicproline-rich phosphoprotein/21 Transferrinreceptorprotein1 Tumornecrosisfactorligandsuperfamily Tumornecrosisfactorreceptorsuperfamily Tumornecrosisfactorreceptorsuperfamily Cysteine-richsecretoryprotein2 Cysteine-richwithEGFlikedomain Interleukin-1familymember5Interleukin-family1member 9 Iggamma-2chainCregion Iggamma-3chainCregion Protein-lysine6oxidase PhospholipaseA2, Phospholipidtransferprotein Pregnancyzoneprotein Haptoglobin-relatedprotein UPF0378proteinKIAA0100 Kininogen-1 Multimerin-1 phospholipaseD Prostaticacidphosphatase Semaphorin-4DSlithomologprotein2 Trefoilfactor3 Acidicmammalianchitinase C-motifchemokine26 C-Xmotifchemokine16 Insulin-like3 Nidogen-1 Perforin-1 Fibrocystin ProrelaxinH2 Alpha-tectorinTenascin-X member6 member1B member5 Thrombopoietin VIPpeptides 11Collectin- protein2 Kallikrein-5 US 2017 /0240590 A1 Aug . 24 , 2017

domainreceptorcysteine-richScavenger Sclerostindomain-containingprotein1 Post-GPIattachmenttoproteinsfactor3 WAPfour-disulfidecoredomainprotein3 Adipocyteplasmamembrane-associated Coiled-coildomaincontainingprotein126 Fibronectintype-IIIdomaincontaining Lysocardiolipinacyltransferase1 C3andPZP-likealpha2macroglobulin Germcell-specificgene1protein Adisintegrinandmetalloproteinasewith thrombospondinmotifs14 Progressiveankylosisproteinhomolog Angiopoietin-relatedprotein5 Alpha-1,3mannosylglycoprotein4beta domain-containingprotein8Retinoicacidreceptor responder Cartilageacidicprotein1 containingprotein4 containinggroupBprotein Tumornecrosisfactorreceptor superfamilymember27 Thioredoxindomain-containing Chitinase-3likeprotein1 Proteincanopyhomolog4 N-acetylglucosaminyltransferaseA PlasmaglutamatecarboxypeptidaseSlithomolog3protein Stanniocalcin-1 Interleukin-21receptorV-setandimmunoglobulindomain Semaphorin-4A Toll-likereceptor7 UPF0672proteinCXorf36ArylsulfataseJ Plateletbasicprotein protein2 protein16Alpha-2antiplasmin Peroxidasinhomolog CD177antigen proteinC4orf31 Interferonepsilon Intelectin-2 Beta-tectorin Prothyroliberin ProteinWFDC9 protein Cortistatin Ceruloplasmin ProteinFAM180A

Secretoglobinfamily1Cmember1 Secretoglobinfamily1Dmember1Secretoglobinfamily1Dmember2 Uncharacterizedaarfdomain-containing Adisintegrinandmetalloproteinasewith thrombospondinmotifs15Sodiumchannelsubunitbeta-2 Metalloproteinaseinhibitor4 Adisintegrinandmetalloproteinasewith motifs10thrombospondin Transmembraneprotein130 Wntprotein-3Protooncogene Zonapellucida-bindingprotein1 Anteriorgradientprotein3homolog Fibroblastgrowthfactor18 Ly6/PLAURdomain-containingprotein6 Chymotrypsin-likeelastasefamily FMRFamide-relatedpeptides domain-containingprotein likeprotein9-related CytokineSCM-1beta UncharacterizedproteinC5orf46 RibonucleaseK6Ribonuclease T2 3GSemaphorin- T-cellimmunomodulatoryprotein Thymicstromallymphopoietin Urocortin-2 Urocortin-3( relatedprotein88 ProteinWnt-10a ProteinWnt-3a ProteinWnt-6 ProteinWnt-9a proteinkinase1 C-Xmotifchemokine11 Repetin protein AER61 SerpinA12 Serpin12 protein ProteinAMBP Amelotin Draxin member1 TABLE1-continued ComplementC1rsubcomponent-likeLeukocyteimmunoglobulinreceptor vonWillebrandfactorCandEGFOsteopetrosis-associatedtransmembrane Uniquecartilagematrix-associatedDisintegrinandmetalloproteinasedomain VonWillebrandfactorDandEGFdomainComplementClqtumornecrosis GrowthinhibitionanddifferentiationAdisintegrinmetalloproteinasewith Pregnancy-specificbeta1glycoprotein9Zymogengranulemembraneprotein16 UncharacterizedglycosyltransferaseLeucine-richrepeatandfibronectintypeIII GPItransamidasecomponentPIG-S Transmembrane9superfamilymember3 Transmembranechannel-likeprotein2Pregnancy-specificbeta1glycoprotein3 Thioredoxindomain-containingprotein5 Tudordomain-containingprotein10 VascularendothelialgrowthfactorD Acidphosphatase-likeprotein2 Interleukin-27receptorsubunitalpha KTELmotif-containingprotein1 domain-containingprotein3 Protocadherinalpha-10 Protocadherinbeta-10 alpha2,6galactoside-Beta Proline-richtransmembraneprotein3 Adisintegrinandmetalloproteinasewith thrombospondinmotifs16 SH2domain-containingprotein3A Transducinbeta-likeprotein2 thrombospondinmotifs17 ApolipoproteinO-like Adisintegrinandmetalloproteinasewith thrombospondinmotifs12 Betagalactosidase-1likeproteinLysozymeg-likeprotein1 Inter-alphatrypsininhibitorheavychain Proenkephalin-AIntegrinalpha-10 subfamilyAmember5 Protocadherin-12 sialyltransferase1 Sulfhydryloxidase2 SHC-transformingprotein4 containingprotein23 Netrin-G1ligand Pannexin-1 containingprotein Tetraspanin-6 Semaphorin-3F Uteroglobin protein1 Tenomodulin Beta-defensin119 ProteinFAM131A ProteinFAM3B H5-likeprotein

Lysophosphatidicacidphosphatasetype6 ApolipoproteinA-II(ApoAII)APOAApolipoproteinA-IV(ApoAIV)APOA ApolipoproteinC-II(ApoCII)ApoC CellsurfaceglycoproteinCD200receptor1 containingdomain-Thrombospondintype1 WNT1inducible-signalingpathwayprotein2 Signalpeptide,CUBandEGF-likedomain ComplementcomponentC8alphachain Bromodomain-containingprotein9 acetylgalactosaminyltransferase14 Leucine-richrepeatcontainingprotein17 Leucine-richrepeatneuronalprotein2 vonWillebrandfactorAdomain-containing C-typelectindomainfamily1memberB NucleotideexchangefactorSILI CD99antigen-likeprotein2 Carbohydratesulfotransferase12ProbableserinecarboxypeptidaseCPVL CUBandzonapellucida-likedomain BifunctionalheparansulfateN deacetylase/N-sulfotransferase3 Disintegrinandmetalloproteinasedomain Apoptosis-relatedprotein3 Histo-bloodgroupABOsystemtransferase chainalpha(VI)Collagen-1 ComplementcomponentC9 UncharacterizedproteinClorf159 containingprotein1 Brainmitochondrialcarrierprotein containingprotein3 Alpha-1acidglycoproteinAlpha-1acidglycoprotein2 containingprotein9 Calcium-activatedchloridechannel Glucose-fructoseoxidoreductasedomain containingprotein2 Beta-2glycoprotein1 PolypeptideN 14-3proteinsigma Beta-secretase2 CathepsinL2 C-motifchemokine3 Matrilin-2 protein4 Mucin-3A Galectin-9 Tuftelin protein1 Angiotensinogen regulator1 Chymase US 2017 /0240590 A1 Aug . 24 , 2017 EC

cDNAFLJ77863,highlysimilartoHomo immunoglobulin-likePutativekillercell Carcinoembryonicantigen-relatedcell Glycosyltransferase8domain-containing Notchhomolog2N-terminallikeprotein sapienssecretedandtransmembrane1 Epididymal-specificlipocalin6 Probablecationtransporting-ATPase SecretoryphospholipaseA2receptor Bonemorphogeneticprotein3 Bonemarrowstromalantigen2 Bactericidal/permeability-increasing GroupIIFsecretoryphospholipaseA2 GDNFfamilyreceptoralpha-like Probableglutathioneperoxidase8 Solutecarrierfamily22member12 Guanylatecyclaseactivator2B Glutathioneperoxidase3 KIR3DP1proteinreceptorlike Proteindpy-19homolog2 Plateletactivating-factoracetylhydrolase Chorionicsomatomammotropinhormone Regulatorofmicrotubuledynamics Catechol-Omethyltransferasedomain Tripeptidyl-peptidase1 Trem-liketranscript1protein Lamininsubunitbeta-2 Matrixextracellularphosphoglycoprotein adhesionmolecule20 2041P450Cytochrome CarboxypeptidaseB Pappalysin-1 Retinoldehydrogenase14 Transcobalamin-2 containingprotein1 InducibleT-cellcostimulator Neuropilin2- (SECTM1),mRNA Claudin-18 protein-like3 protein2 ProteinFAM19A1 Cystatin-D Cystatin-F protein3 Afamin 13A5 Haptoglobin like1 Galanin

Carcinoembryonicantigen-relatedcell Secretoglobinfamily1Dmember4 SisterchromatidcohesionproteinDCC1 Dyneinheavychaindomain-containing ExtracellularsulfataseSulf-2 anchorglycosylphosphatidylinositol Matrixmetalloproteinase-27 Inactiveserineprotease35 UncharacterizedproteinC2orf82 Insulingrowthfactor-likefamily Bonemorphogeneticprotein15 Polymericimmunoglobulinreceptor Hyaluronanandproteoglycanlink domain-containingprotein30 Suppressoroffusedhomolog Collagenalpha-1(XII)chain Collagenalpha-1(XIV)chain Lamininsubunitalpha-3 Erythropoietinreceptor MAMdomain-containing Coiled-coildomaincontaining containingprotein2A Cadherin-likeprotein29 Plasmaserineproteaseinhibitor adhesionmolecule21 Galectin-3bindingprotein Fattyacyl-CoAreductase1Finbudinitiationfactorhomolog Prion-likeproteindoppel Disintegrinandmetalloproteinase Tumornecrosisfactorreceptor superfamilymember14 V-setandtransmembranedomain C-motifchemokine17 C-Xmotifchemokine6 C-Xmotifchemokine10 Folatereceptorbeta Interleukin-21 Interleukin-3 Interleukin-7 Inhibinalphachain protein2 protein134 member1 Alpha-lactalbumin 1protein Beta-defensin1 protein2 Beta-defensin2 Suprabasin ADM Artemin TABLE1-continued

Spermacrosome-associatedprotein5 Multipleepidermalgrowthfactor-like Arylacetamidedeacetylase-like1 vonWillebrandfactorAdomain-containing Dehydrogenase/reductaseSDRfamily CarboxypeptidaseNsubunit2 Collagentriplehelixrepeat-containing likedomain-containingnogoreceptor protein2associatedSurfactant-Adiponectinreceptorprotein1 WAP,kazalimmunoglobulinkunitzandNTRdomain-containingprotein2 Pro-neuregulin3,membranebound Lamininsubunitgamma-1 HEATrepeat-containingproteinC7orf27Collagenalpha-2(IX)chain Collagenalpha-3(IX)chain Collagenalpha-1(XXVII)chain ZincfingerRAD18domain-containing Growthdifferentiationfactor15 Earlyplacentainsulin-likepeptide Leucine-richrepeatandimmunoglobulin interactingprotein2 7B2proteinNeuroendocrine Cathelicidinantimicrobialpeptide Progonadoliberin-1 Interferonalpha-17 Interferonalpha-21Interferonalpha-8 Interferonomega-1 glycoproteinAlpha1B- Agouti-signalingprotein UPF0454proteinC12orf49 Leucine-richrepeattransmembrane4 neuronalprotein Endothelin-2 Fcreceptor-likeB Histatin-3 Claudin-8 5B1protein Cadherin-6 member7B C-motifchemokine16 C-motifchemokine24 protein1 Clorf124protein Glia-derivednexin GranzymeK Widdomains isoform Colipase Fibromodulin

Receptortyrosine-proteinkinaseerbB3 DnajhomologsubfamilyBmember11 Endoplasmicreticulumaminopeptidase1 ComplementfactorH-relatedprotein1 MajorhistocompatibilitycomplexclassI Insulin-likegrowthfactorbindingprotein6 Oxidizedlow-densitylipoproteinreceptor1 Pulmonarysurfactant-associatedproteinDStimulatedbyretinoicacidgene6protein familyphosphodiesterasepyrophosphatase/ Endoplasmicreticulumresidentprotein acetylgalactosaminyltransferase2 Low-densitylipoproteinreceptorrelated JunctionaladhesionmoleculeC FibronectintypeIIIdomain-containing Prostatetumoroverexpressedgene1 Receptor-interactingserine/threonine Equilibrativenucleosidetransporter3 Tissuefactorpathwayinhibitor2 Intercellularadhesionmolecule4 Hepatocytegrowthfactoractivator UncharacterizedproteinKIAA0319Lamininsubunit alpha-5 Matrixmetalloproteinase-9 KinofIRRE-likeprotein1 IgGFc-bindingprotein IgdeltachainCregion Interleukin-1beta proteinkinase2 SelenoproteinP Toll-likereceptor9 Interleukin-19 NPolypeptide relatedgeneprotein LipoproteinlipaseInterstitialcollagenase Trefoilfactor1 Isthmin-2 Kallikrein-10 Ectonucleotide member7 ERp44 Hemopexin protein10 protein4 Mucin-16 Mucin-2 Mucin-5B Myocilin protein homolog Prothrombin US 2017 /0240590 A1 Aug . 24 , 2017 24

Regeneratingislet-derivedprotein4 ComplementClqtumornecrosisfactor Basicsalivaryproline-richprotein4 Dehydrogenasereductase/SDRfamily oligosaccharide1,2-alphamannosidase EGF-containingfibulinlikeextracellular proteintyrosinetype-Receptor Matrixmetalloproteinase-23 Pentraxin-relatedproteinPTX3 Majorfacilitatorsuperfamilydomain Butyrophilin-likeprotein3Butyrophilin-likeprotein9 Lamininsubunitgamma-2 Endoplasmicreticulummannosyl Pancreaticsecretorygranulemembrane Carboxylesterase8 Thioredoxin-relatedtransmembrane containingprotein2 Tumornecrosisfactorreceptor superfamilymember18 Beta-1,4galactosyltransferase GP2glycoproteinmajor Semaphorin-4B matrixprotein1 phosphatasekappa Tachykinin4- relatedprotein5 Pre-small/secretedglycoprotein Kallikrein-12 Brevicancoreprotein C-motifchemokine23 BrotherofCDO Tenascin-R Opticin protein4 Porimin Torsin-1A Testican-3 member9 Eppin Otoancorin Growthfactor ProteinTSPEAR Hephaestin ProteinLMBRIL Mucin-21

Pregnancy-specificbeta1glycoprotein6 proteinderived3Regeneratingislet- Transmembraneandcoiled-coildomain GDNFfamilyreceptoralpha-3 Magnesiumtransporterprotein1 FXYDdomain-containingiontransport VEGFco-regulatedchemokine1 Amiloride-sensitiveamineoxidase TransmembraneproteinC17orf87 chromosomeXmemberon Serineincorporator2 Secretedphosphoprotein1 RINGfingerprotein43 Semenogelin-2 Growth-regulatedalphaprotein containingprotein3 Progonadoliberin-2 Stromelysin-3 Bonesialoprotein2 Delta-likeprotein1 PlateletreceptorGi24 Kallikrein-7 VIP36-likeprotein [copper-containing] modulatorprotein2 regulator6 LACTB,mitochondrial Galectin-3 Pancreaticprohormone member11 Mucin-15 R-spondin2 Ephrin-A1 Fibroblastgrowthfactorreceptor-like1Choriogonadotropinsubunitbetavariant2 Dehydrogenase/reductaseSDRfamilyImmunoglobulinsuperfamilycontaining gamma Lymphotactin ADM2 1-likeprotein ProteinCASC4 TABLE1-continued PeptidoglycanrecognitionproteinI-betaDickkopfrelated3 EGFlatrophilin,andseventransmembraneDehydrogenase/reductaseSDRfamily Serinebeta-lactamaselikeproteinLeucinerichrepeattransmembraneprotein Insulin-likegrowthfactorbindingproteinHydroxysteroid11betadehydrogenase AdisintegrinandmetalloproteinasewithDNAdamage-regulatedautophagy WAPfour-disulfidecoredomainprotein6ApolipoproteinF Transforminggrowthfactorbeta-3 Carcinoembryonicantigen-relatedcell Fibronectintype3andankyrinrepeat TranslocationproteinSEC63homolog kexinsubtilisinconvertase/Proprotein domain-containingprotein1 Lysyloxidasehomolog4 leucine-richrepeatprotein2 containingprotein2 Retinol-bindingprotein3 complexacidlabilechain isoformAmelogenin,Y thrombospondinmotifs1ProteinCOQ10A,mitochondrial domainsprotein1 Nucleobindin-2 PhospholipaseA2 Proenkephalin-B V-setandimmunoglobulindomain ProteinWnt-10b CarboxypeptidaseA4 Olfactomedin-4 ArylsulfataseF adhesionmolecule19 YYPeptide Beta-defensin104 Beta-defensin105 Beta-defensin107 Claudin-6 Lumican Adropin FLRT1 Atherin Renalase type4 ProteinWFDC11 Epigen FAM19A5Protein

Pairedimmunoglobulin-liketype2receptor proteinalphaderived3Regeneratingislet- Scavengerreceptorcysteine-richtype1 Secretedfrizzled-relatedprotein1,isoform WAPfour-disulfidecoredomainprotein5 Latent-transforminggrowthfactorbeta Plasminogen-relatedproteinB Angiopoietin-relatedprotein1 Beta-galactosidase1likeprotein2 PDZdomain-containingprotein11 Shortpalate,lungandnasalepithelium carcinoma-associatedprotein2 Platelet-derivedgrowthfactorC Disintegrinandmetalloproteinasedomain BSDdomain-containingprotein1 DiacylglycerolO-acyltransferase2 Integratorcomplexsubunit1moleculelikeadhesion- Junctional Methionineadenosyltransferase2 Podocalyxin-likeprotein2 E3ubiquitin-proteinligaseRNF5 ProbablepalmitoyltransferaseZDHHC16 ERdegradation-enhancingalphamannosidaselike2 - Interleukin-17receptorE Retinoid-inducibleserinecarboxypeptidase protein33containing Celladhesionmolecule3 Interleukin-17receptorC Interleukin-17receptorD E3ubiquitin-proteinligaseLNX bindingprotein4 Protachykinin-1 UPF0510proteinC19orf63 Interleukin-20 Interleukin-25 CDC45-relatedprotein Leucine-richrepeattransmembraneneuronalprotein3 proteinM160 Relaxin-3 3-ketosteroidreductase alpha CRAa Chondrolectin subunitbeta US 2017 /0240590 A1 Aug . 24 , 2017 S?

ScavengerreceptorclassAmember5 Latent-transforminggrowthfactorbeta Secretedandtransmembrane1precusor Proproteinconvertasesubtilisin/kexin Guanylatebinding-protein5 ProteinRMD5homologB Transmembraneprotein108Sushidomain-containingprotein3 Collagenalpha-1(XX)chain ATP-dependentmetalloproteaseYME1L1 Semaphorin-5B Ectonucleosidetriphosphatediphosphohydrolase6 Semaphorin-6B bindingprotein2Putativeuncharacterizedprotein NetrinreceptorUNC5D Epsilon-sarcoglycan SerpinB3 UNQ6190/PRO20217 Mucin-13 variant type5

ComplementfactorH-relatedprotein5 Transmembraneandubiquitin-like ComplementClqtumornecrosisfactor Sialicacid-bindingIglikelectin11 Secretedandtransmembrane1precusor Cysteine-richsecretoryprotein3 Neuroblastomasuppressorof FK506-bindingprotein7Serine incorporator1 domain-containingprotein1 ComplementC4-A Putativeuncharacterizedprotein Calcium-activatedchloridechannel Toll-likereceptor8 relatedprotein1 UNQ6494/PRO21346 regulator2 variant PRO2829 Cateyesyndromecriticalregionprotein1C-typelectindomainfamily18memberA TABLE1-continued LipomaHMGICfusionpartner-like1proteinProteinERGIC53like Leucine-richrepeatcontainingprotein18Tolllikereceptor10 Ly6/PLAURdomain-containingprotein4Sortingnexin24 AdisintegrinandmetalloproteinasewithPutativeuncharacterizedprotein Leucine-richrepeatcontainingprotein3BSelenoproteinT receptor-likeproteinKIR3DX1(Leukocytetumorigenicity1 Leucine-richrepeatcontainingprotein25 Leucine-richrepeatcontainingprotein3 Putativekillercellimmunoglobulin-like UncharacterizedproteinC19orf41UncharacterizedproteinC21orf63 Cocaine-andamphetamineregulated VitaminKepoxidereductasecomplex thrombospondinmotifs20 Transmembraneandimmunoglobulin domain-containingprotein1 receptorclustermember12) Proteindeltahomolog2 transcriptprotein Putativeuncharacterizedprotein Testis-expressedprotein101 Xylosyltransferase2 subunit1 ENSP00000381830 ProteinFAM20A

Membrane-boundtranscriptionfactorsite1 Lactosylceramidealpha-2,3 SID1transmembranefamilymember2 Transmembraneprotease,serine2 galactosamide-alpha2,3sialyltransferase containing)3(HakataantigenNL3 containing)3(Hakataantigen,isoform Plexindomain-containingprotein2 Sushidomain-containingprotein1 Serine/threonine-proteinkinaseTAO2 UDP-glucuronicaciddecarboxylase1 Transmembraneprotein119Transmembraneprotein98 C14orf144proteinPutativeuncharacterized Ficolin(Collagen/fibrinogendomain Roundabouthomolog4 UncharacterizedproteinC10orf58 Thioredoxin-relatedtransmembrane CMP-Nacetylneuraminatebeta Putativeuncharacterizedprotein Pre-Blymphocyteprotein3 (FicolinCollagen/fibrinogendomain Prominin-2 TIEsialyltransferase protein2 protease CRA_b) ENSP00000380674 US 2017 /0240590 A1 Aug . 24 , 2017 26

[0073 ] The therapeutic proteins provided herein should amounts of its activated form FVIIa . This binding facilitates not be considered to be exclusive . Rather, as is apparent full conversion of FVII to FVIIa and subsequently , in the from the disclosure provided herein , the methods of the presence of calcium and phospholipids, the conversion of invention are applicable to any protein wherein attachment factor IX (FIX ) to factor IXa (FIXa ) and factor X (FX ) to of a water soluble polymer is desired according to the factor Xa ( FXa ) . The association of FVIla with tissue factor invention . For example , therapeutic proteins are described in enhances the proteolytic activity by bringing the binding US 2007 /0026485 , incorporated herein by reference in its sites of FVII for the substrate ( FIX and FX ) into closer entirety . proximity and by inducing a conformational change , which enhances the enzymatic activity of FVIIa . Blood Coagulation Proteins 10080 ] The activation of FX is the common point of the [0074 ] In one aspect , the starting material of the present two pathways. Along with phospholipid and calcium , factors invention is a blood coagulation protein , which can be Va (FVa ) and Xa convert prothrombin to thrombin (pro derived from human plasma, or produced by recombinant thrombinase complex ) , which then cleaves fibrinogen to engineering techniques, as described in U . S . Pat . No . 4 ,757 , form fibrin monomers . The monomers polymerize to form 006 ; U . S . Pat. No . 5 ,733 , 873 ; U . S . Pat. No . 5 , 198 , 349 ; U . S . fibrin strands . Factor XIIIa (FXIIIa ) covalently bonds these Pat . No . 5 , 250 , 421 ; U . S . Pat . No . 5 ,919 , 766 ; and EP 306 strands to one another to form a rigid mesh . 968 . [0081 ] Conversion of FVII to FVIIa is also catalyzed by a [0075 ] Therapeutic polypeptides such as blood coagula number of proteases , including thrombin , FIXa , FXa, factor tion proteins including Factor IX (FIX ) , Factor VIII ( FVIII ) , Xla (FXla ), and factor XIIa (FXIIa ). For inhibition of the Factor VIIa ( FVIIa ) , Von Willebrand Factor (VWF ) , Factor early phase of the cascade, tissue factor pathway inhibitor FV ( FV ) , Factor X ( FX ) , Factor XI ( FXI) , Factor XII ( FXII ) , targets FVIIa / tissue factor/ FXa product complex . thrombin (FII ), protein C , protein S , tPA , PAI- 1 , tissue factor ( TF ) and ADAMTS 13 protease are rapidly degraded by Factor VIIa proteolytic enzymes and neutralized by antibodies. This [0082 ] FVII (also known as stable factor or proconvertin ) reduces their half- life and circulation time, thereby limiting is a vitamin K - dependent serine protease glycoprotein with their therapeutic effectiveness. Relatively high doses and a pivotal role in hemostasis and coagulation (Eigenbrot , frequent administration are necessary to reach and sustain Curr Protein Pept Sci. 2002 ; 3 :287 - 99 ) . the desired therapeutic or prophylactic effect of these coagu [0083 ] FVII is synthesized in the liver and secreted as a lation proteins. As a consequence, adequate dose regulation single - chain glycoprotein of 48 kD . FVII shares with all is difficult to obtain and the need of frequent intravenous vitamin K - dependent serine protease glycoproteins a similar administrations imposes restrictions on the patient ' s way of protein domain structure consisting of an amino - terminal living . gamma- carboxyglutamic acid (Gla ) domain with 9 - 12 resi [ 0076 ] As described herein , blood coagulation proteins dues responsible for the interaction of the protein with lipid including, but not limited to , Factor IX ( FIX ), Factor VIII membranes , a carboxy - terminal serine protease domain ( FVIII ), Factor VIIa (FVIIa ), Von Willebrand Factor (VWF ) , (catalytic domain ) , and two epidermal growth factor - like Factor FV ( FV ) , Factor X (FX ), Factor XI, Factor XII domains containing a calcium ion binding site that mediates (FXII ), thrombin (FII ), protein C , protein S , DPA , PAI- 1, interaction with tissue factor. Gamma- glutamyl carboxylase tissue factor ( TF ) and ADAMTS 13 protease are contem catalyzes carboxylation of Gla residues in the amino - termi plated by the invention . As used herein , the term “ blood nal portion of the molecule. The carboxylase is dependent on coagulation protein ” refers to any Factor IX ( FIX ) , Factor a reduced form of vitamin K for its action , which is oxidized VIII ( FVIII ) , Factor VIIa ( FVIIa ) , Von Willebrand Factor to the epoxide form . Vitamin K epoxide reductase is (VWF ) , Factor FV (FV ), Factor X ( FX ), Factor XII ( FXII ), required to convert the epoxide form of vitamin K back to thrombin ( FII ) , protein C , protein S , tPA , PAI- 1, tissue factor the reduced form . ( TF ) and ADAMTS 13 protease which exhibits biological [ 0084 ] The major proportion of FVII circulates in plasma activity that is associated with that particular native blood in zymogen form , and activation of this form results in coagulation protein . cleavage of the peptide bond between arginine 152 and [ 0077 ] The blood coagulation cascade is divided into three isoleucine 153 . The resulting activated FVIIa consists of a distinct segments : the intrinsic , extrinsic , and common path NH2- derived light chain (20 KD ) and a COOH terminal ways (Schenone et al. , Curr Opin Hematol. 2004 ; 11 :272 - 7 ) . derived heavy chain ( 30 kD ) linked via a single disulfide The cascade involves a series of serine protease enzymes bond (Cys 135 to Cys 262) . The light chain contains the ( zymogens) and protein cofactors . When required , an inac membrane- binding Gla domain , while the heavy chain con tive zymogen precursor is converted into the active form , tains the catalytic domain . which consequently converts the next enzyme in the cas [0085 ] The plasma concentration of FVII determined by cade . genetic and environmental factors is about 0 . 5 mg /mL [0078 ] The intrinsic pathway requires the clotting factors (Pinotti et al. , Blood . 2000 ; 95 :3423 -8 ) . Different FVII VIII, IX , X , XI, and XII . Initiation of the intrinsic pathway genotypes can result in several- fold differences in mean occurs when prekallikrein , high -molecular -weight kinino FVII levels . Plasma FVII levels are elevated during preg gen , factor XI (FXI ) and factor XII ( FXII ) are exposed to a nancy in healthy females and also increase with age and are negatively charged surface . Also required are calcium ions higher in females and in persons with hypertriglyceridemia . and phospholipids secreted from platelets . FVII has the shortest half -life of all procoagulant factors [ 0079 ] The extrinsic pathway is initiated when the vascu ( 3 - 6 h ) . The mean plasma concentration of FVIIa is 3 . 6 lar lumen of blood vessels is damaged . The membrane ng/ mL in healthy individuals and the circulating half - life of glycoprotein tissue factor is exposed and then binds to FVIIa is relatively long ( 2 . 5 h ) compared with other coagu circulating factor VII (FVII ) and to small preexisting lation factors. US 2017 /0240590 A1 Aug . 24 , 2017 27

[0086 ] Hereditary FVII deficiency is a rare autosomal [0093 ] FVIII is synthesized as a single -chain precursor of recessive bleeding disorder with a prevalence estimated to approximately 270 -330 kD with the domain structure be 1 case per 500 , 000 persons in the general population A1- A2 - B - A3- C1 -C2 . When purified from plasma ( e. g. , (Acharya et al . , J Thromb Haemost. 2004 ; 2248 - 56 ) . " plasma- derived ” or “ plasmatic " ) , FVIII is composed of a Acquired FVII deficiency from inhibitors is also very rare . heavy chain (A1 - A2 - B ) and a light chain ( A3 -C1 - C2 ). The Cases have also been reported with the deficiency occurring molecular mass of the light chain is 80 kD whereas, due to in association with drugs such as cephalosporins, penicillins , proteolysis within the B domain , the heavy chain is in the and oral anticoagulants . Furthermore , acquired FVII defi range of 90 -220 kD . ciency has been reported to occur spontaneously or with [0094 ] FVIII is also synthesized as a recombinant protein other conditions, such as myeloma, sepsis, aplastic anemia , for therapeutic use in bleeding disorders . Various in vitro with interleukin - 2 and antithymocyte globulin therapy. assays have been devised to determine the potential efficacy [ 0087 ] Reference polynucleotide and polypeptide of recombinant FVIII (rFVIII ) as a therapeutic medicine . sequences include, e. g ., GenBank Accession Nos. J02933 These assays mimic the in vivo effects of endogenous FVIII . for the genomic sequence , M13232 for the cDNA (Hagen et In vitro thrombin treatment of FVIII results in a rapid al. PNAS 1986 ; 83: 2412 - 6 ) , and P08709 for the polypeptide increase and subsequent decrease in its procoagulant activ sequence (references incorporated herein in their entireties ) . ity , as measured by in vitro assays. This activation and A variety of polymorphisms of FVII have been described , inactivation coincides with specific limited proteolysis both for example see Sabater- Lleal et al. (Hum Genet . 2006 ; in the heavy and the light chains, which alter the availability 118 :741 - 51 ) ( reference incorporated herein in its entirety ) . of different binding epitopes in FVIII , e . g . allowing FVIII to dissociate from VWF and bind to a phospholipid surface or Factor IX altering the binding ability to certain monoclonal antibodies . [ 0088 ] FIX is a vitamin K -dependent plasma protein that [0095 ] The lack or dysfunction of FVIII is associated with participates in the intrinsic pathway of blood coagulation by the most frequent bleeding disorder, hemophilia A . The converting FX to its active form in the presence of calcium treatment of choice for the management of hemophilia A is ions , phospholipids and FVIIIa . The predominant catalytic replacement therapy with plasma derived or rFVIII concen capability of FIX is as a serine protease with specificity for trates . Patients with severe hemophilia A with FVIII levels a particular arginine - isoleucine bond within FX . Activation below 1 % , are generally on prophylactic therapy with the of FIX occurs by FXla which causes excision of the acti aim of keeping FVIII above 1 % between doses. Taking into vation peptide from FIX to produce an activated FIX mol account the average half -lives of the various FVIII products ecule comprising two chains held by one or more disulphide in the circulation , this result can usually be achieved by bonds . Defects in FIX are the cause of recessive X - linked giving FVIII two to three times a week . hemophilia B . [ 0096 ] Reference polynucleotide and polypeptide [0089 ] Hemophilia A and B are inherited diseases charac sequences include, e .g ., UniProtKB /Swiss -Prot P00451 terized by deficiencies in FVIII and FIX polypeptides , ( FA8 _ HUMAN ) ; Gitschier J et al . , Characterization of the respectively . The underlying cause of the deficiencies is human Factor VIII gene , Nature , 312 (5992 ) : 326 - 30 ( 1984 ) ; frequently the result of mutations in FVIII and FIX genes, Vehar G H et al. , Structure of human Factor VIII, Nature , both of which are located on the X chromosome. Traditional 312 (5992 ) : 337 - 42 ( 1984 ); Thompson A R . Structure and therapy for hemophilias often involves intravenous admin Function of the Factor VIII gene and protein , Semin Thromb istration of pooled plasma or semi- purified coagulation Hemost, 2003 :29 ; 11 - 29 (2002 ). proteins from normal individuals . These preparations can be contaminated by pathogenic agents or viruses, such as Von Willebrand Factor infectious prions, HIV , parvovirus , hepatitis A , and hepatitis [0097 ] Von Willebrand factor (VWF ) is a glycoprotein C . Hence , there is an urgent need for therapeutic agents that circulating in plasma as a series ofmultimers ranging in size do not require the use of human serum . from about 500 to 20 , 000 kD . Multimeric forms of VWF are [0090 ] The level of the decrease in FIX activity is directly composed of 250 kD polypeptide subunits linked together proportional to the severity of hemophilia B . The current by disulfide bonds. VWF mediates initial platelet adhesion treatment of hemophilia B consists of the replacement of the to the sub - endothelium of the damaged vessel wall . Only the missing protein by plasma - derived or recombinant FIX larger multimers exhibit hemostatic activity . It is assumed ( so -called FIX substitution or replacement treatment or that endothelial cells secrete large polymeric forms of VWF therapy ) . and those forms of VWF which have a low molecular weight [0091 ] Polynucleotide and polypeptide sequences of FIX ( low molecular weight VWF ) arise from proteolytic cleav can be found for example in the UniProtKB /Swiss -Prot age . The multimers having large molecular masses are Accession No. P00740 , U .S . Pat. No. 6, 531 , 298 and in FIG . stored in the Weibel- Pallade bodies of endothelial cells and 1 (SEQ ID NO : 1 ) . liberated upon stimulation . [0098 ] VWF is synthesized by endothelial cells and mega Factor VIII karyocytes as prepro - VWF that consists to a large extent of [0092 ] Coagulation factor VIII (FVIII ) circulates in repeated domains . Upon cleavage of the signal peptide , plasma at a very low concentration and is bound non pro - VWF dimerizes through disulfide linkages at its C -ter covalently to Von Willebrand factor (VWF ). During hemo minal region . The dimers serve as protomers for multim stasis , FVIII is separated from VWF and acts as a cofactor erization , which is governed by disulfide linkages between for activated factor IX ( FIXa) -mediated FX activation by the free end termini. The assembly to multimers is followed enhancing the rate of activation in the presence of calcium by the proteolytic removal of the propeptide sequence and phospholipids or cellular membranes . (Leyte et al. , Biochem . J . 274 ( 1991 ) , 257 - 261) . US 2017 /0240590 A1 Aug . 24 , 2017

[0099 ] The primary translation product predicted from the herein , “ exogenous therapeutic protein ” includes a blood cloned cDNA of VWF is a 2813 - residue precursor polypep coagulation protein which does not originate from the mam tide (prepro - VWF ) . The prepro - VWF consists of a 22 amino mal intended to receive treatment. acid signal peptide and a 741 amino acid propeptide , with [0107 ] As used herein , “ plasma- derived blood coagulation the mature VWF comprising 2050 amino acids (Ruggeri Z . protein ” or “ plasmatic ” includes all forms of the protein A ., and Ware , J ., FASEB J . , 308 -316 ( 1993 ) . found in blood obtained from a mammal having the property [0100 ] Defects in VWF are causal to Von Willebrand participating in the coagulation pathway . disease (VWD ) , which is characterized by a more or less [0108 ] As used herein “ biologically active derivative ” or pronounced bleeding phenotype . VWD type 3 is the most “ biologically active variant” includes any derivative or severe form , in which VWF is completely missing , and variant of a molecule having substantially the same func VWD type 1 relates to a quantitative loss of VWF and its tional and / or biological properties of said molecule , such as phenotype can be very mild . VWD type 2 relates to quali binding properties, and /or the same structural basis , such as tative defects of VWF and can be as severe as VWD type 3 . a peptidic backbone or a basic polymeric unit. VWD type 2 has many sub forms, some being associated [0109 ] An " analog, ” such as a " variant” or a " derivative, " with the loss or the decrease of high molecular weight is a compound substantially similar in structure and having multimers . Von Willebrand disease type 2a (VWD - 2A ) is the same biological activity , albeit in certain instances to a characterized by a loss of both intermediate and large differing degree, to a naturally - occurring molecule . For multimers . VWD - 2B is characterized by a loss of highest example , a polypeptide variant refers to a polypeptide shar molecular -weight multimers . Other diseases and disorders ing substantially similar structure and having the same related to VWF are known in the art. biological activity as a reference polypeptide . Variants or [0101 ] The polynucleotide and amino acid sequences of analogs differ in the composition of their amino acid prepro - VWF are available at GenBank Accession Nos. sequences compared to the naturally - occurring polypeptide NM _ 000552 and NP _ 000543 , respectively . from which the analog is derived , based on one or more [0102 ] Other blood coagulation proteins according to the mutations involving ( i) deletion of one or more amino acid present invention are described in the art , e . g . Mann KG , residues at one or more termini of the polypeptide and /or one Thromb Haemost , 1999 ; 82 : 165 - 74 . or more internal regions of the naturally -occurring polypep tide sequence ( e . g . , fragments ), ( ii ) insertion or addition of A . Polypeptides one or more amino acids at one or more termini (typically an [ 0103] In one aspect, the starting material of the present " addition ” or “ fusion ” ) of the polypeptide and /or one or invention is a protein or polypeptide . As described herein , more internal regions (typically an " insertion ” ) of the natu the term therapeutic protein refers to any therapeutic protein rally -occurring polypeptide sequence or ( iii ) substitution of molecule which exhibits biological activity that is associated one or more amino acids for other amino acids in the with the therapeutic protein . In one embodiment of the naturally -occurring polypeptide sequence . By way of invention , the therapeutic protein molecule is a full -length example , a “ derivative ” is a type of analog and refers to a protein . polypeptide sharing the same or substantially similar struc [0104 ] Therapeutic protein molecules contemplated ture as a reference polypeptide that has been modified , e . g . , include full - length proteins , precursors of full length pro chemically . teins, biologically active subunits or fragments of full length [0110 ] A variant polypeptide is a type of analog polypep proteins, as well as biologically active derivatives and tide and includes insertion variants, wherein one or more variants of any of these formsof therapeutic proteins . Thus, amino acid residues are added to a therapeutic protein amino therapeutic protein include those that ( 1 ) have an amino acid acid sequence of the invention . Insertions may be located at sequence that has greater than about 60 % , about 65 % , about either or both termini of the protein , and / or may be posi 70 % , about 75 % , about 80 % , about 85 % , about 90 % , about tioned within internal regions of the therapeutic protein 91 % , about 92 % , a , about 94 % 93 % , abbot 94 % , about 95 % , amino acid sequence . Insertion variants , with additional about 96 % , about 97 % , about 98 % or about 99 % or greater residues at either or both termini, include for example , amino acid sequence identity , over a region of at least about fusion proteins and proteins including amino acid tags or 25 , about 50 , about 100 , about 200 , about 300 , about 400 , or other amino acid labels . In one aspect, the blood coagulation more amino acids, to a polypeptide encoded by a referenced protein molecule optionally contains an N - terminal Met , nucleic acid or an amino acid sequence described herein ; especially when the molecule is expressed recombinantly in and / or ( 2 ) specifically bind to antibodies , e . g . , polyclonal or a bacterial cell such as E . coli . monoclonal antibodies, generated against an immunogen [0111 ] In deletion variants , one or more amino acid resi comprising a referenced amino acid sequence as described dues in a therapeutic protein polypeptide as described herein herein , an immunogenic fragment thereof, and /or a conser are removed . Deletions can be effected at one or both termini vatively modified variant thereof. of the therapeutic protein polypeptide , and / or with removal [0105 ] According to the present invention , the term of one or more residues within the therapeutic protein amino " recombinant therapeutic protein ” includes any therapeutic acid sequence . Deletion variants , therefore , include frag protein obtained via recombinant DNA technology . In cer ments of a therapeutic protein polypeptide sequence . tain embodiments , the term encompasses proteins as [0112 ] In substitution variants, one or more amino acid described herein . residues of a therapeutic protein polypeptide are removed [ 0106 ] As used herein , " endogenous therapeutic protein ” and replaced with alternative residues . In one aspect , the includes a therapeutic protein which originates from the substitutions are conservative in nature and conservative mammal intended to receive treatment. The term also substitutions of this type are well known in the art. Alter includes therapeutic protein transcribed from a transgene or natively , the invention embraces substitutions that are also any other foreign DNA present in said mammal. As used non -conservative . Exemplary conservative substitutions are US 2017 /0240590 A1 Aug . 24 , 2017 29 described in Lehninger, [ Biochemistry , 2nd Edition ; Worth about 200 , about 250 , about 500 , about 1000 , or more Publishers , Inc ., New York ( 1975 ) , pp . 71 -77 ] and are set out nucleotides (up to the full length sequence of 1218 nucleo immediately below . tides of the mature protein ) , to a reference nucleic acid sequence as described herein . Exemplary " stringent hybrid Conservative Substitutions ization ” conditions include hybridization at 42° C . in 50 % formamide, 5xSSC , 20 mM Na. P04 , pH 6 .8 ; and washing in [ 0113 ] 1xSSC at 55° C . for 30 minutes . It is understood that variation in these exemplary conditions can be made based SIDE CHAIN on the length and GC nucleotide content of the sequences to CHARACTERISTIC AMINO ACID be hybridized . Formulas standard in the art are appropriate for determining appropriate hybridization conditions . See Non -polar (hydrophobic ): Sambrook et al. , Molecular Cloning: A Laboratory Manual A . Aliphatic ALIVP ( Second ed ., Cold Spring Harbor Laboratory Press, 1989 ) B . Aromatic FW $ 89 . 47 - 9 .51 . C . Sulfur -containing M D . Borderline G [0118 ] A “ naturally - occurring” polynucleotide or poly Uncharged -polar : peptide sequence is typically derived from a mammal A . Hydroxyl STY including , but not limited to , primate , e. g ., human ; rodent, B . Amides NQ e . g ., rat , mouse , hamster ; cow , pig, horse , sheep , or any C . Sulfhydryl mammal. The nucleic acids and proteins of the invention can D . Borderline be recombinant molecules ( e. g ., heterologous and encoding Positively charged (basic ) KRH the wild type sequence or a variant thereof, or non - naturally Negatively charged (acidic ) DE occurring ). [0114 ] Alternatively , exemplary conservative substitutions are set out immediately below . C . Production of Therapeutic Proteins Conservative Substitutions II [0119 ] Production of a therapeutic protein includes any method known in the art for (i ) the production of recombi [0115 ] nant DNA by genetic engineering , ( ii ) introducing recom binant DNA into prokaryotic or eukaryotic cells by , for example and without limitation , transfection , electropora EXEMPLARY tion or microinjection , ( iii ) cultivating said transformed ORIGINAL RESIDUE SUBSTITUTION cells , ( iv ) expressing therapeutic protein , e . g . constitutively Ala ( A ) Val, Leu , Ile or upon induction , and ( v ) isolating said blood coagulation Arg ( R ) Lys , Gln , Asn protein , e . g . from the culture medium or by harvesting the Asn ( N ) Gln , His , Lys , Arg Asp ( D ) Glu transformed cells , in order to obtain purified therapeutic Cys ( C ) Ser protein . Gln ( Q ) Asn Glu ( E ) Asp [0120 ] In other aspects , the therapeutic protein is produced His ( H ) Asn , Gln , Lys , Arg by expression in a suitable prokaryotic or eukaryotic host Ile ( 1 ) Leu , Val, Met, Ala , Phe, system characterized by producing a pharmacologically Leu ( L ) Ile , Val, Met, Ala , Phe acceptable blood coagulation protein molecule . Examples of Lys ( K ) Arg , Gln , Asn Met ( M ) Leu , Phe, Ile eukaryotic cells are mammalian cells , such as CHO , COS , Phe ( F ) Leu , Val , Ile , Ala HEK 293 , BHK , SK -Hep , and HepG2. Pro ( P ) Gly Ser ( S ) Thr 10121 ] A wide variety of vectors are used for the prepa Thr ( T ) Ser ration of the therapeutic protein and are selected from Trp (W ) Tyr eukaryotic and prokaryotic expression vectors . Examples of Tyr ( Y ) Trp, Phe , Thr, Ser vectors for prokaryotic expression include plasmids such as , Val ( V ) Ile , Leu , Met , Phe , Ala and without limitation , PRSET, PET, and PBAD , wherein the promoters used in prokaryotic expression vectors include one or more of, and without limitation , lac , trc , trp , recA , or B . Polynucleotides araBAD . Examples of vectors for eukaryotic expression include : ( i ) for expression in yeast, vectors such as , and [0116 ] Nucleic acids encoding a therapeutic protein of the without limitation , PAO , PPIC , PYES , or pMET, using invention include , for example and without limitation , promoters such as, and without limitation , AOX1 , GAP, genes, pre -mRNAs , mRNAs, cDNAs, polymorphic variants , GAL1, or AUG1; ( ii ) for expression in insect cells , vectors alleles , synthetic and naturally - occurring mutants . such as and without limitation , PMT, PAc5 , PIB , PMIB , or [ 0117 ] Polynucleotides encoding a therapeutic protein of PBAC , using promoters such as and without limitation PH , the invention also include , without limitation , those that ( 1 ) p10 ,MT , Ac5 , OplE2, gp64 , or polh , and ( iii ) for expression specifically hybridize under stringent hybridization condi in mammalian cells , vectors such as and without limitation tions to a nucleic acid encoding a referenced amino acid PSVL , PCMV, pRc/ RSV , pcDNA3 , or pBPV, and vectors sequence as described herein , and conservatively modified derived from , in one aspect, viral systems such as and variants thereof ; ( 2 ) have a nucleic acid sequence that has without limitation vaccinia virus, adeno -associated viruses , greater than about 95 % , about 96 % , about 97 % , about 98 % , herpes viruses , or retroviruses, using promoters such as and about 99 % , or higher nucleotide sequence identity , over a without limitation CMV , SV40 , EF - 1, UbC , RSV , ADV , region of at least about 25 , about 50 , about 100 , about 150 , BPV , and ß -actin . US 2017 /0240590 A1 Aug . 24 , 2017 30

D . Administration ologically acceptable reconstitution solution for the compo [0122 ] In one embodiment a conjugated therapeutic pro sition in the first container . In one aspect , the compound or tein of the present invention may be administered by injec composition is packaged in a unit dosage form . The kit may tion , such as intravenous , intramuscular, or intraperitoneal further include a device suitable for administering the com injection . position according to a specific route of administration . [ 0123] To administer compositions comprising a conju Preferably , the kit contains a label that describes use of the gated therapeutic protein of the present invention to human therapeutic protein or peptide composition . or test animals , in one aspect, the compositions comprise one or more pharmaceutically acceptable carriers . The terms Water Soluble Polymersers " pharmaceutically " or " pharmacologically acceptable ” refer to molecular entities and compositions that are stable , inhibit [0129 ] In one aspect , a therapeutic protein derivative ( i . e . , protein degradation such as aggregation and cleavage prod a conjugated therapeutic protein ) molecule provided is ucts , and in addition do not produce allergic , or other bound to a water - soluble polymer including , but not limited adverse reactions when administered using routes well to , polyethylene glycol (PEG ) , branched PEG , polysialic known in the art , as described below . “ Pharmaceutically acid (PSA ) , hydroxyalkyl starch (HAS ) , hydroxylethyl acceptable carriers” include any and all clinically useful starch (HES ) , carbohydrate , polysaccharides, pullulane , chi solvents , dispersion media , coatings , antibacterial and anti tosan , hyaluronic acid , chondroitin sulfate , dermatan sulfate , fungal agents , isotonic and absorption delaying agents and starch , dextran , carboxymethyl- dextran , polyalkylene oxide the like , including those agents disclosed above . (PAO ) , polyalkylene glycol (PAG ) , polypropylene glycol [0124 ] As used herein , " effective amount” includes a dose (PPG ) polyoxazoline , poly acryloylmorpholine , polyvinyl suitable for treating a disease or disorder or ameliorating a alcohol (PVA ), polycarboxylate , polyvinylpyrrolidone , symptom of a disease or disorder. In one embodiment, polyphosphazene , polyoxazoline , polyethylene -co -maleic " effective amount” includes a dose suitable for treating a acid anhydride, polystyrene - co -maleic acid anhydride, poly mammal having a bleeding disorder as described herein . ( 1 - hydroxymethylethylene hydroxymethylformal) (PHF ) , 10125 ]. The compositions may be administered orally , topi 2 -methacryloyloxy - 2' - ethyltrimethylammoniumphosphate cally , transdermally , parenterally , by inhalation spray , vagi (MPC ) . In one embodiment of the invention , the water nally , rectally , or by intracranial injection . The term paren soluble polymer is consisting of sialic acid molecule having teral as used herein includes subcutaneous injections , a molecular weight range of 350 to 120 ,000 , 500 to 100 ,000 , intravenous , intramuscular, intracisternal injection , or infu 1000 to 80 , 000 , 1500 to 60 ,000 , 2 , 000 to 45 ,000 Da, 3 , 000 sion techniques. Administration by intravenous, intradermal, to 35 ,000 Da, and 5 , 000 to 25 , 000 Da . The coupling of the intramuscular, intramammary , intraperitoneal, intrathecal, water soluble polymer can be carried out by direct coupling retrobulbar , intrapulmonary injection and or surgical to the protein or via linker molecules. One example of a implantation at a particular site is contemplated as well . chemical linker is MBPH ( 4 - [ 4 - N -Maleimidophenyl ] butyric Generally , compositions are essentially free of pyrogens , as acid hydrazide ) containing a carbohydrate - selective hydraz well as other impurities that could be harmful to the recipi ide and a sulfhydryl- reactive maleimide group (Chamow et ent. al. , J Biol Chem 1992 ; 267 : 15916 - 22 ). Other exemplary and [ 0126 ] Single or multiple administrations of the composi preferred linkers are described below . tions can be carried out with the dose levels and pattern being selected by the treating physician . For the prevention [0130 ] In one embodiment, the derivative retains the full or treatment of disease , the appropriate dosage will depend functional activity of native therapeutic protein products , on the type of disease to be treated , as described above , the and provides an extended half -life in vivo , as compared to severity and course of the disease , whether drug is admin native therapeutic protein products . In another embodiment , istered for preventive or therapeutic purposes , previous the derivative retains at least 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , therapy, the patient' s clinical history and response to the 28 , 29 , 30 , 31, 32 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 . drug , and the discretion of the attending physician . 45 , 46 , 47 , 48 , 49 , 50 , 51, 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , [0127 ] The present invention also relates to a pharmaceu 61, 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , tical composition comprising an effective amount of a 77 , 78 , 79 , 80 , 81, 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , conjugated therapeutic protein as defined herein . The phar 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 110 , 120 , 130 , 140 , or 150 maceutical composition may further comprise a pharmaceu percent ( % ) biological activity relative to native blood tically acceptable carrier , diluent, salt , buffer , or excipient . coagulation protein . In a related aspect , the biological activi The pharmaceutical composition can be used for treating the ties of the derivative and native blood coagulation protein above - defined bleeding disorders . The pharmaceutical com are determined by the ratios of chromogenic activity to position of the invention may be a solution or a lyophilized blood coagulation factor antigen value (blood coagulation product . Solutions of the pharmaceutical composition may factor: Chr: blood coagulation factor: Ag ) . In still another be subjected to any suitable lyophilization process. embodiment of the invention , the half - life of the construct is 0128 ] As an additional aspect, the invention includes kits decreased or increased 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 , 1 . 0 , 1 . 1 , 1 . 2 , which comprise a composition of the invention packaged in 1 .3 , 1 .4 , 1. 5 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 -fold relative to the a manner which facilitates its use for administration to in vivo half - life of native therapeutic protein . subjects . In one embodiment, such a kit includes a com pound or composition described herein ( e . g . , a composition A . Sialic Acid and PSA comprising a conjugated therapeutic protein ), packaged in a container such as a sealed bottle or vessel, with a label [0131 ] PSAs consist of polymers ( generally homopoly affixed to the container or included in the package that mers ) of N -acetylneuraminic acid . The secondary amino describes use of the compound or composition in practicing group normally bears an acetyl group , but it may instead the method . In one embodiment, the kit contains a first bear a glycolyl group . Possible substituents on the hydroxyl container having a composition comprising a conjugated groups include acetyl, lactyl, ethyl, sulfate , and phosphate therapeutic protein and a second container having a physi groups . US 2017 /0240590 A1 Aug . 24 , 2017 31

[0137 ] Structure of Colominic Acid (Homopolymer of HO OH N -Acetylneuraminic Acid ) HO COO 101381 Colominic acids (a sub - class ofPSAs ) are homopo çoo lymers of N -acetylneuraminic acid (NANA ) with a (208 ) ou ketosidic linkage , and are produced , inter alia , by particular AcHN7 strains of Escherichia coli possessing K1 antigen . Colomi ÃO OH nic acids have many physiological functions . They are N - Acetylneuraminic acid important as a raw material for drugs and cosmetics . Neu5AC [0139 ] Comparative studies in vivo with polysialylated and unmodified asparaginase revealed that polysialylation [ 0132 ] Structure of Sialic Acid ( N - Acetylneuraminic increased the half - life of the enzyme (Fernandes and Gre Acid ) [0133 ] PSAs and mPSAs generally comprise linear poly goriadis , Biochimica Biophysica Acta 1341 : 26 - 34 , 1997 ) . mers consisting essentially of N -acetylneuraminic acid moi [0140 ] As used herein , “ sialic acid moieties” includes eties linked by 2 , 8 - or 2 , 9 - glycosidic linkages or combina sialic acid monomers or polymers ( polysaccharides ” ) tions of these ( e . g . alternating 2 , 8 - and 2 , 9 - linkages ). In which are soluble in an aqueous solution or suspension and particularly preferred PSAs and mPSAs , the glycosidic have little or no negative impact, such as side effects , to linkages are a - 2 , 8 . Such PSAs and mPSAs are conveniently mammals upon administration of the PSA -blood coagula derived from colominic acids , and are referred to herein as tion protein conjugate in a pharmaceutically effective “ CAs” and “ mCAs” . Typical PSAs and mPSAs comprise at amount . The polymers are characterized , in one aspect , as least 2 , preferably at least 5 , more preferably at least 10 and having 1 , 2 , 3 , 4 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , most preferably at least 20 N -acetylneuraminic acid moi 200 , 300 , 400 , or 500 sialic acid units . In certain aspects , eties . Thus, they may comprise from 2 to 300 N -acetyl different sialic acid units are combined in a chain . neuraminic acid moieties , preferably from 5 to 200 N -acetyl 10141 ] In one embodiment of the invention , the sialic acid neuraminic acid moieties , or most preferably from 10 to 100 portion of the polysaccharide compound is highly hydro N - acetylneuraminic acid moieties. PSAs and CAs preferably are essentially free of sugar moieties other than N -acetyl philic , and in another embodiment the entire compound is neuraminic acid . Thus PSAs and CAs preferably comprise at highly hydrophilic . Hydrophilicity is conferred primarily by least 90 % , more preferably at least 95 % and most preferably the pendant carboxyl groups of the sialic acid units , as well at least 98 % N -acetylneuraminic acid moieties . as the hydroxyl groups . The saccharide unit may contain [ 0134 Where PSAs and CAs comprise moieties other than other functional groups, such as, amine , hydroxyl or sul N - acetylneuraminic acid (as , for example in mPSAS and phate groups , or combinations thereof. These groups may be mCAs) these are preferably located at one or both of the present on naturally - occurring saccharide compounds, or ends of the polymer chain . Such “ other” moieties may, for introduced into derivative polysaccharide compounds . example , be moieties derived from terminal N -acetyl [0142 ] The naturally occurring polymer PSA is available neuraminic acid moieties by oxidation or reduction . as a polydisperse preparation showing a broad size distri [0135 ] For example , WO - A - 0187922 describes such bution ( e .g . Sigma C -5762 ) and high polydispersity (PD ). mPSAs and mCAs in which the non -reducing terminal Because the polysaccharides are usually produced in bacte N - acetylneuraminic acid unit is converted to an aldehyde ria carrying the inherent risk of copurifying endotoxins , the group by reaction with sodium periodate . Additionally, WO purification of long sialic acid polymer chains may raise the 2005 / 016974 describes such mPSAs and mCAs in which the reducing terminal N - acetylneuraminic acid unit is subjected probability of increased endotoxin content. Short PSA mol to reduction to reductively open the ring at the reducing ecules with 1 - 4 sialic acid units can also be synthetically terminal N -acetylneuraminic acid unit , whereby a vicinal prepared (Kang S H et al ., Chem Commun . 2000 ; 227 - 8 ; diol group is formed , followed by oxidation to convert the Ress D K and Linhardt RJ, Current Organic Synthesis . vicinal diol group to an aldehyde group . 2004 ; 1 : 31 - 46 ), thus minimizing the risk of high endotoxin [0136 ] Sialic acid rich glycoproteins bind selectin in levels . However PSA preparations with a narrow size dis humans and other organisms. They play an important role in tribution and low polydispersity , which are also endotoxin human influenza infections. E . g ., sialic acid can hide man free , can now be manufactured . Polysaccharide compounds nose 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 ment, the polysaccharide compounds are substantially free the galactose receptor on the hepatic parenchymal cells . of terminal galactose units.

HO COONa HO COONa HOH. AcHN HO COONa HO HOW . AcHN HO HOH - OH Un AcHN HO US 2017 /0240590 A1 Aug . 24 , 2017 32

B . Polyethylene Glycol (PEG ) and Pegylation mPEG -Succinimidyl a -Methylbutanoate [0143 ] In certain aspects , therapeutic proteins are conju (MPEG - SMB ) gated to a water soluble polymer by any of a varleyvariety of0 (01501 chemical methods ( Roberts J M et al. , Advan Drug Delivery Rev 2002 ; 54 : 459 -76 ) . For example , in one embodiment a therapeutic protein is modified by the conjugation of PEG to free amino groups of the protein using N -hydroxysuccinim ide (NHS ) esters. In another embodiment the water soluble polymer, for example PEG , is coupled to free SH groups mPEG - CH2CH2CH - 0 - 0 - N using maleimide chemistry or the coupling of PEG hydraz CH ides or PEG amines to carbohydrate moieties of the thera peutic protein after prior oxidation . [ 0144 ] The conjugation is in one aspect performed by direct coupling (or coupling via linker systems) of the water soluble polymer to a therapeutic protein under formation of mPEG - CM - HBA -NHS (CM = Carboxymethyl; stable bonds . In addition degradable , releasable or hydroly HBA = Hydroxy Butyric Acid ) sable linker systems are used in certain aspects the present [0151 ] invention ( Tsubery et al. J Biol Chem 2004 ; 279 :38118 - 24 / Greenwald et al ., J Med Chem 1999 ; 42 : 3657 -67 / Zhao et al . , Bioconj Chem 2006 ; 17 : 341- 51/ WO2006 / 138572A2 / U . S . Pat . No. 7 ,259 ,224B2 /U . S . Pat. No. 7 , 060 , 259B2) . 10145 ] In one embodiment of the invention , a therapeutic protein is modified via lysine residues by use of polyethyl mPEG – CH20 - 0 - CHCH2C - 0 - N ene glycol derivatives containing an active N -hydroxysuc CH3 cinimide ester (NHS ) such as succinimidyl succinate , suc cinimidyl glutarate or succinimidyl propionate . These derivatives react with the lysine residues of the therapeutic protein under mild conditions by forming a stable amide bond . In one embodiment of the invention , the chain length Structure of a Branched PEG - Derivative (Nektar of the PEG derivative is 5 , 000 Da. Other PEG derivatives Therapeutics ) with chain lengths of 500 to 2 , 000 Da, 2 ,000 to 5 , 000 Da , greater than 5 ,000 up to 10 , 000 Da or greater than 10 ,000 up Branched PEG N -Hydroxysuccinimide to 20 , 000 Da, or greater than 20 ,000 up to 150 , 000 Da are (mPEG2 - NHS ) used in various embodiments , including linear and branched [0152 ] structures . [0146 ] Alternative methods for the PEGylation of amino groups are , without limitation , the chemical conjugation with PEG carbonates by forming urethane bonds, or the mPEG reaction with aldehydes or ketones by reductive amination forming secondary amide bonds . - C - 0 - N [ 0147 ] In one embodiment of the present invention a mPEG therapeutic protein molecule is chemically modified using PEG derivatives that are commercially available . These PEG derivatives in alternative aspects have linear or branched structures. Examples of PEG -derivatives contain [0153 ] This reagent with branched structure is described in ing NHS groups are listed below . more detail by Kozlowski et al . ( BioDrugs 2001 ; 5 :419 - 29 ) . [0148 ] The following PEG derivatives are non - limiting [0154 ] Other non - limiting examples of PEG derivatives examples of those commercially available from Nektar are commercially available from NOF Corporation ( Tokyo , Therapeutics (Huntsville , Ala . ; see www .nektar . com / PEG Japan ; see www .nof . co . jp /english : Catalogue 2005 ) reagent catalog ; Nektar Advanced PEGylation , price list 2005 - 2006 ): General Structure of Linear PEG - Derivatives (NOF MPEG - Succinimidyl Propionate (mPEG -SPA ) Corp . ) [ 0149 ] [0155 ]

mPEG - CH2CH2 - 0 - 0 - N . CH3O ( CH2CH20 ). — X — N US 2017 /0240590 A1 Aug . 24 , 2017 33

[0156 ] X = carboxymethyl [0162 ] These propane derivatives show a glycerol back bone with a 1 , 2 substitution pattern . In the present invention branched PEG derivatives based on glycerol structures with 1 , 3 substitution or other branched structures described in US2003 /0143596A1 are also contemplated . CH3O (CH2CH2O )n - CH2 - - 0 - N [0163 ] PEG derivatives with degradable ( for example , hydrolysable) linkers as described by Tsubery et al. ( J Biol Chem 2004 ; 279 : 38118 - 24 ) and Shechter et al . (W0004089280A3 ) are also contemplated . [0157 ] X = carboxypentyl [0164 ] Surprisingly , the PEGylated therapeutic protein of this invention exhibits functional activity , combined with an extended half -life in vivo . In addition the PEGylated rFVIII , FVIIa , FIX , or other blood coagulation factor seems to be CH3O ( CH2CH20 ). — (CH2 ) 5 — 0 - 0 - N more resistant against thrombin inactivation . C . Hydroxyalkyl Starch (HAS ) and Hydroxylethyl Starch (HES ) [0158 ] x = succinate [0165 ] In various embodiments of the present invention , a therapeutic protein molecule is chemically modified using hydroxyalkyl starch (HAS ) or hydroxylethyl starch (HES ) or derivatives thereof. CH3O (CH2CH20 )n - O - CH2CH2 - 0 - 0 N [0166 ] HES is a derivative of naturally occurring amylo contactenon pectin and is degraded by alpha -amylase in the body. HES is a substituted derivative of the carbo -hydrate polymer amylopectin , which is present in corn starch at a concentra mPEG Succinimidyl succinate tion of up to 95 % by weight . HES exhibits advantageous [0159 ] x = glutaratePRO Sueci do nos biological properties and is used as a blood volume replace 1915) related seriously ment agent and in hemodilution therapy in the clinics ( Sommermeyer et al. , 1987 , Krankenhauspharmazie , 8 ( 8 ) , 271 - 278 ; and Weidler et al. , 1991, Arzneim . - Forschung/ Drug Res. g 419 494 - 498 ) . CH2O ( CH2CH20 )n — 7 — (CH2 ) 3 — 0 - 0 - N [0167 ] 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 physical - chemical properties of this molecule are MPEG Succinimidyl glutarate mainly determined by the type of glycosidic bonds. Due to the nicked alpha - 1 , 4 - glycosidic bond , helical structures with about six glucose -monomers per turn are produced . The Structures of Branched PEG -Derivatives (NOF physico - chemical as well as the biochemical properties of Corp . ): 2 ,3 -Bis (methylpolyoxyethylene -oxy ) - 1 - (1 , 5 the polymer can be modified via substitution . The introduc dioxo -5 -succinimidyloxy , pentyloxy )propane tion of a hydroxyethyl group can be achieved via alkaline [0160 ] 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 H3CH ( OCH2 - CH2) - 0 - CH respect to a hydroxyethylation . Owing to this fact, the CH2 - 0 - 2 - CH2CH2CH2 - 0 - 0 - N skilled person is able to influence the substitution pattern to a limited extent. [0168 ] HAS refers to a starch derivative which has been substituted by at least one hydroxyalkyl group . Therefore , the term hydroxyalkyl starch is not limited to compounds 2 , 3 - Bis ( methylpolyoxyethylene -oxy ) - 1 - ( succinimidyl where the terminal carbohydrate moiety comprises hydroxy carboxypentyloxy )propane alkyl groups R1, R2 , and/ or R3, but also refers to com [0161 ] pounds in which at least one hydroxy group present any H3C — (OCH2 - CH2) n - 0 – CH2 H3C — (OCH2 – CH2) n - 0 – CH CH2 - O - CH2CH2CH2CH2CH2 - C - 0 US 2017 /0240590 A1 Aug . 24 , 2017 34 where , either in the terminal carbohydrate moiety and /or in polysaccharide. Another linkage by which the therapeutic the remaining part of the starch molecule , HAS ', is substi protein is covalently bonded to the polysaccharide com tuted by a hydroxyalkyl group R1, R2 , or R3. pound is via a Schiff base , between a free amino group on the blood coagulation protein being reacted with an alde hyde group formed at the non - reducing end of the polysac ( 1 ) charide by periodate oxidation ( Jennings H J and Lugowski ? OR, C , J Immunol. 1981 ; 127 : 1011 - 8 ; Fernandes AI and Gre SH goriadis G , Biochim Biophys Acta . 1997 ; 1341 ; 26 -34 ). The HAS' t generated Schiff base is in one aspect stabilized by specific e reduction with NaCNBH3 to form a secondary amine. An R20 mor alternative approach is the generation of terminal free amino HOR3 groups in the PSA by reductive amination with NH4C1 after prior oxidation . Bifunctional reagents can be used for link ing two amino or two hydroxyl groups . For example , PSA [ 0169 ] The alkyl group may be a linear or branched alkyl containing an amino group is coupled to amino groups of the group which may be suitably substituted . Preferably , the protein with reagents like BS3 (Bis ( sulfosuccinimidyl sub hydroxyalkyl group contains 1 to 10 carbon atoms, more erate / Pierce , Rockford , ill. ) . In addition heterobifunctional preferably from 1 to 6 carbon atoms, more preferably from cross linking reagents like Sulfo -EMCS (N -e -Maleimido 1 to 4 carbon atoms, and even more preferably 2 - 4 carbon caproyloxy ) sulfosuccinimide ester / Pierce ) is used for atoms. “ Hydroxyalkyl starch ” therefore preferably com instance to link amine and thiol groups. prises hydroxyethyl starch , hydroxypropyl starch and [0175 ] In another approach , a PSA hydrazide is prepared hydroxybutyl starch , wherein hydroxyethyl starch and and coupled to the carbohydrate moiety of the protein after hydroxypropyl starch are particularly preferred . prior oxidation and generation of aldehyde functions. [ 0170 ] Hydroxyalkyl starch comprising two or more dif [0176 ] As described above, a free amine group of the ferent hydroxyalkyl groups is also comprised in the present therapeutic protein reacts with the 1 -carboxyl group of the invention . The at least one hydroxyalkyl group comprised in sialic acid residue to form a peptidyl bond or an ester linkage HAS may contain two or more hydroxy groups. According is formed between the l -carboxylic acid group and a to one embodiment, the at least one hydroxyalkyl group hydroxyl or other suitable active group on a blood coagu comprised HAS contains one hydroxy group . lation protein . Alternatively, a carboxyl group forms a [0171 ] The term HAS also includes derivatives wherein peptide linkage with deacetylated 5 - amino group , or an the alkyl group is mono - or polysubstituted . In one embodi aldehyde group of a molecule of a therapeutic protein forms ment, the alkyl group is substituted with a halogen , espe a Schiff base with the N -deacetylated 5 -amino group of a cially fluorine , or with an aryl group , provided that the HAS sialic acid residue . remains soluble in water. Furthermore , the terminal hydroxy (0177 ] Alternatively , the polysaccharide compound is group a of hydroxyalkyl group may be esterified or etheri associated in a non - covalent manner with a therapeutic fied . HAS derivatives are described in WO /2004 / 024776 , protein . For example, the polysaccharide compound and the which is incorporated by reference in its entirety . pharmaceutically active compound are in one aspect linked via hydrophobic interactions. Other non - covalent associa D . Methods of Attachment tions include electrostatic interactions, with oppositely [ 0172 ] A therapeutic protein may be covalently linked to charged ions attracting each other. the polysaccharide compounds by any of various techniques [ 0178 ] In various embodiments , the therapeutic protein is known to those of skill in the art . In various aspects of the linked to or associated with the polysaccharide compound in invention , sialic acid moieties are bound to a therapeutic stoichiometric amounts ( e . g . , 1 : 1 , 1 : 2 , 1 : 3 , 1 : 4 , 1 : 5 , 1 : 6 , 1 : 7 , protein , e . g . , FIX , FVIII, FVIIa or VWF, for example by the 1 :7 , 1 :8 , 1: 9 , or 1 : 10 , etc .) . In various embodiments , 1 - 6 , method described in U . S . Pat . No . 4, 356 , 170 , which is 7 - 12 or 13 - 20 polysaccharides are linked to the blood herein incorporated by reference . coagulation protein . In still other embodiments , 1 , 2 , 3 , 4 , 5 , [ 0173 ] Other techniques for coupling PSA to polypeptides 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 or more are also known and contemplated by the invention . For polysaccharides are linked to the blood coagulation protein . example , US Publication No . 2007 / 0282096 describes con [0179 ] In various embodiments , the therapeutic protein is jugating an amine or hydrazide derivative of, e. g ., PSA , to modified to introduce glycosylation sites ( i . e . , sites other proteins. In addition , US Publication No . 2007/ 0191597 than the native glycosylation sites) . Such modification may describes PSA derivatives containing an aldehyde group for be accomplished using standard molecular biological tech reaction with substrates ( e . g . , proteins ) at the reducing end . niques known in the art . Moreover, the therapeutic protein , These references are incorporated by reference in their prior to conjugation to a water soluble polymer via one or entireties. more carbohydrate moieties , may be glycosylated in vivo or [0174 ] Various methods are disclosed at column 7 , line 15 , in vitro . These glycosylated sites can serve as targets for through column 8 , line 5 of U . S . Pat . No . 5 , 846 , 951 ( incor conjugation of the proteins with water soluble polymers (US porated by reference in its entirety ) . Exemplary techniques Patent Application No . 20090028822 , US Patent Applica include linkage through a peptide bond between a carboxyl tion No . 2009/ 0093399 , US Patent Application No . 2009 / group on one of either the blood coagulation protein or 0081188 , US Patent Application No . 2007 / 0254836 , US polysaccharide and an amine group of the blood coagulation Patent Application No . 2006 /0111279 , and DeFrees S . et al. , protein or polysaccharide , or an ester linkage between a Glycobiology, 2006 , 16 , 9 , 833 -43 ). For example , a protein carboxyl group of the blood coagulation protein or polysac - that is not naturally glycoslyated in vivo ( e . g ., a protein that charide and a hydroxyl group of the therapeutic protein or is not a glycoprotein ) may be modified as described above . US 2017 /0240590 A1 Aug . 24 , 2017 35

E . Aminooxy Linkage hydroxymethylethylene hydroxymethylformal ) (PHF ) , 2 -methacryloyloxy - 2 ' - ethyltrimethylammoniumphosphate [0180 ] In one embodiment of the invention , the reaction of hydroxylamine or hydroxylamine derivatives with alde (MPC ) . hydes ( e . g ., on a carbohydrate moiety following oxidation by sodium periodate ) to form an oxime group is applied to Nucleophilic Catalysts the preparation of conjugates of blood coagulation protein . [0184 ] As described herein , the conjugation of water For example , a glycoprotein ( e . g ., a therapeutic protein soluble polymers to therapeutic proteins can be catalyzed by according to the present invention ) is first oxidized with a aniline . Aniline strongly catalyzes aqueous reactions of oxidizing agent such as sodium periodate (Na104 ) (Rothfus aldehydes and ketones with amines to form stable imines JAet Smith E L . , J Biol Chem 1963 , 238 , 1402 - 10 ; and Van such as hydrazones and oximes . The following diagram Lenten L and Ashwell G ., J Biol Chem 1971 , 246 , 1889 - 94 ). compares an uncatalyzed versus the aniline - catalyzed oxime The periodate oxidation of glycoproteins is based on the ligation reaction (Kohler JJ , ChemBioChem 2009 ; 10 :2147 classical Malaprade reaction described in 1928 , the oxida 50 ): tion of vicinal diols with periodate to form an active alde hyde group (Malaprade L . , Analytical application , Bull Soc Chim France , 1928 , 43 , 683 - 96 ) . Additional examples for such an oxidizing agent are lead tetraacetate ( Pb (OAc ) 4 ) , manganese acetate (MnO (Ac ) 3 ), cobalt acetate (Co ( OAc ) 2 ), HR thallium acetate ( TLOAc ) , cerium sulfate (Ce ( SO4 ) 2 ) ( U . S . Pat. No . 4 , 367 , 309 ) or potassium perruthenate (KRu04 ) uncatalized aniline- catalized (Marko et al. , J Am Chem Soc 1997 , 119 , 12661- 2 ) . By reaction + ] reaction " oxidizing agent” a mild oxidizing compound which is capable of oxidizing vicinal diols in carbohydrates, thereby excess generating active aldehyde groups under physiological reac tion conditions is meant. to " HN [0181 ] The second step is the coupling of the polymer NH OH containing an aminooxy group to the oxidized carbohydrate HR moiety to form an oxime linkage . In one embodiment of the HR HR invention , this step can be carried out in the presence of catalytic amounts of the nucleophilic catalyst aniline or -H2O aniline derivatives (Dirksen A et Dawson PE , Bioconjugate Chem . 2008 ; Zeng Y et al. , Nature Methods 2009 ; 6 :207 - 9 ) . The 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 NH2OH alkoxyamine linkage ( FIG . 2 ) . Additional catalysts are described below . HR HR 10182 ] Additional information on aminooxy technology can be found in the following references, each of which is +HT incorporated in their entireties : EP 1681303A1 (HASylated erythropoietin ) ; WO 2005 /014024 ( conjugates of a polymer and a protein linked by an oxime linking group ) ; WO96 / 40662 (aminooxy -containing linker compounds and their protonated aniline 2+ application in conjugates ); WO 2008 / 025856 (Modified Schiff base proteins ) ; 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 . R [0183 ] In various embodiments of the invention , the water NH , 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 ) R include , but are not limited to polyethylene glycol ( PEG ) , ?? ?? branched PEG , polysialic acid ( PSA ), carbohydrate , poly HR saccharides , pullulane, chitosan , hyaluronic acid , chondroi oxime ligation - aniline HR tin sulfate, dermatan sulfate , starch , dextran , carboxym product ethyl- dextran , polyalkylene oxide (PAO ) , polyalkylene glycol ( PAG ) , polypropylene glycol (PPG ) polyoxazoline , poly acryloylmorpholine, polyvinyl alcohol (PVA ) , polycar [0185 ] However , considering the numerous health risks boxylate , polyvinylpyrrolidone , polyphosphazene , polyox associated with aniline , alternative catalysts are desirable . azoline, polyethylene - co -maleic acid anhydride, polysty - The present invention provides aniline derivatives as alter rene - co -maleic acid anhydride , native oxime ligation catalysts . Such aniline derivatives US 2017 /0240590 A1 Aug . 24 , 2017 36 include, but are not limited to , o -amino benzoic acid , primary amines (FIG . 3) . In the first step , one molecule of m - amino benzoic acid , p - amino benzoic acid , sulfanilic 2 , 2 - chlorodiethylether was reacted with two molecules of acid , o -aminobenzamide , o - toluidine , m - toluidine , p - tolui Endo - N -hydroxy -5 -norbornene -2 ,3 - dicarboximide in dim dine , o -anisidine , m - anisidine , and p - anisidine . ethylformamide (DMF ) . The desired homobifunctional (0186 ] In one embodiment of the invention , m - toluidine product was prepared from the resulting intermediate by ( aka meta -toluidine , m -methylaniline , 3 -methylaniline , or hydrazinolysis in ethanol . 3 -amino - 1 -methylbenzene ) is used to catalyze the conjuga tion reactions described herein . M - toluidine and aniline have Example 2 similar physical properties and essentially the same pka value (m - toluidine : pKa 4 .73 , aniline: pKa 4 .63 ). Preparation of the Homobifunctional Linker NH , 10187 ] The nucleophilic catalysts of the invention are [OCH2CH2 ] 4ONH2 useful for oxime ligation ( e . g , using aminooxy linkage ) or [0192 ] The homobifunctional linker NH [OCH CH2] hydrazone formation ( e . g . , using hydrazide chemistry ) . In 40NH, various embodiments of the invention , the nucleophilic catalyst is provided in the conjugation reaction at a concen tration of 0 . 1 , 0 . 2 , 0 . 3 , 0 . 5 , 0 . 5 , 0 . 6 , 0 . 7 , 0 . 8 , 0 . 9 , 1 . 0 , 1 . 5 , H2N NH2 2 . 0 , 2 . 5 , 3 . 0 , 3 . 5 , 4 . 0 , 4 . 5 , 5 . 0 , 5 . 5 , 6 .0 , 6 .5 , 7 . 0 , 7 . 5 , 8 . 0 , 8 . 5 , 9 . 0 , 9 . 5 , 10 . 0 , 11, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 25 , 30 , [0193 ] ( 3 , 6 , 9 -trioxa - undecane - 1 , 11- dioxyamine ) contain 35 , 40 , 45 , or 50 mM . In one embodiment, the nucleophilic ing two active aminooxy groups was synthesized according catalyst is provided between 1 to 10 mM . In various embodi to Boturyn et al. ( Tetrahedron 1997 ; 53 :5485 - 92 ) in a two ments of the invention , the pH range of conjugation reaction step organic reaction employing a modified Gabriel- Synthe is 4 . 5 , 5 . 0 , 5 . 5 , 6 . 0 , 6 . 5 , 7 . 0 and 7 .5 . In one embodiment, the sis of primary amines (FIG . 3 ). In the first step one molecule pH is between 5 . 5 to 6 . 5 . of Bis - ( 2 - ( 2 - chlorethoxy ) - ethyl ) -ether was reacted with two Purification of Conjugated Proteins molecules of Endo - N -hydroxy - 5 - norbornene - 2 , 3 -dicarboxi mide in DMF. The desired homobifunctional product was [0188 ] In various embodiments , purification of a protein prepared from the resulting intermediate by hydrazinolysis that has been incubated with an oxidizing agent and / or a in ethanol. therapeutic protein that has been conjugated with a water soluble polymer according to the present disclosure, is Example 3 desired . Numerous purification techniques are known in the art and include , without limitation , chromatographic meth Preparation of the Homobifunctional Linker NH , ods such as ion - exchange chromatography, hydrophobic [OCH2CH2 ) . ONH2 interaction chromatography, size exclusion chromatography [0194 ] The homobifunctional linker NH [OCH CH2 ] and affinity chromatography or combinations thereof, filtra 60NH ,

HN ^ ° - NH2

tion methods, and precipitation methods (Guide to Protein [0195 ] ( 3 , 6 , 9 , 12, 15 -penatoxa - heptadecane - 1 , 17 - di Purification , Meth . Enzymology Vol 463 (edited by Burgess oxyamine ) containing two active aminooxy groups was R Rand Deutscher MP) , 2nd edition , Academic Press 2009 ) . synthesized according to Boturyn et al. ( Tetrahedron 1997 ; [0189 ] The following examples are not intended to be 53 : 5485 - 92 ) in a two step organic reaction employing a limiting but only exemplary of specific embodiments of the modified Gabriel - Synthesis of primary amines. In the first invention . step one molecule of hexaethylene glycol dichloride was reacted with two molecules of Endo - N -hydroxy - 5 - nor EXAMPLES bornene - 2 ,3 -dicarboximide in DMF. The desired homobi functional product was prepared from the resulting interme Example 1 diate by hydrazinolysis in ethanol. Preparation of the Homobifunctional Linker NH , Example 4 [OCH2CH2 2ONH2 [0190 ] The homobifunctional linker NH [ OCH CH2] Detailed Synthesis of the Aminooxy -PSA Reagent 2ONH2 [0196 ] 3 -oxa -pentane -1 ,5 dioxyamine was synthesized according to Botyryn et al ( Tetrahedron 1997 ; 53 :5485 - 92 ) in a two step organic synthesis as outlined in Example 1. NOH2NH . - NH2 Step 1 : [0191 ] ( 3 -oxa -pentane - 1, 5 -dioxyamine ) containing two [0197 ] To a solution of Endo - N -hydroxy - 5 -norbonene - 2 , active aminooxy groups was synthesized according to Botu 3 -dicarboxiimide (59 . 0 g ; 1 .00 eq ) in 700 ml anhydrous ryn et al. ( Tetrahedron 1997 ; 53 :5485 - 92 ) in a two step N , N - dimetylformamide anhydrous K2CO3 ( 45 .51 g ; 1 . 00 organic reaction employing a modified Gabriel -Synthesis of eq ) and 2, 2 - dichlorodiethylether ( 15 .84 ml; 0 .41 eq ) were US 2017 /0240590 A1 Aug . 24 , 2017 37 added . The reaction mixture was stirred for 22 h at 50° C . formed against Buffer D (20 mM Hepes , 90 mM NaCl, pH The mixture was evaporated to dryness under reduced 7 .4 ). The preparation was analytically characterized by pressure . The residue was suspended in 2 L dichloromethane measuring total PSA (Resorcinol assay ) and total aminooxy and extracted two times with saturated aqueous NaCl groups ( TNBS assay ) to determine the degree of modifica solution ( each 1 L ) . The Dichloromethane layer was dried tion . Furthermore the polydispersity as well as free 3 - oxa over Na SO , and then evaporated to dryness under reduced pentane - 1 , 5 - dioxyamine and cyanide was determined . pressure and dried in high vacuum to give 64 . 5 g of 3 - oxapentane - 1 , 5 - dioxy -endo - 2 ', 3 ' - dicarboxydiimidenor Example 7 bornene as a white - yellow solid ( intermediate 1) . Preparation of Aminooxy - PSA without a Reduction Step 2 : Step 0198 ] To a solution of intermediate 1 (64 .25 g ; 1 .00 eq ) [0201 ] 573 mg of oxidized PSA (MW = 20 kD ) obtained in 800 ml anhydrous Ethanol, 31. 0 ml Hydrazine hydrate from the Serum Institute of India (Pune , India ) was dis ( 4 . 26 eq ) were added . The reaction mixture was then solved in 11. 3 ml 50 mM phosphate buffer pH 6 . 0 (Bufffer refluxed for 2 hrs . The mixture was concentrated to the half A ) . Then 94 mg 3 - oxa - pentane - 1 , 5 - dioxyamine was given to of the starting volume by evaporating the solvent under the reaction mixture . After shaking for 5 h at RT the mixture reduced pressure . The occurring precipitate was filtered off . was then subjected to a weak anion exchange chromatog The remaining ethanol layer was evaporated to dryness raphy step employing a Fractogel EMD DEAE 650 - M under reduced pressure . The residue containing the crude chromatography gel ( column dimension : XK16 / 105 ) . The product 3 -oxa -pentane - 1, 5 -dioxyamine was dried in vacuum reaction mixture was diluted with 50 mlBuffer A and loaded to yield 46 . 3 g . The crude product was further purified by onto the DEAE column pre - equilibrated with Buffer A at a column chromatography (Silicagel 60 ; isocratic elution with flow rate of 1 cm /min . Then the column was washed with 20 Dichloromethane/ Methanol mixture , 9 /1 ) to yield 11. 7 g of CV Buffer B ( 20 mM Hepes, pH 6 . 0 ) to remove free the pure final product 3 - oxa - pentane - 1 , 5 - dioxyamine . 3 - oxa -pentane - 1 , 5 - dioxyamine and cyanide at a flow rate of 2 cm /min . The aminooxy -PSA reagent was the eluted with Example 5 a step gradient consisting of67 % Buffer B and 43 % Buffer C ( 20 mM Hepes , 1 M NaCl, pH 7 . 5 ) . The eluate was Preparation of Aminooxy -PSA concentrated by UF/ DF using a 5 kD membrane made of polyether sulfone ( 50 cm ? ,Millipore ) . The final diafiltration [0199 ] 1000 mg of oxidized PSA (MW = 20 kD ) obtained step was performed against Buffer D (20 mM Hepes, 90 mm from the Serum Institute of India ( Pune , India ) was dis NaCl, pH 7 . 4 ) . The preparation was analytically character solved in 16 ml 50 mM phosphate buffer pH 6 . 0 . Then 170 ized by measuring total PSA (Resorcinol assay ) and total mg 3 -oxa - pentane - 1 , 5 - dioxyamine was given to the reaction aminooxy groups ( TNBS assay ) to determine the degree of mixture . After shaking for 2 hrs at RT 78 . 5 mg sodium cyanoborohydride was added and the reaction was per modification . Furthermore the polydispersity as well as free formed for 18 hours over night. The reaction mixture was 3 - oxapentane - 1 , 5 - dioxyamine was determined . then subjected to a ultrafiltration /diafiltration procedure Example 8 (UF /DF ) using a membrane with a 5 kD cut - off made of regenerated cellulose (50 cm ? , Millipore ). Preparation of Aminooxy - PSA without a Reduction Step in the Presence of the Nucleophilic Catalyst Example 6 m - Toluidine Preparation of Aminooxy - PSA Employing a [0202 ] 573 mg of oxidized PSA (MW = 20 kD ) obtained from the Serum Institute of India (Pune , India ) is dissolved Chromatographic Purification Step in 9 ml 50 mM phosphate buffer pH 6 . 0 (Bufffer A ) . Then [ 0200 ) 1290 mg of oxidized PSA (MW = 20 kD ) obtained 94 mg 3 -oxa -pentane - 1 , 5 -dioxyamine is given to this solu from the Serum Institute of India ( Pune , India ) was dis tion . Subsequently 2 . 3 ml of a 50 mM m -toluidine stock solved in 25 ml 50 mM phosphate buffer pH 6 . 0 (Bufffer A ) . solution are added to this reaction mixture . After shaking for Then 209 mg 3 - oxapentane - 1 , 5 - dioxyamine was given to 2 h at RT the mixture is then subjected to a weak anion the reaction mixture . After shaking for 1 h at RT 101 mg exchange chromatography step employing a Fractogel EMD sodium cyanoborohydride was added and the reaction was DEAE 650 - M chromatography gel (column dimension : performed for 3 hours. Then the mixture was then subjected XK16 / 105 ) . The reaction mixture is diluted with 50 ml to a weak anion exchange chromatography step employing Buffer A and loaded onto the DEAE column pre - equilibrated a Fractogel EMD DEAE 650 - M chromatography gel col- with Buffer A at a flow rate of 1 cm /min . Then the column umn dimension : XK26 / 135 ) . The reaction mixture was is washed with 20 CV Buffer B (20 mM Hepes, pH 6 .0 ) to diluted with 110 ml Buffer A and loaded onto the DEAE remove free 3 - oxa -pentane - 1 , 5 -dioxyamine and cyanide at a column pre - equilibrated with Buffer A at a flow rate of 1 flow rate of 2 cm /min . The aminooxy - PSA reagent is the cm /min . Then the column was washed with 20 CV Buffer B eluted with a step gradient consisting of 67 % Buffer B and ( 20 mM Hepes, pH 6 . 0 ) to remove free 3 -oxa -pentane - 1 , 5 43 % Buffer C ( 20 mM Hepes, 1 M NaCl, pH 7 . 5 ) . The eluate dioxyamine and cyanide at a flow rate of 2 cm /min . The is concentrated by UF /DF using a 5 kD membrane made of aminooxy - PSA reagent was then eluted with a step gradient polyether sulfone ( 50 cm ? , Millipore ). The final diafiltration consisting of 67 % Buffer B and 43 % Buffer C ( 20 mM step is performed against Buffer D ( 20 mM Hepes , 90 mm Hepes , 1M NaC1, pH 7 . 5 ) . The eluate was concentrated by NaCl, pH 7 . 4 ) . The preparation is analytically characterized UF /DF using a 5 kD membrane made of polyether sulfone by measuring total PSA (Resorcinol assay ) and total ami (50 cm ? , Millipore ). The final diafiltration step was per nooxy groups ( TNBS assay ) to determine the degree of US 2017 /0240590 A1 Aug . 24 , 2017 38 modification . Furthermore the polydispersity as well as free Example 11 3 -oxa -pentane -1 , 5 -dioxyamine is determined . Polysialylation of rFIX Using Aminooxy - PSA and Example 9 m - Toluidine as a Nucleophilic Catalyst Preparation of Aminooxy -PSA Reagent Method 1 : [0203 ] An Aminooxy — PSA reagent was prepared accord [0209 ] 12 .3 mg rFIX was dissolved in 6 . 1 ml histidine ing to the Examples 4 - 8 . After diafiltration , the product was buffer , pH 6 .0 ( 20 mM L -histidine , 150 mM NaC1, 5 mM frozen at - 80° C . and lyophilized . After lyophilization the CaC12 ) . 254 ul of an aqueous sodium periodate solution ( 5 reagent was dissolved in the appropriate volume of water mM ) was then added and the reaction mixture is incubated and used for preparation of PSA - protein conjugates via for 1 h in the dark at 4° C . under gentle stirring and quenched carbohydrate modification . for 15 min at room temperature by the addition of 6 . 5 ul of a 1 M aqueous cysteine solution . The mixture was subse Example 10 quently subjected to UF /DF employing Vivaspin 15R 10 kD centrifugal filtrators to remove excess periodate , quencher Evaluation of the Efficacy of Different Alternative and the byproducts thereof. Nucleophilic Catalysts [ 0210 ] The retentate (8 . 8 ml) , containing oxidized rFIX 10204 ) rFIX was incubated with sodium periodate , ami was mixed with 2 .46 ml of an aqueous m - toluidine solution nooxy - PSA reagent under standardized conditions ( 1 mg/ ml (50 mm ) and incubated for 30 min at room temperature . rFIX in 20 mM L -histidine , 150 mM NaCl, 5 mM CaCl2 , pH Then aminooxy -PSA reagent with a MW of 20 kD (de 6 .0 , 5 - fold molar aminooxy -PSA reagent excess, 100 UM scribed above ) was added to give a 5 - fold molar reagent NaI04) using different nucleophilic catalysts (aniline , excess. This mixture was incubated for 2 . 5 h at RT in the m - toluidine , o -anisidine , m - anisidine , o -aminobenzoic acid , dark under gentle stirring . m -aminobenzoic acid , p - aminobenzoic acid , p - aminobenz [0211 ] The free rFIX was removed by means of anion amide , sulfanilic acid / standard concentration : 10 mM ) The exchange chromatography ( AEC ) . The reaction mixture was reaction was carried out for 2 hrs in the dark at room diluted with 15 ml Buffer A (50 mM Hepes , 5 mM CaC12 , temperature under gentle stirring and quenched for 15 min pH 7 . 5 ) and loaded onto a 20 ml HiPrep QFF 16 / 10 column at room temperature by the addition of aqueous cysteine (GE Healthcare , Fairfield , Conn .) pre - equilibrated with Buf solution with a final concentration of 1 mM . fer A . The column was then eluted with Buffer B (50 mM [ 0205 ] The coupling efficiency was determined by SDS Hepes , 1 M NaCl, 5 mM CaCl2 , pH 7 . 5 ) . Free rFIX elutes PAGE using an Invitrogen X -cell mini system . Samples at a conductivity between 12 - 25 mS/ cm and the conjugate were spiked with lithium dodecyl sulfate (LDS ) buffer and between 27 -45 mS/ cm . The conductivity of the conjugate denatured for 10 min at 70° C . Then the samples were containing fractions was subsequently raised to 190 mS/ cm applied on 3 - 8 % TRIS - acetate gels and ran at 150 V for 60 with Buffer C (50 mM Hepes , 5M NaCl, 5 mM CaC12 , pH min . Subsequently the gels were stained with Coomassie . 6 . 9 ) and loaded onto a 20 ml HiPrep Butyl FF 16 / 10 column [0206 ] In addition the samples were characterized by use (GE Healthcare , Fairfield , Conn .) pre - equilibrated with Buf of a SEC -HPLC system using a Agilent 1200 HPLC system fer D ( 50 mM Hepes, 3 M NaCl, 5 mM CaCl2 , pH 6 . 9 ) . Free equipped with a Shodex KW 803 column under conditions aminooxy -PSA reagent was washed out within 5 CV Buffer as previously described (Kolarich et al, Transfusion 2006 ; D . Subsequently the conjugate is eluted with 100 % Buffer E 46 : 1959 - 77) . (50 mM Hepes, 5 mM CaCl2 , pH 7 . 4 ) . The conjugate [ 0207] 50 ul of samples were injected undiluted and eluted containing fractions were concentrated by UF /DF using isocratically with a 0 . 22 m filtered solution of 20 mM Vivaspin 15R 10 kD centrifugal filtrator . The final diafiltra NaH2PO4 , 50 mM Na2SO4 , pH 6 . 1 at a flow rate of 0 . 5 tion step was performed against histidine buffer, pH 7 . 2 ml/min . The elution pattern was recorded at 280 nm . containing 150 mM NaCl and 5 mM CaC12 . The preparation [ 0208 ] The results are summarized in FIGS . 5A - C and 6 was analytically characterized by measuring total protein (SDS PAGE ) and Table 2 (SEC -HPLC results ) . The catalytic (Bradford ) and FIX chromogenic activity . The PSA -rFIX effect of the different preparations is demonstrated . It is conjugate showed a specific activity of > 50 % in comparison shown that the use of m - toluidine leads to equivalent results to native rFIX is determined . as obtained with aniline . Method 2 : TABLE 2 [0212 ] 12 . 3 mg rFIX is dissolved in L -histidine buffer, pH di- PSAylated mono free 6 . 0 ( 20 mM L -histidine , 150 mM NaCl, 5 mM CaCl2 ) to get nucleophilic catalysts rFIX PSAylated rFIX rFIX a final protein concentration of 1 mg rFIX /ml . A 5 mM aqueous sodium periodate solution is added to get a final no catalyst 4 . 5 % 24 . 9 % 70 . 6 % 10 mM aniline 47. 7 % 33 . 6 % 18 . 7 % concentration of 100 uM and the reaction mixture is incu 10 mM m -toluidine 31. 4 % 40 . 8 % 27. 8 % bated for 1 hour in the dark at 4° C . under gentle stirring at 10 mM o -aminobenzioc acid 30 . 9 % 38 . 5 % 30 . 6 % pH 6 . 0 and quenched for 15 min at room temperature by the 10 mM m -aminobenzioc acid 27 . 6 % 38 . 0 % 34 . 4 % addition of an 1 M aqueous L -cysteine solution (or other 10 mM p - aminobenzioc acid 18 . 1 % 39 . 3 % 42 .6 % 10 mM o -aminobenzamide 15 . 9 % 38 . 4 % 45 . 7 % quenching reagents ) to get a final concentration of 10 mM . 10 mM sulfanilic acid 11 . 8 % 35 . 8 % 52 . 4 % The mixture is subsequently subjected to UF /DF employing Vivaspin 15R 10 kD centrifugal filtrators to remove excess periodate , quencher and the byproducts thereof. US 2017 /0240590 A1 Aug . 24 , 2017 39

[0213 ] The obtained retentate (8 . 8 ml) , containing oxi- (GE Healthcare , Fairfield , Conn .) pre -equilibrated with Buf dized rFIX , is mixed with an aqueous m -toluidine solution fer D (50 mM Hepes, 3 M NaC1, 5 mM CaC12 , pH 6 . 9 ) . Free ( 50 mm ) to give a final concentration of 10 mM and aminooxy - PSA reagent was washed out within 5 CV Buffer incubated for 30 min at room temperature . Then aminooxy - D . Subsequently , the conjugate was eluted with 100 % Buffer PSA reagent with a MW of 20 kD ( described above ) is added E ( 50 mM Hepes , 5 mM CaCl2 , pH 7 . 4 ) . The conjugate to give a 5 - fold molar reagent excess . This mixture was containing fractions were concentrated by UF /DF using a 10 incubated at pH 6 . 0 for 2 . 5 hours at room temperature; 0 . 5 kD membrane made of regenerated cellulose (88 cm2, hours to 18 hours at + 4° C . ) in the dark under gentle stirring . cut -off 10 kD , Millipore ) . The final diafiltration step was [ 0214 ] The free rFIX is removed by means of anion performed against histidine buffer, pH 7 . 2 containing 150 exchange chromatography (AEC ) . The reaction mixture is mM NaCl and 5 mM CaC12 . The preparation was analyti diluted with appropriate amounts of Buffer A (50 mM cally characterized by measuring total protein (Bradford ) Hepes, 5 mM CaC12 , pH 7 . 5 ) to correct the solutions and FIX chromogenic activity . For the PSA -rFIX conjugate conductivity and pH prior to load onto a 20 ml HiPrep QFF a specific activity of > 50 % in comparison to native rFIX was 16 / 10 column (GE Healthcare , Fairfield , Conn .) pre - equili determined . The conjugate was additionally analytically brated with buffer A . Then the column is eluted with Buffer characterized by Size Exclusion HPLC using a Agilent 1200 B (50 mM Hepes , 1 M NaC1, 5 mM CaC12 , pH 7 . 5 ). Free HPLC system equipped with a Shodex KW 803 column rFIX is eluted by a step gradient using 25 % of Buffer B , under conditions as previously described (Kolarich et al , which results in a conductivity between 12 -25 mS/ cm in the Transfusion 2006 ; 46 : 1959 -77 ) . It was shown that the prepa obtained fraction and the conjugate using a step gradient of ration contains no free FIX . The conjugate consisted of 57 % 50 % Buffer B , which results in a conductivity between mono -polysialylated and 31 % di- polysialylated and 12 % 27 -45 mS/ cm in the conjugate fraction . The conductivity of tri -polysialyated product . the conjugate containing fraction is subsequently raised to 190 mS/ cm with Buffer C (50 mM Hepes , 5 M NaC1, 5 mM Method 4 : CaC12 , pH 6 .9 or by use of anti -chaotropic salts e .g . ammo nium sulphate, ammonium acetate etc .) and loaded onto a 20 [0217 ] 25 .4 mg rFIX was dissolved in L -histidine buffer, ml HiPrep Butyl FF 16 / 10 column (GE Healthcare , Fairfield , pH 6 .0 ( 20 mM L -histidine , 150 mM NaCl, 5 mM CaC12 ) Conn . or comparable HIC media ) pre - equilibrated with to get a final protein concentration of 2 mg rFIX /ml . Buffer D ( 50 mM Hepes , 3 M NaCl, 5 mM CaC12 , pH 6 . 9 ) . Subsequently an 5 mM aqueous sodium periodate solution Free aminooxy - PSA reagent is washed out within 5 CV was added within 15 minutes to give a final concentration of Buffer D . Subsequently , the conjugate is eluted with 100 % 100 uM , followed by addition of an 50 mM aqueous Buffer E (50 mM Hepes , 5 mM CaCl2 , pH 7 . 4 ) . The m - toluidine solution to get a final concentration of 10 mm conjugate containing fractions are concentrated by UF /DF within a time period of 30 minutes . Then the aminooxy -PSA using a 10 kD membrane made of regenerated cellulose (88 reagent with a MW of 20 kD (described above ) was added cm2, cutoff 10 kD , Millipore ). The final diafiltration step is to give a 5 - fold molar reagent excess. After correction of the performed against L - histidine buffer , pH 7 . 2 containing 150 pH to 6 . 0 the mixture was incubated for 2 h in the dark at mM NaCl and 5 mM CaC12 . The preparation is analytically room temperature under gentle stirring and quenched for 15 characterized by measuring total protein ( Bradford and BCA min at room temperature by the addition of a 1 M aqueous procedure ) and FIX chromogenic - and clotting activity . For L - cysteine solution to give a final concentration of 10 mM . [0218 ] The free rFIX was removed by means of ion the PSA - rFIX conjugate a specific activity of > 50 % in exchange chromatography ( IEC ) . The reaction mixture was comparison to native rFIX is determined . diluted with appropriate amounts of Buffer A (50 mM Hepes , 5 mM CaC12 , pH 7 . 5 ) to correct the solutions Method 3 : conductivity and pH value prior to load onto a 20 mlHiPrep 02151 25 . 4 mg rFIX was dissolved in 18 .7 ml histidine QFF 16 / 10 column (GE Healthcare , Fairfield , Conn .) pre buffer , pH 6 . 0 (20 mM L -histidine , 150 mM NaCl, 5 mM equilibrated with Buffer A . Then the column was eluted with CaCl2 ) . 531 ul of an aqueous sodium periodate solution ( 5 Buffer B (50 mM Hepes , 1 M NaCl, 5 mM CaCl2 , pH 7 .5 ) . mM ) and 5 .07 ml of an aqueous m - toluidine solution (50 Free rFIX was eluted by a step gradient using 25 % of Buffer mM ) were then added . Subsequently , the aminooxy - PSA B , which results in a conductivity between 12 - 25 mS/ cm in reagent with a MW of 20 kD (described above ) was added the obtained fraction and the conjugate using a step gradient to give a 5 - fold molar reagent excess . The mixture was of 50 % Buffer B , which results in a conductivity between incubated for 2 h in the dark at room temperature under 27 -45 mS/ cm in the conjugate fraction . The conductivity of gentle stirring and quenched for 15 min at room temperature the conjugate containing fraction was subsequently raised to by the addition of 25 ul of 1 M aqueous cysteine solution . 190 mS/ cm with Buffer C (50 mM Hepes, 5 M NaC1, 5 mM [0216 ] The free rFIX was removed by means of anion CaCl2 , pH 6 . 9 ; by use of anti -chaotropic salts e . g . ammo exchange chromatography (AEC ). The reaction mixture was nium acetate ) and loaded onto a 20 ml HiPrep Butyl FF diluted with 20 ml Buffer A ( 50 mM Hepes, 5 mM CaCl2 , 16 / 10 column (GE Healthcare , Fairfield , Conn . ; or compa pH 7 . 5 ) and loaded onto a 20 mlHiPrep QFF 16 / 10 column rable HIC media ) pre - equilibrated with Buffer D (50 mM (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with Buf Hepes , 3 M NaC1, 5 mM CaC12 , pH 6 . 9 ) . Free aminooxy fer A . Then the column was eluted with Buffer B (50 mM PSA reagent was washed out within 5 CV Buffer D . Sub Hepes, 1 M NaC1, 5 mM CaC12 , pH 7 . 5 ) . Free rFIX eluted sequently the conjugate was eluted with 100 % Buffer E (50 at a conductivity between 12 -25 mS/ cm and the conjugate mM Hepes, 5 mM CaC12 , pH 7 .4 ) . The conjugate containing between 27 - 45 mS/ cm . The conductivity of the conjugate fractions were concentrated by UF/ DF using a 10 kD containing fractions was subsequently raised to 190 mS / cm membranemade of regenerated cellulose ( 88 cm2, cut -off 10 with Buffer C (50 mM Hepes, 5 M NaCl, 5 mM CaC12 , pH kD , Millipore ). The final diafiltration step was performed 6 . 9 ) and loaded onto a 20 ml HiPrep Butyl FF 16 / 10 column against L -histidine buffer , pH 7 . 2 containing 150 mM NaCl US 2017 /0240590 A1 Aug . 24 , 2017 40 and 5 mM CaC12 . The preparation was analytically charac - Polysorbate 80 , pH 7. 4 ) is dissolved in reaction buffer (50 terized by measuring total protein ( Bradford and BCA mM Hepes, 350 mM sodium chloride, 5 mM calcium procedure ) and FIX chromogenic - and clotting activity . For chloride , pH 6 . 0 ) to get a final protein concentration of the PSA -rFIX conjugate a specific activity of > 50 % in 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is corrected comparison to native rFIX was determined . The conjugate to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 was additionally analytically characterized by Size Exclu - solution . Subsequently , a 40 mM aqueous sodium periodate sion HPLC using a Agilent 1200 HPLC system equipped solution is added within 10 minutes to give a concentration with a Shodex KW 803 column under conditions as previ of 200 uM . The oxidation reaction is carried out for 30 + / - 5 ously described (Kolarich et al, Transfusion 2006 ; 46 : 1959 min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction 77 ) . It was shown that the preparation contains no free FIX . is stopped by addition of an aqueous L - cysteine solution ( 1 The conjugate consisted of 57 % mono - polysialylated and M ) within 15 minutes at T = + 22 + / - 2° C . to give a final 31 % di- polysialylated and 12 % tri -polysialyated product . concentration of 10 mM in the reaction mixture and incu bation for 60 + / - 5 min . Example 12 [0221 ] The oxidized rFVIII is further purified by anion exchange chromatography on EMD TMAE ( M ) (Merck ). Polysialylation of rFVIII Using Aminooxy -PSA and The mixture is diluted with Buffer A (20 mM Hepes , 5 mM m - Toluidine as a Nucleophilic Catalyst CaCl , , pH 6 . 5 ) to give a conductivity of 5 ms/ cm . This solution is loaded onto the IEX column (bed height: 5 .4 cm ) Method 1 : with a column volume of 10 ml using a flow rate of 1 . 5 10219 ] 50 mg rFVIII was transferred into reaction buffer cm /min . This column is subsequently washed ( flow rate : 1 . 5 (50 mM Hepes, 350 mM sodium chloride , 5 mM calcium cm /min ) with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and chloride, pH 6 .0 ) and diluted to obtain a protein concentra Buffer B ( 20 mM Hepes , 5 mM CaCl2 , 1 . 0 M NaCl, pH 7 . 0 ) . tion of 1 mg/ ml . To this solution , NalO4 was added to give Then the oxidized rFVIII is eluted with a 50 :50 ( w / w ) a final concentration of 200 uM . The oxidation was carried mixture of Buffer A and Buffer B followed by a postelution at RT for 30 min in the dark under gentle shaking . Then the step with 5 CV of Buffer B . The elution steps are carried out reaction was quenched with cysteine ( final concentration : 10 by use of a flow rate of 1 . 0 cm /min . mM ) for 60 min at RT. The solution was subjected to an IEX [0222 ] Subsequently , the aminooxy - polysialic acid (PSA column with a volume of 20 ml (Merck EMD TMAE ( M ) ) ONH2) reagent is added in a 50 - fold molar excess to the which was equilibrated with Buffer A (20 mM Hepes , 5 mM eluate containing the purified oxidized rFVIII within a CaCl2 , pH 7. 0 ). The column was equilibrated with 5 CV maximum timeperiod ( t ) of 15 minutes under gentle stirring . Buffer A . Then the oxidized rFVIII was eluted with Buffer Then an aqueous m - toluidine solution (50 mM is added B (20 mM Hepes , 5 mM CaCl2, 1M NaCl, pH 7 . 0 ). The within 15 minutes to get a final concentration of 10 mM . The rFVIII containing fractions were collected . The protein reaction mixture is incubated for 120 +/ – 10 min . in the dark content was determined ( Coomassie , Bradford ) and adjusted at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . to 1 mg/ ml with reaction buffer and adjusted to pH 6 . 0 by [0223 ] The obtained PSA - rFVIII conjugate is purified by dropwise addition of 0 . 5 M HC1. Then a 50 -fold molar Hydrophobic Interaction Chromatography (HIC ) using a excess of a aminooxy - PSA reagent with a MW of 20 kD Phenyl Sepharose FF low sub resin (GE Healthcare ) packed ( described above ) was added followed by m - toluidine as a into a column manufactured by GE Healthcare with a bed nucleophilic catalyst ( final concentration : 10 mM ) . The height (h ) of 15 cm and a resulting column volume ( CV ) of coupling reaction was performed for 2 hours in the dark 81 ml. under gentle shaking at room temperature . The excess of [0224 ] The reaction mixture is spiked with ammonium aminooxy - PSA reagent was removed by means of HIC . The acetate by addition of 50 mM Hepes buffer, containing 350 conductivity of the reaction mixture was raised to 130 mM sodium chloride , 8 M ammonium acetate , 5 mM mS / cm by adding a buffer containing ammonium acetate (50 calcium chloride, pH 6 . 9 . Two volumes of the reaction mM Hepes , 350 mM sodium chloride, 5 mM calcium mixture are mixed with 1 volume of the ammonium acetate chloride , 8 M ammonium acetate , pH 6 . 9 ) and loaded onto containing buffer system and the pH value is corrected to pH a column filled with 80 ml Phenyl Sepharose FF (GE 6 . 9 by drop wise addition of a 0 . 5 N aqueous NaOH solution . Healthcare , Fairfield , Conn . ) pre - equilibrated with 50 mM This mixture is loaded onto the HIC column at flow rate of Hepes, 2 . 5 M ammonium acetate , 350 mM sodium chloride , 1 cm /min followed by a washing step using > 3 CV equili 5 mM calcium chloride, pH 6 . 9 . Subsequently , the conjugate bration buffer (50 mM Hepes, 350 mM sodium chloride, 2 . 5 was eluted with 50 mM Hepes buffer pH 7 .5 containing 5 M ammonium acetate , 5 mM calcium chloride, pH 6 . 9 ). mM CaCl2 . Finally , the PSA -rFVIII containing fractions [0225 ] For removal of reaction by -products and anti were collected and subjected to UF /DF by use of a 30 kD chaotropic salt a second washing step is performed with > 5 membrane made of regenerated cellulose ( 88 cm², Milli CV washing buffer 1 (50 mM Hepes, 3 M sodium chloride , pore ). The preparation was analytically characterized by 5 mM calcium chloride , pH 6 . 9 ) in upflow mode at a flow measuring total protein (Coomassie , Bradford ) and FVIII rate of 2 cm /min . Then elution of purified PSA -rFVIII chromogenic activity. The PSA - rFVIII conjugate showed a conjugate is performed in down flow mode using a step specific activity of > 70 % in comparison to native rFVIII was gradient of 40 % washing buffer 2 (50 mM Hepes , 1 . 5 M determined . sodium chloride , 5 mM calcium chloride, pH 6 . 9 ) and 60 % elution buffer (20 mM Hepes, 5 mM calcium chloride, pH Method 2 : 7 . 5 ) at a flow rate of 1 cm /min . The elution of the PSA [0220 ] 58 mg of recombinant factor VIII ( rFVIII ) derived rFVIII conjugate is monitored at UV 280 nm and the eluate from the ADVATE process in Hepes buffer ( 50 mM HEPES , containing the conjugate is collected within < 4 CV. The post - 350 mM sodium chloride , 5 mM calcium chloride , 0 . 1 % elution step is performed with > 3 CV elution buffer under US 2017 /0240590 A1 Aug . 24 , 2017

the same conditions to separate minor and /or non modified tion of an aqueous L - cysteine solution ( 1 M ) to give a final rFVIII from the main product . concentration of 10 mM in the reaction mixture and incu [0226 ] Finally the purified conjugate is concentrated by bation for 60 + / - 5 min . ultra - / diafiltration (UF /DF ) using a membrane made of [0232 ] The obtained PSA -rFVIII conjugate was purified regenerated cellulose with a molecular weight cut off 30 kD by Hydrophobic Interaction Chromatography (HIC ) using a (88 cm², Millipore ). Phenyl Sepharose FF low sub resin (GE Healthcare ) packed [ 0227 ] The conjugate prepared by use of this procedure into a column manufactured by GE Healthcare with a bed are analytically characterized by measuring total protein , height ( h ) of 15 cm and a resulting column volume (CV ) of FVIII chromogenic activity and determination of the poly 81 ml. sialyation degree by measuring the PSA content ( resorcinol 0233 ] The reaction mixture was spiked with ammonium assay ) . For the conjugate obtained a specific activity > 50 % acetate by addition of 50 mM Hepes buffer, containing 350 and a PSA degree > 5 . 0 is calculated . mM sodium chloride , 8 M ammonium acetate , 5 mM calcium chloride , pH 6 . 9 . Two volumes of the reaction Method 3 : mixture was mixed with 1 volume of the ammonium acetate containing buffer system and the pH value was corrected to [ 0228 ] 50 mg rFVIII was transferred into reaction buffer pH 6 . 9 by drop wise addition of an 0 . 5 N aqueous NaOH ( 50 mM Hepes, 350 mM sodium chloride , 5 mM calcium solution . This mixture was loaded onto the HIC column chloride , pH 6 .0 ) and diluted to obtain a protein concentra using a flow rate of 1 cm /min followed by a washing step tion of 1 mg/ ml . A 50 - fold molar excess of aminooxy -PSA using > 3 CV equilibration buffer (50 mM Hepes , 350 mM reagent with a MW of 20 kD (described above ) was added sodium chloride, 2 . 5 M ammonium acetate, 5 mM calcium followed by m - toluidine as a nucleophilic catalyst ( final chloride , pH 6 . 9 ) . concentration : 10 mM ) and Nalo , ( final concentration : 400 [0234 ] For removal of reaction by - products and anti UM ). The coupling reaction was performed for 2 hours in the chaotropic salt a second washing step was performed with dark under gentle shaking at room temperature . Subse > 5 CV washing buffer 1 (50 mM Hepes, 3 M sodium quently , the reaction was quenched with cysteine for 60 min chloride , 5 mM calcium chloride , pH 6 . 9 ) in upflow mode at at RT ( final concentration : 10 mM ). Then the conductivity of a flow rate of 2 cm /min . Then elution of purified rFVIII the reaction mixture was raised to 130 mS / cm by adding a conjugate was performed in down flow mode using a step buffer containing ammonium acetate (50 mM Hepes , 350 gradient of 40 % washing buffer 2 (50 mM Hepes, 1 . 5 M mM sodium chloride , 5 mM calcium chloride , 8 M ammo sodium chloride , 5 mM calcium chloride, pH 6 . 9 ) and 60 % nium acetate , pH 6 . 9 ) and loaded onto a column filled with elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 80 ml Phenyl Sepharose FF (GE Healthcare , Fairfield , 7 . 5 ) at a flow rate of 1 cm /min . The elution of the PSA Conn . ) pre - equilibrated with 50 mM Hepes , 2 . 5 M ammo rFVIII conjugate was monitored at UV 280 nm and the nium acetate , 350 mM sodium chloride , 5 mM calcium eluate containing the conjugate was collected within < 4 CV . chloride , 0 .01 % Tween 80 , pH 6 . 9 . Subsequently , the con The post elution step was performed with > 3 CV elution jugate was eluted with 50 mM Hepes , 5 mM calcium buffer under the same conditions to separate minor and /or chloride , pH 7 . 5 . Finally , the PSA - rFVIII containing frac non modified rFVIII from the main product. tions were collected and subjected to UF /DF by use of a 30 [0235 ] Finally , the purified conjugate was concentrated by kD membrane made of regenerated cellulose ( 88 cm " , ultra - /diafiltration (UF /DF ) using a membrane made of Millipore ) . The preparation was analytically characterized regenerated cellulose with a molecular weight cut off 30 kD by measuring total protein (Bradford ) and FVIII chromoge (88 cm2, Millipore ) . nic activity . For the PSA - rFVIII conjugate a specific activity [0236 ] The conjugates prepared by use of this procedure of > 70 % in comparison to native rFVIII was determined . were analytically characterized by measuring total protein , FVIII chromogenic activity and determination of the poly Method 4 : sialyation degree by measuring the PSA content ( resorcinol [ 0229 ] 50 mg recombinant factor VIII ( rFVIII ) derived assay ) . from the ADVATE process in 50 mM Hepes buffer (50 mM [ 0237 ] Analytical data (mean of 6 consecutive batches) : HEPES , ~ 350 mM sodium chloride , 5 mM calcium chloride , [0238 ] Process yield ( Bradford ) : 58 . 9 % 0 . 1 % Polysorbate 80 , pH 7 . 4 ) was dissolved in reaction [0239 ] Process yield (FVIII chrom . ) : 46 . 4 % buffer (50 mM Hepes , 350 mM sodium chloride , 5 mM [0240 ] Specific activity : (FVIII chrom ./ mg protein ): 4148 calcium chloride , pH 6 . 0 ) to get a final protein concentration IU /mg of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution was [0241 ] Specific activity ( % of starting material ): 79. 9 % corrected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous [ 0242 ] PSA degree (mol / mol ) : 8 . 1 HCl solution . [ 0230 ] Subsequently , the aminooxy -polysialic acid (PSA Example 13 ONH2 ) reagent was added in a 50 -fold molar excess to this rFVIII solution within a maximum time period ( t ) of 15 PEGylation of r FVIII Using an Aminooxy - PEG minutes under gentle stirring. Then an aqueous m - toluidine Reagent and m - Toluidine as a Nucleophilic solution ( 50 mM ) was added within 15 minutes to get a final Catalyst concentration of 10 mM . Finally , a 40 mM aqueous sodium periodate solution was added to give a concentration of 400 Method 1: UM . [0243 ] rFVIII is PEGylated by use of a linear 20 kD [ 0231 ] The reaction mixture was incubated for 120 + / - 10 PEGylation reagent containing an aminooxy group . An min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under example of this type of reagent is the Sunbright® CA series gentle shaking. Then the reaction was stopped by the addi from NOF (NOF Corp ., Tokyo , Japan ). 14 .7 mg rFVIII is US 2017 /0240590 A1 Aug . 24 , 2017 dissolved in 7 . 0 ml histidine buffer, pH 6 . 0 (20 mM L -his maximum time period (t ) of 15 minutes under gentle stirring . tidine, 150 mM NaC1, 5 mM CaC12 ) . Then 296 ul of an Then an aqueous m - toluidine solution ( 50 mM ) is added aqueous sodium periodate solution (5 mM ) is added and the within 15 minutes to get a final concentration of 10 mM . The reaction mixture is incubated for 1 h in the dark at 4° C . reaction mixture is incubated for 120 + / – 10 min . in the dark under gentle stirring and quenched for 15 min at room at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking. temperature by the addition of 7 . 5 ul of a 1 M aqueous [0249 ] The obtained PEG -rFVIII conjugate is purified by cysteine solution . The mixture was subsequently subjected Hydrophobic Interaction Chromatography (HIC ) using a to UF /DF employing Vivaspin 15R 10 kD centrifugal fil - Phenyl Sepharose FF low sub resin (GE Healthcare ) packed trators to remove excess periodate , quencher and the byprod - into a column manufactured by GE Healthcare with a bed ucts thereof . height ( h ) of 15 cm and a resulting column volume ( CV ) of [ 0244 ] The retentate ( 10 . 9 ml) , containing oxidized 81 ml. rFVIII , is mixed with 2 .94 ml of an aqueous m - toluidine (0250 ). The reaction mixture is spiked with ammonium solution (50 mM ) and incubated for 30 min at room tem acetate by addition of 50 mM Hepes buffer , containing 350 perature . Then aminooxy - PEG reagent with a MW of 20 kD mM sodium chloride , 8 M ammonium acetate , 5 mM is added to give a 5 - fold molar reagent excess . This mixture calcium chloride , pH 6 . 9 . Two volumes of the reaction was incubated for 2 . 5 h at room temperature in the dark mixture are mixed with 1 volume of the ammonium acetate under gentle stirring . containing buffer system and the pH value is corrected to pH [0245 ] Finally , the PEG - rFVIII conjugate is purified by 6 . 9 by drop wise addition of a 0 . 5 N aqueous NaOH solution . ion -exchange chromatography on Q Sepharose FF. 1 . 5 mg This mixture is loaded onto the HIC column using a flow protein /ml gel is loaded on the column equilibrated with 50 rate of 1 cm /min followed by a washing step using > 3 CV mM Hepes buffer , pH 7 .4 containing 5 mM CaC12 . The equilibration buffer (50 mM Hepes , 350 mM sodium chlo conjugate is eluted with 50 mM Hepes buffer containing 5 ride , 2 . 5 M ammonium acetate , 5 mM calcium chloride , pH mM CaC12 and 500 mM sodium chloride, pH 7 . 4 and is then 6 . 9 ) . subjected to UF /DF using a 30 kD membrane (50 cm2, [0251 ] For removal of reaction by -products and anti Millipore ). The preparation is analytically characterized by chaotropic salt a second washing step is performed with > 5 measuring total protein (Coomassie , Bradford ) and FVIII CV washing buffer 1 (50 mM Hepes, 3 M sodium chloride, chromogenic activity . It is expected that the PEG - rFVIII 5 mM calcium chloride , pH 6 . 9 ) in upflow mode at a flow conjugate will demonstrate a specific activity of > 70 % in rate of 2 cm /min . Then elution of purified rFVIII conjugate comparison to native rFVIII was determined . is performed in down flow mode using a step gradient of 40 % washing buffer 2 (50 mM Hepes , 1 . 5 M sodium Method 2 : chloride , 5 mM calcium chloride, pH 6 . 9 ) and 60 % elution [0246 ] [FVIII is PEGylated by use of a linear 20 kD buffer ( 20 mM Hepes, 5 mM calcium chloride , pH 7 . 5 ) at a PEGylation reagent containing an aminooxy group . An flow rate of 1 cm /min . The elution of the PEG -rFVIII example of this type of reagent is the Sunbright® CA series conjugate is monitored at UV 280 nm and the eluate from NOF (NOF Corp ., Tokyo , Japan ). A starting weight or containing the conjugate is collected within < 4 CV . The post concentration of rFVIII is dissolved in or transferred to a elution step is performed with > 3 CV elution buffer under reaction buffer (50 mM Hepes , 350 mM sodium chloride , 5 the same conditions to separate minor and /or non modified mM calcium chloride , pH 6 . 0 ) to get a final protein con rFVIII from the main product . centration of 1 . 0 + / - 0 . 25 mg/ml . Then the pH of the solution [0252 ] Finally , the purified conjugate is concentrated by is corrected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous ultra - /diafiltration (UF /DF ) using a membrane made of HCl solution . Subsequently a 40 mM aqueous sodium regenerated cellulose with a molecular weight cut off 30 kD periodate solution is added within 10 minutes to give a (Millipore ). concentration of 200 uM . The oxidation reaction is carried [0253 ] The conjugate prepared by use of this procedure out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . are analytically characterized by measuring total protein and Then the reaction is stopped by addition of an aqueous biological activity according to methods known in the art . L - cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° C . to give a final concentration of 10 mM in the reaction Method 3: mixture and incubation for 60 + / - 5 min . [0254 ) rFVIII is PEGylated by use of a linear 20 kD [0247 ] The oxidized rFVIII is further purified by anion PEGylation reagent containing an aminooxy group . An exchange chromatography on EMD TMAE ( M ) (Merck ). example of this type of reagent is the Sunbright @ CA series The mixture is diluted with Buffer A (20 mM Hepes, 5 mM from NOF (NOF Corp ., Tokyo , Japan ). 7 .84 mg rFVIII , CaCl2 , pH 6 . 5 ) to give a conductivity of 5 ms/ cm . This dissolved in 6 ml Hepes buffer (50 mM Hepes , 150 mM solution is loaded onto the IEX column (bed height: 5 . 4 cm ) sodium chloride, 5 mM calcium chloride , pH 6 .0 ) are mixed with a column volume of 10 ml using a flow rate of 1 . 5 with 314 ul of an aqueous sodium periodate solution ( 10 cm /min . This column is subsequently washed ( flow rate : 1 .5 mM ) , and 1 .57 ml of an aqueous m - toluidine solution ( 50 cm /min ) with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and mM ) . Subsequently the aminooxy reagent is added to give Buffer B ( 20 mM Hepes, 5 mM CaCl2 , 1 . 0 M NaCl, pH 7 . 0 ) . a 20 -fold molar reagent excess . The mixture is incubated for Then the oxidized rFVIII is eluted with a 50 :50 ( w / w ) 2 h in the dark at room temperature under gentle stirring and mixture of Buffer A and Buffer B followed by a postelution quenched for 15 min at room temperature by the addition of step with 5 CV of Buffer B . The elution steps are carried out 8 ul of aqueous cysteine solution ( 1 M ) . by use of a flow rate of 1 .0 cm /min . [0255 ] Finally the PEG - rFVIII conjugate is purified by [0248 ] Subsequently , the aminooxy -PEG reagent with a ion -exchange chromatography on Q -Sepharose FF . 1. 5 mg MW of 20 kD reagent is added in a 50 - fold molar excess to protein /ml gel is loaded on the column pre equilibrated with the eluate containing the purified oxidized rFVIII within a 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaCl2 . The US 2017 /0240590 A1 Aug . 24 , 2017 43 conjugate is eluted with 50 mM Hepes buffer containing 5 buffer (50 mM Hepes , 350 mM sodium chloride , 5 mM mM CaCl2 and 500 mM sodium chloride, pH 7 . 4 and is then calcium chloride , pH 6 . 0 ) to get a final protein concentration subjected to UF /DF using a 30 kD membrane (88 cm ” , of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is cor Millipore ) . The analytical characterization of the conjugate rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous NaOH by FVIII chromogenic assay and determination of total solution . Subsequently , a 40 mM aqueous sodium periodate protein (Bradford ) shows a specific activity of > 60 % com solution is added within 10 minutes to give a concentration pared to the rFVIII starting material . of 50 JM . The oxidation reaction is carried out for 30 + / - 5 Method 4 : min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction [ 0256 ] FVIII is PEGylated by use of a linear 20 kD is stopped by addition of an aqueous L - cysteine solution ( 1 PEGylation reagent containing an aminooxy group . An M ) within 15 minutes at T = + 22 + / - 2° C . to give a final example of this type of reagent is the Sunbright® CA series concentration of 10 mM in the reaction mixture and incu from NOF (NOF Corp ., Tokyo , Japan ) . An initial concen bation for 60 + / - 5 min . tration or weight of rFVIII is transferred or dissolved in [0260 ] The oxidized rFVIIa is further purified by anion Hepes buffer (50 mM Hepes, 150 mM sodium chloride , 5 exchange chromatography on EMD TMAE ( M ) (Merck ). mM calcium chloride , pH 6 .0 ) to get a final protein con The mixture is diluted with Buffer A ( 20 mM Hepes , 5 mM centration of 2 mg rFVIII /ml . Subsequently , an 5 mM CaCl , , pH 6 . 5 ) to give a conductivity of 5 ms/ cm . This aqueous sodium periodate solution is added within 15 min solution is loaded onto the IEX column (bed height: 5 . 4 cm ) utes to give a final concentration of 100 UM , followed by with a column volume of 10 ml using a flow rate of 1 . 5 addition of an 50 mM aqueous m - toluidine solution to get a cm /min . This column is subsequently washed ( flow rate : 1 . 5 final concentration of 10 mM within a time period of 30 minutes . Then the aminooxy -PEG reagent with a MW of 20 cm /min ) with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and kD (described above ) is added to give a 20 - fold molar Buffer B ( 20 mM Hepes , 5 mM CaCl2 , 1 . 0 M NaCl, pH 7 . 0 ) . excess . After correction of the pH to 6 . 0 the mixture is Then the oxidized rFVIIa is eluted with a 50 :50 ( w / w ) incubated for 2 h in the dark at room temperature under mixture of Buffer A and Buffer B followed by a postelution gentle stirring and quenched for 15 min at room temperature step with 5 CV of Buffer B . The elution steps are carried out by the addition of a 1 Maqueous L - cysteine solution to give by use of a flow rate of 1. 0 cm /min . a final concentration of 10 mM . [0261 ] Subsequently , the aminooxy -polysialic acid (PSA [0257 ] The free rFVIII is removed by means of ion ONH2) reagent is added in a 50 - fold molar excess to the exchange chromatography ( IEC ) . The reaction mixture was eluate containing the purified oxidized rFVIIa within a diluted with appropriate amounts of Buffer A ( 50 mM maximum time period (t ) of 15 minutes under gentle stirring . Hepes, 5 mM CaC12 , pH 7 . 5 ) to correct the solutions Then an aqueous m - toluidine solution (50 mM ) is added conductivity and pH value prior to load onto a 20 ml HiPrep within 15 minutes to get a final concentration of 10 mM . The QFF 16 / 10 column (GE Healthcare , Fairfield , Conn . ) pre reaction mixture is incubated for 120 + / – 10 min . in the dark equilibrated with Buffer A . Then the column was eluted with at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . Buffer B (50 mM Hepes , 1 M NaC1, 5 mM CaC12 , PH 7 . 5 ) . [0262 ] The obtained PSA -rFVIIa conjugate is purified by Free rFVIII was eluted by a step gradient using 25 % of Hydrophobic Interaction Chromatography (HIC ) using a Buffer B , which results in a conductivity between 12 - 25 Phenyl Sepharose FF low sub resin (GE Healthcare ) packed mS/ cm in the obtained fraction and the conjugate using a into a column manufactured by GE Healthcare with a bed step gradient of 50 % Buffer B , which results in a conduc height ( h ) of 15 cm and a resulting column volume (CV ) of tivity between 27 -45 mS/ cm in the conjugate fraction . The 81 ml. conductivity of the conjugate containing fraction is subse 10263 ] The reaction mixture is spiked with ammonium quently raised with Buffer C ( 50 mM Hepes , 5 M NaCl, 5 acetate by addition of 50 mM Hepes buffer , containing 350 mM CaC12 , pH 6 . 9 ; by use of anti - chaotropic salts e . g . mM sodium chloride , 8 M ammonium acetate , 5 mM ammonium acetate , ammonium sulphate etc . ) and loaded calcium chloride , pH 6 . 9 . Two volumes of the reaction onto a 20 ml HiPrep Butyl FF 16 / 10 column (GE Healthcare , mixture are mixed with 1 volume of the ammonium acetate Fairfield , Conn . ; or comparable HIC media ) pre - equilibrated containing buffer system and the pH value is corrected to pH with Buffer D (50 mM Hepes , 3 M NaCl, 5 mM CaC12 , pH 6 . 9 by drop wise addition of a 0 . 5 N aqueous NaOH solution . 6 . 9 ) . Free PEG - reagent was washed out within 5 CV Buffer This mixture is loaded onto the HIC column using a flow D . Subsequently , the conjugate was eluted with 100 % Buffer rate of 1 cm /min followed by a washing step using > 3 CV E (50 mM Hepes, 5 mM CaCl2 , pH 7 . 4 ) . The conjugate equilibration buffer (50 mM Hepes, 350 mM sodium chlo containing fractions are concentrated by UF/ DF using a 10 ride , 2 . 5 M ammonium acetate , 5 mM calcium chloride, pH kD membrane made of regenerated cellulose (88 cm2, 6 . 9 ) . cut -off 10 kD , Millipore ) . The final diafiltration step is [0264 ] For removal of reaction by - products and anti performed against Hepes buffer (50 mM Hepes, 5 mM chaotropic salt a second washing step is performed with > 5 CaCl2 , pH 7. 5) . CV washing buffer 1 (50 mM Hepes, 3 M sodium chloride , [ 0258 ] The preparation is analytically characterized by 5 mM calcium chloride , pH 6 . 9 ) in upflow mode at a flow measuring total protein (Bradford and BCA procedure ) and rate of 2 cm /min . Then elution of purified rFVIIa conjugate biological activity according to known methods. is performed in down flow mode using a step gradient of Example 14 40 % washing buffer 2 (50 mM Hepes, 1 . 5 M sodium Polysialylation of rFVIIa Using Aminooxy -PSA chloride , 5 mM calcium chloride , pH 6 . 9 ) and 60 % elution and m - Toluidine as a Nucleophilic Catalyst buffer ( 20 mM Hepes, 5 mM calcium chloride, pH 7 . 5 ) at a flow rate of 1 cm /min . The elution of the PSA -rFVIIa Method 1 : conjugate is monitored at UV 280 nm and the eluate [ 0259 ] A starting concentration or weight of recombinant containing the conjugate is collected within < 4 CV. The post factor VIIa (rFVIIa ) is transferred or dissolved in reaction elution step is performed with > 3 CV elution buffer under US 2017 /0240590 A1 Aug . 24 , 2017 44 the same conditions to separate minor and / or non modified under the same conditions to separate minor and /or non rFVIIa from the main product . modified rFVIII from the main product. [0265 ] Finally , the purified conjugate is concentrated by [0273 ] Finally, the purified conjugate is concentrated by ultra -/ diafiltration (UF /DF ) using a membrane made of ultra -/ diafiltration (UF /DF ) using a membrane made of regenerated cellulose with an appropriate molecular weight regenerated cellulose (Millipore ) . cut off ( e. g . 10 kD MWCO , 88 cm², Millipore ). [ 0274 ] The conjugates prepared by use of this procedure [ 0266 ] The conjugate prepared by use of this procedure is are analytically characterized by measuring total protein , analytically characterized by measuring total protein , bio biological activity according to methods known in the art, logical activity, and determination of the polysialyation and determination of the polysialyation degree by measuring degree by measuring the PSA content (resorcinol assay ) . the PSA content ( resorcinol assay ) . Method 2 : Example 15 [0267 ] A starting weight or concentration of rFVIIa is PEGylation of rFIX Using an Aminooxy - PEG dissolved in or transferred to a reaction buffer (50 mM Reagent and m - Toluidine as a Nucleophilic Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 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 NaOH solution . [0275 ) rFIX is PEGylated by use of a linear 20 kD [ 0268 ] Subsequently , the aminooxy -polysialic acid (PSA PEGylation reagent containing an aminooxy group . An ONH , ) reagent is added in a 50 - fold molar excess to this example of this type of reagent is the Sunbright® CA series rFVIIa solution within a maximum time period (t ) of 15 from NOF (NOF Corp . , Tokyo , Japan ). A starting weight or minutes under gentle stirring . Then an aqueous m - toluidine concentration of rFIX is dissolved in or transferred to a solution (50 mM ) is added within 15 minutes to get a final reaction buffer (50 mM Hepes , 350 mM sodium chloride , 5 concentration of 10 mM . Finally a 40 mM aqueous sodium mM calcium chloride , pH 6 .0 ) to get a final protein con periodate solution is added to give a concentration of 150 centration of 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution UM . is corrected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous [0269 ] The reaction mixture is incubated for 120 + / - 10 HCl solution . Subsequently , a 40 mM aqueous sodium min . in the dark at a temperature ( T ) ofT = + 22 + / - 2° C . under periodate solution is added within 10 minutes to give a gentle shaking . Then the reaction is stopped by the addition concentration of 200 UM . The oxidation reaction is carried of an aqueous L - cysteine solution ( 1 M ) to give a final out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . concentration of 10 mM in the reaction mixture and incu Then the reaction is stopped by addition of an aqueous bation for 60 + / - 5 min . L -cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° [0270 ] The obtained PSA - rFVIIa conjugate is purified by C . to give a final concentration of 10 mM in the reaction Hydrophobic Interaction Chromatography (HIC ) using a mixture and incubation for 60 + / - 5 min . Phenyl Sepharose FF low sub resin (GE Healthcare ) packed 0276 ] The oxidized rFVIII is further purified by anion into a column manufactured by GE Healthcare with a bed exchange chromatography on EMD TMAE ( M ) (Merck ). height ( h ) of 15 cm and a resulting column volume (CV ) of The mixture is diluted with Buffer A ( 20 mM Hepes, 5 mM 81 ml. CaCl2 , pH 6 . 5 ) to give a conductivity of 5 mS/ cm . This [0271 ] The reaction mixture is spiked with ammonium solution is loaded onto the IEX column (bed height : 5 . 4 cm ) acetate by addition of 50 mM Hepes buffer , containing 350 with a column volume of 10 ml using a flow rate of 1 . 5 mM sodium chloride , 8 M ammonium acetate , 5 mM cm /min . This column is subsequently washed ( flow rate : 1 . 5 calcium chloride , pH 6 . 9 . Two volumes of the reaction cm /min ) with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and mixture is mixed with 1 volume of the ammonium acetate Buffer B ( 20 mM Hepes , 5 mM CaC12 , 1 . 0 M NaCl, pH 7 . 0 ) . containing buffer system and the pH value is corrected to pH Then the oxidized rFIX is eluted with a 50 : 50 ( w / w ) mixture 6 .9 by drop wise addition of an 0 . 5 N aqueous NaOH of Buffer A and Buffer B followed by a postelution step with solution . This mixture is loaded onto the HIC column using 5 CV of Buffer B . The elution steps are carried out by use a flow rate of 1 cm /min followed by a washing step using > 3 of a flow rate of 1 . 0 cm /min . CV equilibration buffer ( 50 mM Hepes, 350 mM sodium [0277 ] Subsequently , the aminooxy -PEG reagent with a chloride , 2 . 5 M ammonium acetate , 5 mM calcium chloride , MW of 20 kD reagent is added in a 50 - fold molar excess to pH 6 . 9 ). the eluate containing the purified oxidized rFIX within a [0272 ] For removal of reaction by - products and anti maximum time period ( t ) of 15 minutes under gentle stirring. chaotropic salt a second washing step is performed with > 5 Then an aqueous m - toluidine solution (50 mM ) is added CV washing buffer 1 (50 mM Hepes , 3 M sodium chloride , within 15 minutes to get a final concentration of 10 mM . The 5 mM calcium chloride , pH 6 . 9 ) in upflow mode at a flow reaction mixture is incubated for 120 + / – 10 min . in the dark rate of 2 cm /min . Then elution of purified rFVIIa conjugate at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . is performed in down flow mode using a step gradient of [ 0278 ] The obtained PEG -rFIX conjugate is purified by 40 % washing buffer 2 (50 mM Hepes , 1 . 5 M sodium Hydrophobic Interaction Chromatography (HIC ) using a chloride, 5 mM calcium chloride , pH 6 . 9 ) and 60 % elution Phenyl Sepharose FF low sub resin (GE Healthcare ) packed buffer ( 20 mM Hepes , 5 mM calcium chloride , pH 7 . 5 ) at a into a column manufactured by GE Healthcare with a bed flow rate of 1 cm /min . The elution of the PSA -rFVIIa height (h ) of 15 cm and a resulting column volume ( CV ) of conjugate is monitored at UV 280 nm and the eluate 81 ml. containing the conjugate was collected within < 4 CV. The f0279 ] The reaction mixture is spiked with ammonium post elution step is performed with > 3 CV elution buffer acetate by addition of 50 mM Hepes buffer, containing 350 US 2017 /0240590 A1 Aug . 24 , 2017 45 mM sodium chloride, 8 M ammonium acetate , 5 mM of 50 % Buffer B , which results in a conductivity between calcium chloride , pH 6 . 9 . Two volumes of the reaction 27 -45 mS/ cm in the conjugate fraction . The conductivity of mixture are mixed with 1 volume of the ammonium acetate the conjugate containing fraction is subsequently raised with containing buffer system and the pH value is corrected to pH Buffer C (50 mM Hepes , 5 M NaC1, 5 mM CaC12 , pH 6 . 9 ; 6 . 9 by drop wise addition of a 0 . 5 N aqueous NaOH solution . by use of anti - chaotropic salts e . g . ammonium acetate , etc ) This mixture is loaded onto the HIC column using a flow and loaded onto a 20 ml HiPrep Butyl FF 16 /10 column (GE rate of 1 cm /min followed by a washing step using > 3 CV Healthcare , Fairfield , Conn . ; or comparable HIC media ) equilibration buffer (50 mM Hepes , 350 mM sodium chlo pre -equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 ride , 2 . 5 M ammonium acetate , 5 mM calcium chloride , pH mM CaC12 , pH 6 . 9 ) . Free aminooxy -PEG reagent was 6 . 9 ) . washed out within 5 CV Buffer D . Subsequently , the con [0280 ] For removal of reaction by - products and anti jugate was eluted with 100 % Buffer E ( 50 mM Hepes, 5 mM chaotropic salt a second washing step is performed with > 5 CaC12 , pH 7 . 4 ) . The conjugate containing fractions are CV washing buffer 1 (50 mM Hepes , 3 M sodium chloride , concentrated by UF /DF using a 10 kD membrane made of 5 mM calcium chloride , pH 6 . 9 ) in upflow mode at a flow regenerated cellulose ( 88 cm2, cut -off 10 kD , Millipore ) . rate of 2 cm /min . Then elution of purified FIX conjugate is The final diafiltration step is performed against Hepes buffer performed in down flow mode using a step gradient of 40 % ( 50 mM Hepes , 5 mM CaCl2 , pH 7 . 5 ). washing buffer 2 (50 mM Hepes , 1 . 5 M sodium chloride , 5 [0285 ] The preparation is analytically characterized by mM calcium chloride , pH 6 . 9 ) and 60 % elution buffer ( 20 measuring total protein (Bradford and BCA procedure ) and mM Hepes, 5 mM calcium chloride, pH 7 . 5 ) at a flow rate biological activity according to known methods. of 1 cm / min . The elution of the PEG - rFIX conjugate is monitored at UV 280 nm and the eluate containing the conjugate is collected within < 4 CV . The post elution step is Example 16 performed with > 3 CV elution buffer under the same con ditions to separate minor and / or non modified rFIX from the PEGylation of rFVIIa Using an Aminooxy - PEG main product. Reagent and m - Toluidine as a Nucleophilic [ 0281] Finally , the purified conjugate is concentrated by Catalyst ultra - / diafiltration (UF /DF ) using a membrane made of regenerated cellulose with a molecular weight cut off 10 kD Method 1 : (88 cm2, Millipore ). [0286 ] rFVIIa is PEGylated by use of a linear 20 kD [0282 ] The conjugate prepared by use of this procedure PEGylation reagent containing an aminooxy group . An are analytically characterized by measuring total protein and example of this type of reagent is the Sunbright® CA series biological activity according to methods known in the art. from NOF (NOF Corp . , Tokyo , Japan ). A starting weight or concentration of rFVIIa is dissolved in or transferred to a Method 2 : reaction buffer ( 50 mM Hepes , 350 mM sodium chloride , 5 [ 0283] rFIX is PEGylated by use of a linear 20 kD mM calcium chloride , pH 6 .0 ) to get a final protein con PEGylation reagent containing an aminooxy group . An centration of 1. 0 + / - 0 .25 mg/ ml . Then the pH of the solution example of this type of reagent is the Sunbright® CA series is corrected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous from NOF (NOF Corp . , Tokyo , Japan ) . An initial concen NaOH solution . Subsequently, a 40 mM aqueous sodium tration or weight of rFIX is transferred or dissolved in Hepes periodate solution is added within 10 minutes to give a buffer (50 mM Hepes, 150 mM sodium chloride , 5 mM concentration of 50 uM . The oxidation reaction is carried out calcium chloride , pH 6 . 0 ) to get a final protein concentration for 30 + / – 5 min at a temperature ( T ) of T = + 22 + / - 2° C . Then of 2 mg rFIX / ml. Subsequently , an 5 mM aqueous sodium the reaction is stopped by addition of an aqueous L - cysteine periodate solution is added within 15 minutes to give a final solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° C . to give concentration of 100 uM , followed by addition of an 50 mM a final concentration of 10 mM in the reaction mixture and aqueous m -toluidine solution to get a final concentration of incubation for 60 + / – 5 min . 10 mM within a time period of 30 minutes . Then the [0287 ] The oxidized rFVIIa is further purified by anion aminooxy -PEG reagent with a MW of 20 kD (described exchange chromatography on EMD TMAE (M ) (Merck ). above ) is added to give a 20 - fold molar reagent excess . After The mixture is diluted with Buffer A ( 20 mM Hepes , 5 mM correction of the pH to 6 . 0 the mixture is incubated for 2 h CaC12 , pH 6 . 5 ) to give a conductivity of 5 mS/ cm . This in the dark at room temperature under gentle stirring and solution is loaded onto the IEX column (bed height: 5 . 4 cm ) quenched for 15 min at room temperature by the addition of with a column volume of 10 ml using a flow rate of 1 . 5 a 1 M aqueous L - cysteine solution to give a final concen cm /min . This column is subsequently washed ( flow rate : 1 . 5 tration of 10 mM . cm /min ) with 5 CV of a 92 : 8 mixture ( w / w ) of Buffer A and 102841. The free rFIX is removed by means of ion Buffer B (20 mM Hepes, 5 mM CaC12 , 1. 0 M NaCl, pH 7 .0 ). exchange chromatography ( IEC ) . The reaction mixture was Then the oxidized rFVIIa is eluted with a 50 :50 ( w / w ) diluted with appropriate amounts of Buffer A ( 50 mM mixture of Buffer A and Buffer B followed by a postelution Hepes, 5 mM CaCl2 , pH 7 . 5 ) to correct the solutions step with 5 CV of Buffer B . The elution steps are carried out conductivity and pH value prior to load onto a 20 ml HiPrep by use of a flow rate of 1 . 0 cm /min . OFF 16 / 10 column (GE Healthcare , Fairfield , Conn . ) pre 0288 ] Subsequently , the aminooxy - PEG reagent with a equilibrated with Buffer A . Then the column was eluted with MW of 20 kD reagent is added in a 50 - fold molar excess to Buffer B (50 mM Hepes , 1 M NaCl, 5 mM CaCl2 , pH 7 . 5 ) . the eluate containing the purified oxidized rFVIIa within a Free FIX was eluted by a step gradient using 25 % of Buffer maximum time period (t ) of 15 minutes under gentle stirring. B , which results in a conductivity between 12 - 25 mS/ cm in Then an aqueous m - toluidine solution (50 mM ) is added the obtained fraction and the conjugate using a step gradient within 15 minutes to get a final concentration of 10 mM . The US 2017 /0240590 A1 Aug . 24 , 2017 46 reaction mixture is incubated for 120 + / – 10 min . in the dark [0295 ] The free rFVIIa is removed by means of ion at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . exchange chromatography ( IEC ) . The reaction mixture was [0289 ] The obtained PEG - rFVIIa conjugate is purified by diluted with appropriate amounts of Buffer A (50 mM Hydrophobic Interaction Chromatography (HIC ) using a Hepes, 5 mM CaC12 , pH 7 .5 ) to correct the solutions Phenyl Sepharose FF low sub resin (GE Healthcare ) packed conductivity and pH value prior to load onto a 20 ml HiPrep into a column manufactured by GE Healthcare with a bed QFF 16 / 10 column (GE Healthcare, Fairfield , Conn .) pre height ( h ) of 15 cm and a resulting column volume (CV ) of equilibrated with Buffer A . Then the column was eluted with 81 ml. Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaC12 , pH 7 . 5 ) . [0290 ] The reaction mixture is spiked with ammonium Free rFVIIa was eluted by a step gradient using 25 % of acetate by addition of 50 mM Hepes buffer , containing 350 Buffer B , which results in a conductivity between 12 -25 mM sodium chloride, 8 M ammonium acetate , 5 mM mS / cm in the obtained fraction and the conjugate using a calcium chloride , pH 6 . 9. Two volumes of the reaction step gradient of 50 % Buffer B , which results in a conduc mixture are mixed with 1 volume of the ammonium acetate tivity between 27 - 45 mS/ cm in the conjugate fraction . The containing buffer system and the pH value is corrected to pH conductivity of the conjugate containing fraction is subse 6 . 9 by drop wise addition of a 0 . 5 N aqueous NaOH solution . quently raised with Buffer C (50 mM Hepes , 5 M NaC1, 5 This mixture is loaded onto the HIC column using a flow mM CaCl2 , pH 6 . 9 ; by use of anti- chaotropic salts e . g . rate of 1 cm /min followed by a washing step using > 3 CV ammonium acetate ) and loaded onto a 20 ml HiPrep Butyl equilibration buffer (50 mM Hepes , 350 mM sodium chlo FF 16 / 10 column (GE Healthcare, Fairfield , Conn .; or com ride, 2 . 5 M ammonium acetate , 5 mM calcium chloride , pH parable HIC media ) pre - equilibrated with Buffer D (50 mM 6 . 9 ) . Hepes , 3 M NaCl, 5 mM CaCl2 , pH 6 . 9 ) . Free PEG - reagent [0291 ] For removal of reaction by -products and anti was washed out within 5 CV Buffer D . Subsequently the chaotropic salt a second washing step is performed with > 5 conjugate was eluted with 100 % Buffer E (50 mM Hepes , 5 CV washing buffer 1 (50 mM Hepes , 3 M sodium chloride , mM CaCl2 , pH 7 . 4 ) . The conjugate containing fractions are 5 mM calcium chloride, pH 6 . 9 ) in upflow mode at a flow concentrated by UF/ DF using a 10 kD membrane made of rate of 2 cm /min . Then elution of purified rFVIIa conjugate regenerated cellulose (88 cm2, cut -off 10 kD , Millipore ) . is performed in down flow mode using a step gradient of The final diafiltration step is performed against Hepes buffer 40 % washing buffer 2 (50 mM Hepes , 1 . 5 M sodium ( 50 mM Hepes , 5 mM CaC12 , pH 7 . 5 ) . chloride , 5 mM calcium chloride , pH 6 . 9 ) and 60 % elution [0296 ] The preparation is analytically characterized by buffer (20 mM Hepes , 5 mM calcium chloride , pH 7 .5 ) at a measuring total protein ( Bradford and BCA procedure ) and flow rate of 1 cm /min . The elution of the PEG -rFVIIa biological activity according to known methods. conjugate is monitored at UV 280 nm and the eluate containing the conjugate is collected within < 4 CV . The post Example 17 elution step is performed with > 3 CV elution buffer under Polysialylation of rFIX in the Presence of o - Amino the same conditions to separate minor and / or non modified rFVIIa from the main product . Benzoic Acid [0292 ] Finally , the purified conjugate is concentrated by Method 1 : ultra - / diafiltration (UF /DF ) using a membrane made of regenerated cellulose with a molecular weight cut off 10 kD [0297 ] 8 . 2 mg rFIX is dissolved in 4 .0 ml histidine buffer , pH 6 . 0 ( 20 mM L -histidine , 150 mM NaC1, 5 mM CaCl , ) . (Millipore ) . Then 82 ul of an aqueous sodium periodate solution ( 5 mM ) [ 0293 ] The conjugate prepared by use of this procedure is added and the reaction mixture is incubated for 1 h in the are analytically characterized by measuring total protein and dark at 4° C . under gentle stirring and quenched for 15 min biological activity according to methods known in the art . at room temperature by the addition of 4 ul of a 1 M aqueous cysteine solution . The mixture is subsequently subjected to Method 2 : UF /DF employing Vivaspin 6 10 kD centrifugal filtrators to [0294 ) rFVIIa is PEGylated by use of a linear 20 kD remove excess periodate , quencher and the byproducts PEGylation reagent containing an aminooxy group . An thereof . example of this type of reagent is the Sunbright® CA series [0298 ] The retentate (6 . 5 ml) , containing oxidized rFIX , is from NOF (NOF Corp ., Tokyo , Japan ). An initial concen mixed with 1 .64 ml of an aqueous o - amino benzoic acid (50 tration or weight of rFVIIa is transferred or dissolved in mM ) and incubated for 30 min at room temperature . Then Hepes buffer ( 50 mM Hepes , 150 mM sodium chloride , 5 aminooxy - PSA reagent with a MW of 20 kD (described mM calcium chloride , pH 6 . 0 ) to get a final protein con above ) is added to give a 5 - fold molar reagent excess. This centration of 2 mg rFVIIa /ml . Subsequently an 5 mM mixture was incubated for 2 .5 h at room temperature in the aqueous sodium periodate solution is added within 15 min dark under gentle stirring . utes to give a final concentration of 100 uM , followed by [0299 ] The further purification of the conjugate is carried addition of an 50 mM aqueous m -toluidine solution to get a out as described herein . final concentration of 10 mM within a time period of 30 minutes . Then the aminooxy - PEG reagent with a MW of 20 Method 2 : kD (described above ) is added to give a 20 - fold molar [0300 ] A solution of 1 mg rFIX in 0 .65 ml sodium reagent excess. After correction of the pH to 6 .0 the mixture phosphate buffer, pH 6 .0 containing a 5 - fold molar excess of is incubated for 2 h in the dark at room temperature under aminooxy - PSA reagent with a MW of 20 kD (described gentle stirring and quenched for 15 min at room temperature above ) was prepared . Then 333 ul of an aqueous o -amino by the addition of a 1 M aqueous L - cysteine solution to give benzoic acid solution ( 30 mM ) was added as nucleophilic a final concentration of 10 mM . catalyst to give a final concentration of 10 mM . Subse US 2017 /0240590 A1 Aug . 24 , 2017 47 quently 20 ul of an aqueous solution of NaI04 (5 mM ) was UF/ DF employing Vivaspin 15R 10 kD centrifugal filtrators added yielding in a final concentration of 100 uM . The to remove excess periodate , quencher and the byproducts coupling process was performed for 2 hours in the dark thereof. under gentle shaking at room temperature and quenched for [0306 ] The retentate ( approx . 7 ml) , containing oxidized 15 min at room temperature by the addition of 1 ul of EPO , is mixed with 2 ml of an aqueous m - toluidine solution aqueous cysteine solution ( 1 M ). The further purification of (50 mM ) and incubated for 30 min at room temperature . the conjugate is carried out as described herein . Then aminooxy - PSA reagent with a MW of 20 kD (de scribed above ) is added to give a 5 - fold molar reagent Example 18 excess . This mixture is incubated for 2 . 5 h at RT in the dark under gentle stirring . Polysialylation of EPO Using Aminooxy -PSA and [0307 ) The free EPO is removed by means of anion m - Toluidine as a Nucleophilic Catalyst exchange chromatography (AEC ) . The reaction mixture is diluted with 20 ml Buffer A ( 50 mM Hepes , pH 7 . 5 ) and Method 1: loaded onto a 20 ml HiPrep QFF 16 / 10 column (GE Health [ 0301 ] A starting concentration of erythropoietin (EPO ) is care , Fairfield , Conn . ) pre - equilibrated with Buffer A . Then transferred into a reaction buffer ( e . g . 50 mM Hepes , 350 the column is eluted with Buffer B (50 mM Hepes, 1 M mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and NaC1, pH 7 . 5 ) . Free EPO is eluted by washing the column diluted to obtain a protein concentration of 1 mg /ml . To this with 25 % Buffer B and the conjugate at 50 % Buffer B . The solution , NalO2 is added to give a final concentration of 200 conductivity of the conjugate containing fractions is subse uM . The oxidation is carried at RT for 30 min in the dark quently raised to - 190 mS/ cm with Buffer C ( 50 mM Hepes , under gentle shaking . The reaction is then quenched with 5 M NaCl, pH 6 . 9 ) and loaded onto a 20 ml HiPrep Butyl FF cysteine ( final concentration : 10 mM ) for 60 min at RT. 16 / 10 column (GE Healthcare , Fairfield , Conn . ) pre - equili [0302 ] The solution is next subjected to UF /DF employing brated with Buffer D (50 mM Hepes , 3 M NaCl, pH 6 . 9 ) . Vivaspin centrifugal filtrators to remove excess periodate , Free PSA - reagent is washed out within 5 CV Buffer D . quencher and the byproducts thereof or, in the alternative , to Subsequently , the conjugate is eluted with 100 % Buffer E an IEX column with a volume of 20 ml (Merck EMD TMAE ( 50 mM Hepes, pH 7 . 4 ) . The conjugate containing fractions ( M ) ) which is equilibrated with Buffer A (20 mM Hepes, 5 are concentrated by UF /DF using a 10 kD membrane made mM CaCl, , pH 7 . 0 ) . The column is equilibrated with 5 CV of regenerated cellulose (88 cm2, cutoff 10 kD /Millipore ) . Buffer A . The oxidized EPO is eluted with Buffer B (20 mM The final diafiltration step is performed against histidine Hepes, 5 mM CaCl2 , 1M NaCl, pH 7 . 0 ) . The EPO contain buffer , pH 7 . 2 containing 150 mM NaCl. The preparation is ing fractions are collected . The protein content is determined analytically characterized by measuring total protein (Brad (Coomassie , Bradford ) and adjusted to 1 mg/ml with reac ford ) and biological activity according to methods known in tion buffer and adjusted to pH 6 . 0 by dropwise addition of the art. For the PSA - EPO conjugate a specific activity of 0 .5M HCl. > 50 % in comparison to native EPO is determined . The [0303 ] A 50 - fold molar excess of a aminooxy - PSA reagent conjugate is additionally analytically characterized by Size with a MW of 20 kD ( described above ) is added followed by Exclusion HPLC using a Agilent 1200 HPLC system m -toluidine as a nucleophilic catalyst ( final concentration : equipped with a Shodex KW 803 column under conditions 10 mM ) . The coupling reaction is performed for 2 hours in as previously described (Kolarich et al, Transfusion 2006 ; the dark under gentle shaking at room temperature . The 46 : 1959 - 77 ) . It is shown that the preparation contains no excess of aminooxy - PSA reagent is removed by means of free EPO . HIC . The conductivity of the reaction mixture is adjusted by adding a buffer containing ammonium acetate ( 50 mM Method 2 : Hepes, 350 mM sodium chloride , 5 mM calcium chloride , 8 [0308 ] EPO is transferred or dissolved in reaction buffer M ammonium acetate , pH 6 . 9 ) and loaded onto a column ( e . g . 50 mM Hepes, 350 mM sodium chloride , 5 mM filled with 80 ml Phenyl Sepharose FF (GE Healthcare , calcium chloride, pH 6 . 0 ) to get a final protein concentration Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes, 2 . 5 M of 1 . 0 + / - 0 . 25 mg /ml . Then the pH of the solution is cor ammonium acetate , 350 mM sodium chloride , 5 mM cal rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HCI cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted solution . Subsequently , a 40 mM aqueous sodium periodate with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaCl2 . solution is added within 10 minutes to give a concentration Finally the PSA - EPO containing fractions are collected and of 200 uM . The oxidation reaction is carried out for 30 + / - 5 subjected to UF /DF by use of a membrane made of regen min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction erated cellulose (MWCO 10 kD , 50 cm ?, Millipore ). The is stopped by addition of an aqueous L - cysteine solution ( 1 preparation is next analytically characterized by measuring M ) within 15 minutes at T = + 22 + / - 2° C . to give a final total protein ( Coomassie , Bradford ) and biological activity concentration of 10 mM in the reaction mixture and incu according to methods known in the art . bation for 60 + / – 5 min . [0304 ] In an alternative embodiment, Method 1 is carried [0309 ] The oxidized EPO is further purified by ion out as follows. exchange chromatography. The oxidized EPO containing [ 0305 ] 10 mg EPO is dissolved in 5 mlhistidine buffer , pH fractions of the eluate are collected and used for the conju 6 . 0 ( 20 mM L - histidine , 150 mM NaCl) . 100 ul of an gation reaction . aqueous sodium periodate solution ( 5 mm ) is then added [ 0310 ] The aminooxy -polysialic acid (PSA -ONH2 ) and the reaction mixture is incubated for 1 h in the dark at reagent is added in a 50 - fold molar excess to the eluate 4° C . under gentle stirring and quenched for 15 min at room containing the purified oxidized EPO within a maximum temperature by the addition of 50 ul of a 1 M aqueous time period ( t ) of 15 minutes under gentle stirring . Then an cysteine solution . The mixture is subsequently subjected to aqueous m - toluidine solution (50 mM ) is added within 15 US 2017 /0240590 A1 Aug . 24 , 2017 minutes to get a final concentration of 10 mM . The reaction 16 / 10 column (GE Healthcare , Fairfield , Conn . ) pre - equili mixture is incubated for 120 + / - 10 min . at pH 6 . 0 in the dark brated with Buffer D ( 50 mM Hepes , 3 M NaCl, pH 6 . 9 ) . at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking Free PSA - reagent is washed out within 5 CV Buffer D . (protein concentration : 1 mg/ ml ). Subsequently , the conjugate is eluted with 100 % Buffer E [0311 ] The obtained PSA - EPO conjugate is further puri (50 mM Hepes, pH 7 . 4 ). The conjugate containing fractions fied by ion exchange chromatography . The PSA - EPO con are concentrated by UF /DF using a 10 kD membrane made jugate containing fractions are collected and concentrated by of regenerated cellulose (88 cm2, cut -off 10 kD , Millipore ) . ultra - /diafiltration (UF /DF ) using a membrane made of The final diafiltration step is performed against histidine regenerated cellulose with an appropriate molecular weight buffer , pH 7 . 2 containing 150 mM NaCl. The preparation is cut off (Millipore ). analytically characterized by measuring total protein (Brad [ 0312] The conjugate prepared by use of this procedure is ford ) and biological activity according to methods known in analytically characterized by measuring total protein , bio the art. For the PSA - EPO conjugate a specific activity of logical activity , and determination of the polysialyation > 50 % in comparison to native EPO is determined . The degree by measuring the PSA content ( resorcinol assay ). conjugate is additionally analytically characterized by Size Exclusion HPLC using a Agilent 1200 HPLC system Method 3 : equipped with a Shodex KW 803 column under conditions [0313 ] Erythropoietin (EPO ) is transferred into reaction as previously described (Kolarich et al, Transfusion 2006 ; buffer (50 mM Hepes , 350 mM sodium chloride, 5 mM 46 : 1959 - 77 ). It is shown that the preparation contains no calcium chloride , pH 6 . 0 ) and diluted to obtain a protein free EPO . concentration of 1 mg/ ml . A 50 fold molar excess of a aminooxy - PSA reagent with a MW of 20 kD ( described Method 4 : above ) is added followed by m - toluidine as a nucleophilic [0316 ] EPO is dissolved in or transferred to a reaction catalyst ( 10 mM final concentration ) and NalO4 ( final buffer ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 mM concentration : 400 UM ) . The coupling reaction is performed calcium chloride , pH 6 . 0 ) to get a final protein concentration for 2 hours in the dark under gentle shaking at room of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is cor temperature . Subsequently , the reaction is quenched with rected to 6 .0 by drop wise addition of a 0 .5 N aqueous HC1 cysteine for 60 min at RT ( cysteine concentration : 10 mM ) . solution . Then the conductivity of the reaction mixture is adjusted by [0317 ] Subsequently , the aminooxy -polysialic acid (PSA adding a buffer containing ammonium acetate ( 50 mM ONH2) reagent is added in a 50 - fold molar excess to this Hepes, 350 mM sodium chloride , 5 mM calcium chloride , 8 EPO solution within a maximum time period ( t ) of 15 M ammonium acetate , pH 6 . 9 ) and loaded onto a column minutes under gentle stirring. Then an aqueous m - toluidine filled with Phenyl Sepharose FF (GE Healthcare , Fairfield , solution ( 50 mM ) is added within 15 minutes to get a final Conn . ) pre - equilibrated with 50 mM Hepes, 2 . 5 M ammo concentration of 10 mM . Finally a 40 mM aqueous sodium nium acetate , 350 mM sodium chloride , 5 mM calcium periodate solution is added to give a concentration of 400 chloride, 0 .01 % Tween 80 , pH 6 .9 . Subsequently , the con UM . jugate is eluted with 50 mM Hepes , 5 mM calcium chloride , [0318 ] The reaction mixture is incubated for 120 + / - 10 pH 7 .5 . Finally , the PSA -EPO containing fractions are min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under collected and subjected to UF /DF by use of a membrane gentle shaking . Then the reaction is stopped by the addition made of regenerated cellulose (MWCO 10 kD , 88 cm2, of an aqueous L - cysteine solution ( 1 M ) to give a final Millipore ) . The preparation is analytically characterized by concentration of 10 mM in the reaction mixture and incu measuring total protein (Bradford ) and biological activity bation for 60 + / - 5 min . according to methods known in the art. [0319 ] The obtained PSA - EPO conjugate is purified by [0314 ] In an alternative embodiment, Method 3 is carried ion - exchange chromatography . The PSA - EPO containing out as follows . 10 mg EPO is dissolved in 8 ml histidine fractions of the eluate are collected and concentrated by buffer, pH 6 . 0 ( 20 mM L - histidine , 150 mM NaCl) . 200 ul ultra - /diafiltration (UF /DF ) using a membrane made of of an aqueous sodium periodate solution (5 mM ) and 2 ml regenerated cellulose (MWCO 10 kD , 88 cm2, Millipore ). of an aqueous m - toluidine solution (50 mM ) are then added . (0320 ] The conjugates prepared by use of this procedure Subsequently , the aminooxy - PSA reagent with a MW of 20 are analytically characterized by measuring total protein , kD ( described above ) is added to give a 5 - fold molar reagent biological activity according to methods known in the art, excess . The mixture is incubated for 2 h in the dark at room and determination of the polysialyation degree by measuring temperature under gentle stirring and quenched for 15 min the PSA content (resorcinol assay ) . at room temperature by the addition of 100 ul of 1 M aqueous cysteine solution . Example 19 [ 0315 ] The free EPO is removed by means of anion exchange chromatography (AEC ) . The reaction mixture is Polysialylation of Ang - 2 Using Aminooxy -PSA and diluted with 20 ml Buffer A ( 50 mM Hepes , pH 7 . 5 ) and m - Toluidine as a Nucleophilic Catalyst loaded onto a 20 mlHiPrep QFF 16 / 10 column (GE Health care , Fairfield , Conn . ) pre -equilibrated with Buffer A . Then Method 1 : the column is eluted with Buffer B (50 mM Hepes , 1 M [0321 ] A starting concentration of angiopoietin - 2 ( Ang - 2 ) NaCl, pH 7 .5 ) . Free EPO is eluted by washing the column is transferred into a reaction buffer ( e . g . 50 mM Hepes, 350 with 25 % Buffer B and the conjugate at 50 % Buffer B . The mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and conductivity of the conjugate containing fractions is subse diluted to obtain a protein concentration of 1 mg/ml . To this quently raised to ~ 190 mS/ cm with Buffer C (50 mM Hepes , solution , NalO4 is added to give a final concentration of 200 5 M NaCl, pH 6 . 9 ) and loaded onto a 20 ml HiPrep Butyl FF UM . The oxidation is carried at RT for 30 min in the dark US 2017 /0240590 A1 Aug . 24 , 2017 49 under gentle shaking . The reaction is then quenched with of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor cysteine ( final concentration : 10 mM ) for 60 min at RT. rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 [ 0322 ] The solution is next subjected to UF /DF employing solution . Subsequently , a 40 mM aqueous sodium periodate Vivaspin centrifugal filtrators to remove excess periodate , solution is added within 10 minutes to give a concentration quencher and the byproducts , or, in the alternative , subjected of 200 uM . The oxidation reaction is carried out for 30 + / - 5 to an IEX column with a volume of 20 ml (Merck EMD min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction TMAE ( M ) ) which is equilibrated with Buffer A (20 mM is stopped by addition of an aqueous L -cysteine solution ( 1 Hepes , 5 mM CaC12 , pH 7 . 0 ) . The column is equilibrated M ) within 15 minutes at T = + 22 + / - 2° C . to give a final with 5 CV Buffer A . The oxidized Ang -2 is eluted with concentration of 10 mM in the reaction mixture and incu Buffer B (20 mM Hepes, 5 mM CaCl2 , 1 M NaCl, pH 7 . 0 ) . bation for 60 + / - 5 min . The Ang - 2 containing fractions are collected . The protein [0328 ] The oxidized Ang - 2 is further purified by ion content is determined ( Coomassie , Bradford ) and adjusted to exchange chromatography. The oxidized Ang - 2 containing 1 mg/ ml with reaction buffer and adjusted to pH 6 .0 by fractions of the eluate are collected and used for the conju dropwise addition of 0 . 5 M HC1. gation reaction . [ 0323 ] A 50 - fold molar excess of aminooxy -PSA reagent [0329 ] The aminooxy -polysialic acid ( PSA - ONH2 ) with a MW of 20 kD (described above ) is added followed by reagent is added in a 50 - fold molar excess to the eluate m - toluidine as a nucleophilic catalyst ( final concentration : containing the purified oxidized Ang - 2 within a maximum 10 mM ) . The coupling reaction is performed for 2 hours in time period ( t ) of 15 minutes under gentle stirring . Then an the dark under gentle shaking at room temperature . The aqueous m - toluidine solution (50 mM ) is added within 15 excess of aminooxy reagent is removed by means of HIC . minutes to get a final concentration of 10 mM . The reaction The conductivity of the reaction mixture is adjusted by mixture is incubated for 120 + / - 10 min . at pH 6 . 0 in the dark adding a buffer containing ammonium acetate ( 50 mM at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking Hepes, 350 mM sodium chloride , 5 mM calcium chloride , 8 (protein concentration : 1 mg/ ml ) . M ammonium acetate , pH 6 . 9 ) and loaded onto a column (0330 ] The obtained PSA - Ang - 2 conjugate is further puri filled with 80 ml Phenyl Sepharose FF (GE Healthcare , fied by ion - exchange chromatographyn Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes, 2 . 5 M 0331 ] The PSA - Ang - 2 conjugate containing fractions are ammonium acetate , 350 mM sodium chloride, 5 mM cal collected and concentrated by ultra - / diafiltration (UF /DF ) cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted using a membrane made of regenerated cellulose with an with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . appropriate molecular weight cut off (Millipore ). Finally , the PSA - Ang - 2 - containing fractions are collected [0332 ] The conjugate prepared by use of this procedure is and subjected to UF /DF by use of a membrane made of analytically characterized by measuring total protein , bio regenerated cellulose (Millipore ) . The preparation is next logical activity , and determination of the polysialyation analytically characterized by measuring total protein (Coo degree by measuring the PSA content ( resorcinol assay ) . massie , Bradford ) and biological activity according to meth ods known in the art. Method 3 : [ 0324 ] In an alternative embodiment, Method 1 is carried out as follows. Angiopoietin -2 (Ang - 2 ) is transferred into a [0333 ] Angiopoietin - 2 (Ang - 2 ) is transferred into reaction reaction buffer ( e . g ., 50 mM Hepes , 350 mM sodium chlo buffer (50 mM Hepes , 350 mM sodium chloride, 5 mM ride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to obtain calcium chloride, pH 6 . 0 ) and diluted to obtain a protein concentration of 1 mg/ ml . A 50 fold molar excess of a PSA a protein concentration of 1 mg/ ml . To this solution , Nal04 aminooxy reagent with a MW of 20 kD ( described above ) is is added to give a final concentration of 200 uM . The added followed by m -toluidine as a nucleophilic catalyst ( 10 oxidation is carried at RT for 30 min in the dark under gentle mM final concentration ) and NalO4 ( final concentration : shaking . The reaction is then quenched with cysteine ( final 400 UM ). The coupling reaction is performed for 2 hours in concentration : 10 mM ) for 60 min at R . T . the dark under gentle shaking at room temperature . Subse [0325 ] The solution is next subjected to UF /DF employing quently , the reaction is quenched with cysteine for 60 min at Vivaspin centrifugal filtrators to remove excess periodate , RT ( cysteine concentration : 10 mM ) . Then the conductivity quencher and the byproducts thereof. of the reaction mixture is adjusted by adding a buffer [0326 ] A 50 - fold molar excess of aminooxy - PSA reagent containing ammonium acetate (50 mM Hepes, 350 mM with a MW of 20 kD (described above ) is added followed by sodium chloride , 5 mM calcium chloride , 8 M ammonium m - toluidine as a nucleophilic catalyst ( final concentration : acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl 10 mM ) . The coupling reaction is performed for 2 hours in Sepharose FF (GE Healthcare , Fairfield , Conn .) pre - equili the dark under gentle shaking at room temperature. The brated with 50 mM Hepes, 2 . 5 M ammonium acetate , 350 excess of aminooxy reagent is removed by means of ion mM sodium chloride , 5 mM calcium chloride, 0 .01 % Tween exchange chromatography . The PSA - Ang - 2 conjugate - con 80 , pH 6 . 9 . Subsequently, the conjugate is eluted with 50 taining fractions of the eluate are collected and subjected to mM Hepes , 5 mM calcium chloride , pH 7 .5 . Finally , the UF /DF by use of a membrane made of regenerated cellulose PSA Ang -2 - containing fractions are collected and subjected (Millipore ). The preparation is next analytically character to UF /DF by use of a membrane made of regenerated ized by measuring total protein (Coomassie , Bradford ) and cellulose (Millipore ). The preparation is analytically char biological activity according to methods known in the art . acterized by measuring total protein (Bradford ) and biologi cal activity according to methods known in the art. Method 2 : [0334 ] In an alternative embodiment, Method 3 is carried [0327 ] Ang- 2 is transferred or dissolved in reaction buffer out as follows. Angiopoietin - 2 ( Ang - 2 ) is transferred into ( e . g . 50 mM Hepes , 350 mM sodium chloride, 5 mM reaction buffer ( e . g . 50 mM Hepes, 350 mM sodium chlo calcium chloride, pH 6 . 0 ) to get a final protein concentration ride, 5 mM calcium chloride, pH 6 . 0 ) and diluted to obtain US 2017 /0240590 A1 Aug . 24 , 2017 50 a protein concentration of 1 mg/ml . A 50 - fold molar excess [0341 ] The solution is next subjected to UF /DF employing of a PSA aminooxy reagent with a MW of 20 kD ( described Vivaspin centrifugal filtrators to remove excess periodate , above ) is added followed by m - toluidine as a nucleophilic quencher and the byproducts thereof or, in the alternative , to catalyst ( 10 mM final concentration ) and NalO4 ( final an IEX column with a volume of 20 ml (Merck EMD TMAE concentration : 400 ?M ). The coupling reaction is performed ( M )) which is equilibrated with Buffer A ( 20 mM Hepes, 5 for 2 hours in the dark under gentle shaking at room mM CaCl2 , pH 7 .0 ). The column is equilibrated with 5 CV temperature . Subsequently, the reaction is quenched with Buffer A . The oxidized VEGF is eluted with Buffer B ( 20 cysteine for 60 min at RT ( cysteine concentration : 10 mM ) mM Hepes , 5 mM CaCl2 , 1 M NaCl, pH 7 . 0 ) . The VEGF and the conjugate is purified by ion exchange chromatog containing fractions are collected . The protein content is raphy. PSA Ang - 2 - containing fractions of the eluate are determined (Coomassie , Bradford ) and adjusted to 1 mg/ ml collected and subjected to UF/ DF by use of a membrane with reaction buffer and adjusted to pH 6 . 0 by dropwise made of regenerated cellulose (Millipore ). The preparation addition of 0 .5M NaOH . is analytically characterized by measuring total protein [ 0342 ] A 50 - fold molar excess of aminooxy -PSA reagent (Bradford ) and biological activity according to methods with a MW of 20 kD ( described above ) is added followed by known in the art . m -toluidine as a nucleophilic catalyst ( final concentration : 10 mM ) . The coupling reaction is performed for 2 hours in Method 4 : the dark under gentle shaking at room temperature . The excess of aminooxy reagent is removed by means of HIC . [ 0335 ] Ang - 2 is dissolved in or transferred to a reaction 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 . Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes , 2 . 5 M [0336 ] Subsequently , the aminooxy -polysialic acid (PSA ammonium acetate , 350 mM sodium chloride , 5 mM cal ONH2) reagent is added in a 50 - fold molar excess to this cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted Ang - 2 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 - VEGF -containing fractions are collected solution (50 mM ) is added within 15 minutes to get a final 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 [0337 ] The reaction mixture is incubated for 120 + / – 10 ods known in the art . min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under 0343 ] In an alternative embodiment, Method 1 is carried gentle shaking . Then the reaction is stopped by the addition out as follows. Vascular endothelial growth factor (VEGF ) is of an aqueous L - cysteine solution ( 1 M ) to give a final transferred into a reaction buffer ( e . g ., 50 mM Hepes , 350 concentration of 10 mM in the reaction mixture and incu mM sodium chloride, 5 mM calcium chloride , pH 6 . 0 ) and bation for 60 + / – 5 min . diluted to obtain a protein concentration of 1 mg/ ml . To this [0338 ] The obtained PSA -Ang - 2 conjugate is purified by solution , NalO4 is added to give a final concentration of 200 ion -exchange chromatography . The PSA - Ang - 2 containing UM . The oxidation is carried at RT for 30 min in the dark fractions of the eluate are collected and concentrated by under gentle shaking . The reaction is then quenched with ultra - / diafiltration (UF / DF ) using a membrane made of cysteine ( final concentration : 10 mM ) for 60 min at RT. regenerated cellulose (Millipore ). 103441. The solution is next subjected to UF /DF employing [ 0339 ] The conjugates prepared by use of this procedure Vivaspin centrifugal filtrators to remove excess periodate , are analytically characterized by measuring total protein , quencher and the byproducts thereof. biological activity according to methods known in the art, [0345 ] A 50 - fold molar excess of aminooxy - PSA reagent and determination of the polysialyation degree by measuring with a MW of 20 kD ( described above ) is added followed by the PSA content (resorcinol assay ) . m - toluidine as a nucleophilic catalyst ( final concentration : 10 mM ) . The coupling reaction is performed for 2 hours in Example 20 the dark under gentle shaking at room temperature . The excess of aminooxy reagent is removed by means of ion Polysialylation of VEGF Using Aminooxy -PSA and exchange chromatography. The PSA - VEGF -containing m - Toluidine as a Nucleophilic Catalyst fractions of the eluate are collected and subjected to UF /DF by use of a membrane made of regenerated cellulose (Mil Method 1: lipore ) . The preparation is next analytically characterized by [ 0340 ] A starting concentration of vascular endothelial measuring total protein (Coomassie , Bradford ) and biologi growth factor (VEGF ) is transferred into a reaction buffer cal activity according to methods known in the art . ( e. g ., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride , pH 6 . 0 ) and diluted to obtain a protein Method 2 : concentration of 1 mg/ ml . To this solution , NalO4 is added [ 0346 ] VEGF is transferred or dissolved in reaction buffer to give a final concentration of 200 uM . The oxidation is ( e . g . 50 mM Hepes , 350 mM sodium chloride, 5 mM carried at RT for 30 min in the dark under gentle shaking . calcium chloride , pH 6 . 0 ) to get a final protein concentration The reaction is then quenched with cysteine ( final concen of 1 . 0 + / - 0 . 25 mg /ml . Then the pH of the solution is cor tration : 10 mM ) for 60 min at RT. rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 US 2017 /0240590 A1 Aug . 24 , 2017 solution . Subsequently a 40 mM aqueous sodium periodate 50 - fold molar excess of aminooxy - PSA reagent with a MW solution is added within 10 minutes to give a concentration of 20 kD (described above) is added followed by m - tolui of 200 uM . The oxidation reaction is carried out for 30 + / - 5 dine as a nucleophilic catalyst ( 10 mM final concentration ) min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction and NalO4 ( final concentration : 400 uM ) . The coupling is stopped by addition of an aqueous L - cysteine solution ( 1 reaction is performed for 2 hours in the dark under gentle M ) within 15 minutes at T = + 22 + / - 2° C . to give a final shaking at room temperature . Subsequently , the reaction is concentration of 10 mM in the reaction mixture and incu quenched with cysteine for 60 min at RT ( cysteine concen bation for 60 + / - 5 min . tration : 10 mM ) and the conjugate is purified by ion [0347 ] The oxidized VEGF is further purified by ion exchange chromatography. The PSA - VEGF containing frac exchange chromatography. The oxidized VEGF containing tions of the eluate are collected and subjected to UF /DF by fractions of the eluate are collected and used for the conju use of a membrane made of regenerated cellulose (Milli gation reaction . pore ). The preparation is analytically characterized by mea [ 0348 ] The aminooxy - polysialic acid (PSA -ONH2 ) suring total protein (Bradford ) and biological activity reagent is added in a 50 - fold molar excess to the eluate according to methods known in the art . containing the purified oxidized VEGF within a maximum time period (t ) of 15 minutes under gentle stirring . Then an Method 4 : aqueous m - toluidine solution (50 mM ) is added within 15 [ 0353 ] VEGF is dissolved in or transferred to a reaction minutes to get a final concentration of 10 mM . The reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chloride, 5 mM mixture is incubated for 120 + / – 10 min . at pH 6 . 0 in the dark calcium chloride, pH 6 . 0 ) to get a final protein concentration at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking of 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is cor ( protein concentration : 1 mg/ ml ) . rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 10349 ] The obtained PSA - VEGF conjugate is further puri solution . fied by ion exchange chromatography . The PSA -VEGF [0354 ] Subsequently , the aminooxy -polysialic acid (PSA conjugate containing fractions are collected and concen ONH2 ) reagent is added in a 50 - fold molar excess to this trated by ultra - /diafiltration (UF /DF ) using a membrane VEGF solution within a maximum time period ( t) of 15 made of regenerated cellulose with an appropriate molecular minutes under gentle stirring . Then an aqueous m - toluidine weight cut off (Millipore ) . solution ( 50 mM ) is added within 15 minutes to get a final [0350 ] The conjugate prepared by use of this procedure is concentration of 10 mM . Finally a 40 mM aqueous sodium analytically characterized by measuring total protein , bio periodate solution is added to give a concentration of 400 logical activity , and determination of the polysialyation UM . degree by measuring the PSA content ( resorcinol assay ). [0355 ) The reaction mixture is incubated for 120 + / - 10 min . in the dark at a temperature ( T ) of T = + 22 +/ - 2° C . under Method 3 : gentle shaking . Then the reaction is stopped by the addition [ 0351] Vascular endothelial growth factor (VEGF ) is of an aqueous L - cysteine solution ( 1 M ) to give a final transferred into reaction buffer (50 mM Hepes, 350 mm concentration of 10 mM in the reaction mixture and incu sodium chloride, 5 mM calcium chloride, pH 6 . 0 ) and bation for 60 + / - 5 min . diluted to obtain a protein concentration of 1 mg/ ml . A [ 0356 ] The obtained VEGF - conjugate is purified by ion 50 - fold molar excess of a PSA aminooxy reagent with a MW exchange chromatography . The PSA - VEGF containing frac of 20 kD ( described above ) is added followed by m - tolui tions of the eluate are collected and concentrated by ultra -/ dine as a nucleophilic catalyst ( 10 mM final concentration ) diafiltration (UF / DF ) using a membrane made of and Nal04 ( final concentration : 400 UM ). The coupling regenerated cellulose (Millipore ). reaction is performed for 2 hours in the dark under gentle [0357 ] The conjugates prepared by use of this procedure shaking at room temperature . Subsequently , the reaction is are analytically characterized by measuring total protein , quenched with cysteine for 60 min at RT ( cysteine concen biological activity according to methods known in the art, tration : 10 mM ) . Then the conductivity of the reaction and determination of the polysialyation degree by measuring mixture is adjusted by adding a buffer containing ammo the PSA content ( resorcinol assay ) . nium acetate ( 50 mM Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 M ammonium acetate , pH 6 . 9 ) and Example 21 loaded onto a column filled with Phenyl Sepharose FF (GE Healthcare , Fairfield , Conn . ) pre - equilibrated with 50 mM Polysialylation of EGF Using Aminooxy - PSA and Hepes, 2 . 5 M ammonium acetate , 350 mM sodium chloride , m - Toluidine as a Nucleophilic Catalyst 5 mM calcium chloride, 0 .01 % Tween 80 , pH 6 . 9 . Subse quently the conjugate is eluted with 50 mM Hepes , 5 mM Method 1: calcium chloride , pH 7 .5 . Finally , the PSA - VEGF contain [ 0358 ] A starting concentration of epidermal growth factor ing fractions are collected and subjected to UF /DF by use of (EGF ) is transferred into a reaction buffer ( e . g ., 50 mM a membrane made of regenerated cellulose (Millipore ). The Hepes, 350 mM sodium chloride , 5 mM calcium chloride , preparation is analytically characterized by measuring total pH 6 . 0 ) and diluted to obtain a protein concentration of 1 protein (Bradford ) and biological activity according to meth mg/ ml . To this solution , NalO4 is added to give a final ods known in the art . concentration of 200 uM . The oxidation is carried at RT for [0352 ] In an alternative embodiment, Method 3 is carried 30 min in the dark under gentle shaking . The reaction is then out as follows. Vascular endothelial growth factor (VEGF ) is quenched with cysteine ( final concentration : 10 mM ) for 60 transferred into reaction buffer ( e . g . 50 mM Hepes, 350 mM min at R . T . sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and [0359 ] The solution is next subjected to UF /DF employing diluted to obtain a protein concentration of 1 mg/ ml . A Vivaspin centrifugal filtrators to remove excess periodate , US 2017 /0240590 A1 Aug . 24 , 2017 quencher and the byproducts thereof or, in the alternative, to of 200 °M . The oxidation reaction is carried out for 30 + / - 5 an IEX column with a volume of 20 ml (Merck EMD TMAE min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction ( M )) which is equilibrated with Buffer A ( 20 mM Hepes, 5 is stopped by addition of an aqueous L - cysteine solution ( 1 mM CaC12 , pH 7 . 0 ) . The column is equilibrated with 5 CV M ) within 15 minutes at T = + 22 + / - 2° C . to give a final Buffer A . The oxidized EGF is eluted with Buffer B ( 20 mM concentration of 10 mM in the reaction mixture and incu Hepes, 5 mM CaC12 , 1M NaCl, pH 7 . 0 ) . The EGF contain bation for 60 + / - 5 min . ing fractions are collected . The protein content is determined [0365 ] The oxidized EGF is further purified by ion (Coomassie , Bradford ) and adjusted to 1 mg/ml with reac exchange chromatography . The oxidized EGF containing tion buffer and adjusted to pH 6 . 0 by dropwise addition of fractions of the eluate are collected and used for the conju 0 .5M HCl. gation reaction . [0360 ] A 50 - fold molar excess of aminooxy - PSA reagent [0366 ] The aminooxy -polysialic acid (PSA -ONH2 ) with a MW of 20 kD (described above) is added followed by reagent is added in a 50 - fold molar excess to the eluate m - toluidine as a nucleophilic catalyst ( final concentration : containing the purified oxidized EGF within a maximum 10 mM ) . The coupling reaction is performed for 2 hours in time period (t ) of 15 minutes under gentle stirring . Then an the dark under gentle shaking at room temperature . The aqueous m - toluidine solution (50 mM ) is added within 15 excess of aminooxy reagent is removed by means of HIC . minutes to get a final concentration of 10 mM . The reaction The conductivity of the reaction mixture is adjusted by mixture is incubated for 120 + / – 10 min . at pH 6 . 0 in the dark adding a buffer containing ammonium acetate ( 50 mM at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking Hepes, 350 mM sodium chloride, 5 mM calcium chloride, 8 (protein concentration : 1 mg/ ml ) . M ammonium acetate , pH 6 . 9 ) and loaded onto a column [0367 ] The obtained PSA - EGF conjugate is further puri filled with 80 ml Phenyl Sepharose FF (GE Healthcare , fied by ion exchange chromatography . The PSA - EGF con Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes, 2 . 5 M jugate containing fractions are collected and concentrated by ammonium acetate , 350 mM sodium chloride , 5 mM vdcal ultra - /diafiltration (UF /DF ) using a membrane made of cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted regenerated cellulose with an appropriate molecular weight with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . cut off (Millipore ). Finally , the PSA - EGF containing fractions are collected and [0368 ] The conjugate prepared by use of this procedure is subjected to UF /DF by use of a membrane made of regen analytically characterized by measuring total protein , bio erated cellulose (Millipore ) . The preparation is next analyti logical activity, and determination of the polysialyation cally characterized by measuring total protein (Coomassie , degree by measuring the PSA content ( resorcinol assay ). Bradford ) and biological activity according to methods known in the art . Method 3 : [0361 ] In an alternative embodiment, Method 1 is carried [0369 ] Epidermal growth factor (EGF ) is transferred into out as follows . Epidermal growth factor (EGF ) is transferred reaction buffer (50 mM Hepes , 350 mM sodium chloride , 5 into a reaction buffer ( e. g . , 50 mM Hepes , 350 mM sodium mM calcium chloride , pH 6 . 0 ) and diluted to obtain a protein chloride, 5 mM calcium chloride , pH 6 . 0 ) and diluted to concentration of 1 mg/ ml . A 50 - fold molar excess of a PSA obtain a protein concentration of 1 mg/ ml . To this solution , aminooxy reagent with a MW of 20 kD (described above ) is NalO4 is added to give a final concentration of 200 uM . The added followed by m - toluidine as a nucleophilic catalyst ( 10 oxidation is carried at RT for 30 min in the dark under gentle mM final concentration ) and NalO4 ( final concentration : shaking . The reaction is then quenched with cysteine ( final 400 uM ) . The coupling reaction is performed for 2 hours in concentration : 10 mM ) for 60 min at R . T . the dark under gentle shaking at room temperature . Subse [ 0362] The solution is next subjected to UF/ DF employing quently , the reaction is quenched with cysteine for 60 min at Vivaspin centrifugal filtrators to remove excess periodate , RT ( cysteine concentration : 10 mM ) . Then the conductivity quencher and the byproducts thereof. of the reaction mixture is adjusted by adding a buffer [ 0363] A 50 - fold molar excess of aminooxy - PSA reagent containing ammonium acetate (50 mM Hepes, 350 mm with a MW of 20 kD ( described above ) is added followed by sodium chloride , 5 mM calcium chloride , 8 M ammonium m - toluidine as a nucleophilic catalyst ( final concentration : acetate , pH 6 . 9) and loaded onto a column filled with Phenyl 10 mM ). The coupling reaction is performed for 2 hours in Sepharose FF (GE Healthcare , Fairfield , Conn . ) pre - equili the dark under gentle shaking at room temperature . The brated with 50 mM Hepes, 2 . 5 M ammonium acetate , 350 excess of aminooxy reagent is removed by means of ion mM sodium chloride , 5 mM calcium chloride , 0 . 01 % Tween exchange chromatography . The PSA - EGF containing frac 80 , pH 6 . 9 . Subsequently the conjugate is eluted with 50 tions of the eluate are collected and subjected to UF /DF by mM Hepes , 5 mM calcium chloride , pH 7 . 5 . Finally the use of a membrane made of regenerated cellulose (Milli PSA - EGF containing fractions are collected and subjected to pore ). The preparation is next analytically characterized by UF /DF by use of a membrane made of regenerated cellulose measuring total protein (Coomassie , Bradford ) and biologi (Millipore ) . The preparation is analytically characterized by cal activity according to methods known in the art. measuring total protein ( Bradford ) and biological activity according to methods known in the art. Method 2 : [0370 ] In an alternative embodiment, Method 3 is carried [0364 ] EGF is transferred or dissolved in reaction buffer out as follows. Epidermal growth factor ( EGF) is transferred ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 mM into reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium calcium chloride , pH 6 . 0 ) to get a final protein concentration chloride , 5 mM calcium chloride, pH 6 .0 ) and diluted to of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor obtain a protein concentration of 1 mg/ ml . A 50 - fold molar rected to 6 . 0 by drop wise addition of a 0 .5 N aqueous HC1 excess of a PSA aminooxy reagent with a MW of 20 kD solution . Subsequently , a 40 mM aqueous sodium periodate (described above ) is added followed by m - toluidine as a solution is added within 10 minutes to give a concentration nucleophilic catalyst (10 mM final concentration ) and US 2017 /0240590 A1 Aug . 24 , 2017 53

NaI04 ( final concentration : 400 uM ). The coupling reaction mM CaC12 , pH 7 .0 ). The column is equilibrated with 5 CV is performed for 2 hours in the dark under gentle shaking at Buffer A . The oxidized NGF is eluted with Buffer B (20 mM room temperature . Subsequently , the reaction is quenched Hepes , 5 mM CaCl2 , 1M NaC1, pH 7 . 0 ) . The NGF contain with cysteine for 60 min at RT (cysteine concentration : 10 ing fractions are collected . The protein content is determined mM ) and the conjugate is purified by ion exchange chro (Coomassie , Bradford ) and adjusted to 1 mg/ ml with reac matography. The conjugate containing fractions of the eluate tion buffer and adjusted to pH 6 . 0 by dropwise addition of are collected and subjected to UF /DF by use of a membrane 0 . 5M HC1. made of regenerated cellulose (Millipore ) . The preparation [0378 ] 50 - fold molar excess of aminooxy -PSA reagent is analytically characterized by measuring total protein with a MW of 20 kD (described above) is added followed by (Bradford ) and biological activity according to methods m - toluidine as a nucleophilic catalyst ( final concentration : known in the art . 10 mM ) . The coupling reaction is performed for 2 hours in the dark under gentle shaking at room temperature . The Method 4 : excess of aminooxy reagent is removed by means of HIC . [0371 ] EGF is dissolved in or transferred to a reaction 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 HCI filled with 80 ml Phenyl Sepharose FF (GE Healthcare , solution . Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes , 2 . 5 M [0372 ] Subsequently the aminooxy -polysialic acid (PSA ammonium acetate , 350 mM sodium chloride, 5 mM cal ONH2 ) reagent is added in a 50 - fold molar excess to this cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted EGF -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 -NGF containing fractions are collected and solution (50 mM ) is added within 15 minutes to get a final subjected to UF/ DF by use of a membrane made of regen concentration of 10 mM . Finally a 40 mM aqueous sodium erated cellulose (Millipore ) . The preparation is next analyti periodate solution is added to give a concentration of 400 cally characterized by measuring total protein ( Coomassie , UM . Bradford ) and biological activity according to methods [ 0373 ] The reaction mixture is incubated for 120 + / - 10 known in the art . min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under [0379 ] In an alternative embodiment, Method 1 is carried gentle shaking . Then the reaction is stopped by the addition out as follows. Nerve growth factor (NGF ) is transferred into of an aqueous L -cysteine solution (1 M ) to give a final a reaction buffer ( e . g ., 50 mM Hepes , 350 mM sodium concentration of 10 mM in the reaction mixture and incu chloride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to bation for 60 + / - 5 min . obtain a protein concentration of 1 mg/ ml . To this solution , [ 0374 ] The obtained EGF- conjugate is purified by ion NalO4 is added to give a final concentration of 200 uM . The exchange chromatography . The PSA -EGF containing frac oxidation is carried at RT for 30 min in the dark under gentle tions of the eluate are collected and concentrated by ultra - / shaking. The reaction is then quenched with cysteine ( final diafiltration (UF /DF ) using a membrane made of concentration : 10 mM ) for 60 min at RT . regenerated cellulose (Millipore ) . [0380 ] The solution is next subjected to UF /DF employing [0375 ] The conjugates prepared by use of this procedure Vivaspin centrifugal filtrators to remove excess periodate , are analytically characterized by measuring total protein , quencher and the byproducts thereof . biological activity according to methods known in the art, [0381 ] A 50 - fold molar excess of aminooxy - PSA reagent and determination of the polysialyation degree by measuring with a MW of 20 kD (described above ) is added followed by the PSA content (resorcinol assay ) . m - toluidine as a nucleophilic catalyst ( final concentration : 10 mM ) . The coupling reaction is performed for 2 hours in Example 22 the dark under gentle shaking at room temperature . The excess of aminooxy reagent is removed by means of ion Polysialylation of NGF Using Aminooxy - PSA and exchange chromatography . The PSA -NGF containing frac m - Toluidine as a Nucleophilic Catalyst tions of the eluate are collected and subjected to UF /DF by use of a membrane made of regenerated cellulose (Milli Method 1 : pore ). The preparation is next analytically characterized by [ 0376 ] A starting concentration of nerve growth factor measuring total protein (Coomassie , Bradford ) and biologi ( NGF) is transferred into a reaction buffer ( e . g . , 50 mM cal activity according to methods known in the art . Hepes, 350 mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to obtain a protein concentration of 1 Method 2 : mg/ml . To this solution , NalO4 is added to give a final [0382 ] NGF is transferred or dissolved in reaction buffer concentration of 200 uM . The oxidation is carried at RT for ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 mM 30 min in the dark under gentle shaking . The reaction is then calcium chloride , pH 6 . 0 ) to get a final protein concentration quenched with cysteine ( final concentration : 10 mM ) for 60 of 1 . 0 + / - 0 .25 mg /ml . Then the pH of the solution is cor min at RT. rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 [ 03771. The solution is next subjected to UF /DF employing solution . Subsequently , a 40 mM aqueous sodium periodate Vivaspin centrifugal filtrators to remove excess periodate , solution is added within 10 minutes to give a concentration quencher and the byproducts thereof or, in the alternative , to of 200 uM . The oxidation reaction is carried out for 30 + / - 5 an IEX column with a volume of 20 ml (Merck EMD TMAE min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes , 5 is stopped by addition of an aqueous L - cysteine solution ( 1 US 2017 /0240590 A1 Aug . 24 , 2017 54

M ) within 15 minutes at T = + 22 + / - 2° C . to give a final cysteine for 60 min at RT ( cysteine concentration : 10 mm ) concentration of 10 mM in the reaction mixture and incu and the conjugate is purified by ion exchange chromatog bation for 60 + / - 5 min . raphy . Then the PSA - NGF containing fractions of the eluate [0383 ] The oxidized NGF is further purified by ion are collected and subjected to UF /DF by use of a membrane exchange chromatography . The oxidized NGFcontaining made of regenerated cellulose (Millipore ) . The preparation fractions of the eluate are collected and used for the conju is analytically characterized by measuring total protein gation reaction . (Bradford ) and biological activity according to methods [ 0384 ] The aminooxy - polysialic acid (PSA -ONH2 ) known in the art. reagent is added in a 50 - fold molar excess to the eluate containing the purified oxidized NGF within a maximum Method 4 : time period ( t ) of 15 minutes under gentle stirring. Then an [ 0389 ] NGF is dissolved in or transferred to a reaction aqueous m - toluidine solution (50 mM ) is added within 15 buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride , 5 mM minutes to get a final concentration of 10 mM . The reaction calcium chloride , pH 6 . 0 ) to get a final protein concentration mixture is incubated for 120 + / – 10 min . at pH 6 . 0 in the dark of 1 . 0 + / - 0 . 25 mg/ml . Then the pH of the solution is cor at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 ( protein concentration : 1 mg/ ml ) . solution . [ 0385 ]. The obtained PSA - NGF conjugate is further puri 10390 ) Subsequently , the aminooxy -polysialic acid ( PSA fied by ion exchange chromatography . The PSA -NGF con ONH2 ) reagent is added in a 50 - fold molar excess to this jugate containing fractions are collected and concentrated by NGF - solution within a maximum time period (t ) of 15 ultra - / diafiltration (UF /DF ) using a membrane made of minutes under gentle stirring . Then an aqueous m -toluidine regenerated cellulose with an appropriate molecular weight solution (50 mM ) is added within 15 minutes to get a final cut off (Millipore ) . concentration of 10 mM . Finally a 40 mM aqueous sodium [ 0386 ] The conjugate prepared by use of this procedure is periodate solution is added to give a concentration of 400 analytically characterized by measuring total protein , bio UM . logical activity , and determination of the polysialyation [ 0391 ] The reaction mixture is incubated for 120 + 1 - 10 degree by measuring the PSA content ( resorcinol assay ) . min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . Then the reaction is stopped by the addition Method 3 : of an aqueous L - cysteine solution ( 1 M ) to give a final [ 0387] Nerve growth factor (NGF ) is transferred into concentration of 10 mM in the reaction mixture and incu reaction buffer (50 mM Hepes, 350 mM sodium chloride , 5 bation for 60 + / - 5 min . mM calcium chloride , pH 6 . 0 ) and diluted to obtain a protein 10392 ] The obtained NGF - conjugate is purified by ion concentration of 1 mg/ ml . A 50 -fold molar excess of ami exchange chromatography . The PSA -NGF containing frac nooxy -PSA reagent with a MW of 20 kD (described above ) tions of the eluate are collected and concentrated by ultra is added followed by m -toluidine as a nucleophilic catalyst diafiltration (UF /DF ) using a membrane made of ( 10 mM final concentration ) and Nal04 ( final concentration : regenerated cellulose (Millipore ). 400 UM ) . The coupling reaction is performed for 2 hours in [0393 ] The conjugates prepared by use of this procedure the dark under gentle shaking at room temperature . Subse are analytically characterized by measuring total protein , quently , the reaction is quenched with cysteine for 60 min at biological activity according to methods known in the art , RT ( cysteine concentration : 10 mM ) . Then the conductivity and determination of the polysialyation degree by measuring of the reaction mixture is adjusted by adding a buffer the PSA content (resorcinol assay ) . containing ammonium acetate (50 mM Hepes , 350 mm sodium chloride , 5 mM calcium chloride , 8 M ammonium Example 23 acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl Sepharose FF (GE Healthcare , Fairfield , Conn . ) pre - equili Polysialylation of HGH Using Aminooxy - PSA and brated with 50 mM Hepes , 2 . 5 M ammonium acetate , 350 m - Toluidine as a Nucleophilic Catalyst mM sodium chloride , 5 mM calcium chloride , 0 .01 % Tween 80 , pH 6 . 9 . Subsequently the conjugate is eluted with 50 Method 1 : mM Hepes, 5 mM calcium chloride , pH 7 . 5 . Finally , the [0394 ] As described herein , the amino acid sequence of PSA NGF - containing fractions are collected and subjected human growth hormone (HGH ) is first modified to incor to UF /DF by use of a membrane made of regenerated porate at least one glycosylation site . Following purification , cellulose (Millipore ). The preparation is analytically char HGH is glycosylated in vitro according to methods known acterized by measuring total protein (Bradford ) and biologi in the art. cal activity according to methods known in the art . [0395 ] A starting concentration of human growth hormone [ 0388 ] In an alternative embodiment, Method 3 is carried (HGH ) is transferred into a reaction buffer ( e . g . , 50 mM outas follows. Nerve growth factor (NGF ) is transferred into Hepes, 350 mM sodium chloride , 5 mM calcium chloride , reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chlo pH 6 . 0 ) and diluted to obtain a protein concentration of 1 ride , 5 mM calcium chloride, pH 6 . 0 ) and diluted to obtain mg/ml . To this solution , NalO4 is added to give a final a protein concentration of 1 mg/ml . A 50 - fold molar excess concentration of 200 uM . The oxidation is carried at RT for of aminooxy - PSA reagent with a MW of 20 kD ( described 30 min in the dark under gentle shaking . The reaction is then above ) is added followed by m -toluidine as a nucleophilic quenched with cysteine ( final concentration : 10 mM ) for 60 catalyst ( 10 mM final concentration ) and Nal04 ( final min at RT. concentration : 400 uM ) . The coupling reaction is performed [0396 ] The solution is next subjected to UF /DF employing for 2 hours in the dark under gentle shaking at room Vivaspin centrifugal filtrators to remove excess periodate , temperature . Subsequently , the reaction is quenched with quencher and the byproducts thereof or, in the alternative , to US 2017 /0240590 A1 Aug . 24 , 2017 55 an IEX column with a volume of 20 ml (Merck EMD TMAE porate at least one glycosylation site . Following purification , ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes , 5 HGH is glycosylated in vitro according to methods known mM CaC12 , pH 7 . 0 ) . The column is equilibrated with 5 CV in the art. Buffer A . The oxidized HGH is eluted with Buffer B (20 mM [0402 ) HGH is transferred or dissolved in reaction buffer Hepes , 5 mM CaCl2 , 1 M NaCl, pH 7 .0 ). The HGH (e . g. 50 mM Hepes, 350 mM sodium chloride, 5 mM containing fractions are collected . The protein content is calcium chloride , pH 6 .0 ) to get a final protein concentration determined (Coomassie , Bradford ) and adjusted to 1 mg/ml of 1 . 0 + / - 0 .25 mg/ml . Then the pH of the solution is cor with reaction buffer and adjusted to pH 6 . 0 by dropwise rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 addition of 0 . 5 M HCl. solution . Subsequently , a 40 mM aqueous sodium periodate [03971 A 50 - fold molar excess of aminooxy - PSA reagent solution is added within 10 minutes to give a concentration with a MW of 20 kD (described above ) is added followed by of 200 uM . The oxidation reaction is carried out for 30 + / - 5 m - toluidine as a nucleophilic catalyst ( final concentration : min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction 10 mM ) . The coupling reaction is performed for 2 hours in is stopped by addition of an aqueous L - cysteine solution ( 1 the dark under gentle shaking at room temperature . The M ) within 15 minutes at T = + 22 + / - 2° C . to give a final concentration of 10 mM in the reaction mixture and incu excess of aminooxy reagent is removed by means of HIC . bation for 60 + / - 5 min . The conductivity of the reaction mixture is adjusted by [0403 ] The oxidized HGH is further purified by ion adding a buffer containing ammonium acetate (50 mM exchange chromatography . The oxidized HGH containing Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 fractions of the eluate are collected and used for the conju M ammonium acetate , pH 6 .9 ) and loaded onto a column gation reaction . filled with 80 ml Phenyl Sepharose FF (GE Healthcare , [0404 ] The aminooxy -polysialic acid (PSA -ONH2 ) Fairfield , Conn . ) pre - equilibrated with 50 mM Hepes , 2 . 5 M reagent is added in a 50 - fold molar excess to the eluate ammonium acetate , 350 mM sodium chloride , 5 mM cal containing the purified oxidized HGH within a maximum cium chloride , pH 6 .9 . Subsequently the conjugate is eluted time period ( t ) of 15 minutes under gentle stirring . Then an with 50 mM Hepes buffer pH 7 .5 containing 5 mM CaC12 . aqueous m - toluidine solution (50 mm ) is added within 15 Finally , the PSA -HGH containing fractions are collected and minutes to get a final concentration of 10 mM . The reaction subjected to UF/ DF by use of a membrane made of regen mixture is incubated for 120 + / - 10 min . at pH 6 . 0 in the dark erated cellulose (Millipore ) . The preparation is next analyti at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking cally characterized by measuring total protein ( Coomassie , (protein concentration : 1 mg/ml ) . Bradford ) and biological activity according to methods [04051 . The obtained PSA - HGH conjugate is further puri known in the art. fied by ion exchange chromatography . The PSA -HGH con 10398 ] In an alternative embodiment, Method 1 is carried jugate containing fractions are collected and concentrated by out as follows. As described herein , the amino acid sequence ultra - / diafiltration (UF /DF ) using a membrane made of of human growth hormone (HGH ) is first modified to regenerated cellulose with an appropriate molecular weight incorporate at least one glycosylation site . Following puri cut off (Millipore ) . fication , HGH is glycosylated in vitro according to methods [0406 ] The conjugate prepared by use of this procedure is known in the art . HGH is transferred into a reaction buffer analytically characterized by measuring total protein , bio ( e . g ., 50 mM Hepes , 350 mM sodium chloride, 5 mM logical activity , and determination of the polysialyation calcium chloride, pH 6 .0 ) and diluted to obtain a protein degree by measuring the PSA content (resorcinol assay ) . concentration of 1 mg/ ml . To this solution , NalO4 is added to give a final concentration of 200 uM . The oxidation is carried at RT for 30 min in the dark under gentle shaking . Method 3 : The reaction is then quenched with cysteine ( final concen [0407 ] As described herein , the amino acid sequence of tration : 10 mM ) for 60 min at RT. human growth hormone (HGH ) is first modified to incor porate at least one glycosylation site . Following purification , [0399 ] The solution is next subjected to UF /DF employing HGH is glycosylated in vitro according to methods known Vivaspin centrifugal filtrators to remove excess periodate , in the art. quencher and the byproducts thereof. [0408 ] Human growth hormone (HGH ) is transferred into [0400 ] A 50 - fold molar excess of aminooxy - PSA reagent reaction buffer ( 50 mM Hepes, 350 mM sodium chloride, 5 with a MW of 20 kD (described above ) is added followed by mM calcium chloride , pH 6 . 0 ) and diluted to obtain a protein m -toluidine as a nucleophilic catalyst ( final concentration : concentration of 1 mg/ml . A 50 fold molar excess of 10 mM ) . The coupling reaction is performed for 2 hours in aminooxy - PSA reagent with a MW of 20 kD (described the dark under gentle shaking at room temperature . The above ) is added followed by m - toluidine as a nucleophilic excess of aminooxy reagent is removed by means of ion catalyst ( 10 mM final concentration ) and NaI04 ( final exchange chromatography. The PSA - HGH containing frac concentration : 400 UM ). The coupling reaction is performed tions of the eluate are collected and subjected to UF /DF by for 2 hours in the dark under gentle shaking at room use of a membrane made of regenerated cellulose (Milli temperature . Subsequently , the reaction is quenched with pore ). The preparation is next analytically characterized by cysteine for 60 min at RT ( cysteine concentration : 10 mM ) . measuring total protein (Coomassie , Bradford ) and biologi Then the conductivity of the reaction mixture is adjusted by cal activity according to methods known in the art . adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride , 5 mM calcium chloride, 8 Method 2 : M ammonium acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl Sepharose FF (GE Healthcare, Fairfield , [0401 ] As described herein , the amino acid sequence of Conn .) pre - equilibrated with 50 mM Hepes, 2 . 5 M ammo human growth hormone (HGH ) is first modified to incor- nium acetate , 350 mM sodium chloride , 5 mM calcium US 2017 /0240590 A1 Aug . 24 , 2017 56 chloride , 0 .01 % Tween 80 , pH 6 .9 . Subsequently the con [0415 ] The conjugates prepared by use of this procedure jugate is eluted with 50 mM Hepes , 5 mM calcium chloride , are analytically characterized by measuring total protein , pH 7 . 5 . Finally, the PSA HGH - containing fractions are biological activity according to methods known in the art, collected and subjected to UF /DF by use of a membrane and determination of the polysialyation degree by measuring made of regenerated cellulose (Millipore ) . The preparation the PSA content ( resorcinol assay ). is analytically characterized by measuring total protein (Bradford ) and biological activity according to methods Example 24 known in the art . [0409 ] In an alternative embodiment, Method 3 is carried Polysialylation of TNF - Alpha Using out as follows. As described herein , the amino acid sequence Aminooxy - PSA and m - Toluidine as a Nucleophilic of human growth hormone (HGH ) is first modified to Catalyst incorporate at least one glycosylation site . Following puri [0416 ] A starting concentration of tumor necrosis factor fication , HGH is glycosylated in vitro according to methods alpha ( TNF - alpha ) is transferred into a reaction buffer ( e . g . , known in the art. HGH is transferred into reaction buffer 50 mM Hepes , 350 mM sodium chloride , 5 mM calcium ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 mM chloride , pH 6 . 0 ) and diluted to obtain a protein concentra calcium chloride, pH 6 . 0 ) and diluted to obtain a protein tion of 1 mg/ ml . To this solution , NalO4 is added to give a concentration of 1 mg/ ml . A 50 fold molar excess of final concentration of 200 uM . The oxidation is carried at RT aminooxy - PSA reagent with a MW of 20 kD (described for 30 min in the dark under gentle shaking . The reaction is above ) is added followed by m - toluidine as a nucleophilic then quenched with cysteine ( final concentration : 10 mm ) catalyst ( 10 mM final concentration ) and NalO4 ( final for 60 min at RT. concentration : 400 UM ) . The coupling reaction is performed [ 0417 ] The solution is next subjected to UF /DF employing for 2 hours in the dark under gentle shaking at room Vivaspin centrifugal filtrators to remove excess periodate , temperature . Subsequently , the reaction is quenched with quencher and the byproducts thereof or, in the alternative , to cysteine for 60 min at RT ( cysteine concentration : 10 mM ) an IEX column with a volume of 20 ml (Merck EMD TMAE and the conjugate is purified by ion exchange chromatog ( M ) which is equilibrated with Buffer A ( 20 mM Hepes , 5 raphy . Then the PSA -HGH - containing fractions of the eluate mM CaC12 , pH 7 . 0 ) . The column is equilibrated with 5 CV are collected and subjected to UF /DF by use of a membrane Buffer A . The oxidized TNF -alpha is eluted with Buffer B made of regenerated cellulose (Millipore ). The preparation ( 20 mM Hepes, 5 mM CaC12 , 1M NaC ) , pH 7 . 0 ) . The is analytically characterized by measuring total protein TNF- alpha containing fractions are collected . The protein (Bradford ) and biological activity according to methods content is determined (Coomassie , Bradford ) and adjusted to known in the art. 1 mg/ ml with reaction buffer and adjusted to pH 6 . 0 by dropwise addition of 0 .5M HCI. Method 4 : 10418 ) A 50 - fold molar excess of aminooxy -PSA reagent [0410 ] As described herein , the amino acid sequence of with a MW of 20 kD (described above ) is added followed by human growth hormone (HGH ) is first modified to incor m - toluidine as a nucleophilic catalyst ( final concentration : porate at least one glycosylation site . Following purification , 10 mM ) . The coupling reaction is performed for 2 hours in the dark under gentle shaking at room temperature . The HGH is glycosylated in vitro according to methods known excess of aminooxy reagent is removed by means of HIC . in the art . The conductivity of the reaction mixture is adjusted by [0411 ] HGH is dissolved in or transferred to a reaction adding a buffer containing ammonium acetate (50 mM buffer ( e . g . 50 mM Hepes , 350 mM sodium chloride, 5 mM Hepes , 350 mM sodium chloride , 5 mM calcium chloride , 8 calcium chloride , pH 6 . 0 ) to get a finalprotein concentration M ammonium acetate , pH 6 . 9 ) and loaded onto a column of 1 . 0 + / - 0 . 25 mg/ml . Then the pH of the solution is cor filled with 80 ml Phenyl Sepharose FF (GE Healthcare , rected to 6 . 0 by drop wise addition of a 0 .5 N aqueous HC1 Fairfield , Conn .) pre -equilibrated with 50 mM Hepes , 2 .5 M solution . ammonium acetate , 350 mM sodium chloride, 5 mM cal [ 0412 ] Subsequently , the aminooxy - polysialic acid (PSA cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted ONH2) reagent is added in a 50 - fold molar excess to this with 50 mM Hepes buffer pH 7 .5 containing 5 mM CaC12 . HGH - solution within a maximum time period ( t ) of 15 Finally the PSA - TNF - alpha - containing fractions are col minutes under gentle stirring. Then an aqueous m -toluidine lected and subjected to UF /DF by use of a membrane made solution (50 mM ) is added within 15 minutes to get a final of regenerated cellulose (Millipore ) . The preparation is next concentration of 10 mM . Finally a 40 mM aqueous sodium analytically characterized by measuring total protein (Coo periodate solution is added to give a concentration of 400 massie , Bradford ) and biological activity according to meth uM . ods known in the art . [ 0413 ] The reaction mixture is incubated for 120 + / - 10 [0419 ] In an alternative embodiment , Method 1 is carried min . in the dark at a temperature ( T ) ofT = + 22 + / - 2° C . under out as follows. Tumor necrosis factor- alpha ( TNF - alpha ) is gentle shaking . Then the reaction is stopped by the addition transferred into a reaction buffer ( e . g . 50 mM Hepes , 350 of an aqueous L - cysteine solution ( 1 M ) to give a final mM sodium chloride , 5 mM calcium chloride, pH 6 .0 ) and concentration of 10 mM in the reaction mixture and incu diluted to obtain a protein concentration of 1 mg/ ml . To this bation for 60 + / – 5 min . solution , NalO4 is added to give a final concentration of 200 [0414 ] The obtained HGH - conjugate is purified by ion UM . The oxidation is carried at RT for 30 min in the dark exchange chromatography. The PSA -HGH containing frac under gentle shaking. The reaction is then quenched with tions of the eluate are collected and concentrated by ultra -/ cysteine ( final concentration : 10 mM ) for 60 min at RT. diafiltration (UF / DF ) using a membrane made of [0420 ] The solution is next subjected to UF/ DF employing regenerated cellulose (Millipore ) . Vivaspin centrifugal filtrators to remove excess periodate , US 2017 /0240590 A1 Aug . 24 , 2017 57

Method 3 : of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HCI [0445 ] As described herein , the amino acid sequence of solution . insulin is first modified to incorporate at least one glycosy [0451 ] Subsequently , the aminooxy -polysialic acid (PSA lation site . Following purification , insulin is glycosylated in ONH2) reagent is added in a 50 - fold molar excess to this vitro according to methods known in the art . insulin - solution within a maximum time period ( t ) of 15 [0446 ] Insulin is transferred into reaction buffer (50 mM minutes under gentle stirring . Then an aqueous m - toluidine Hepes, 350 mM sodium chloride, 5 mM calcium chloride , solution ( 50 mm ) is added within 15 minutes to get a final pH 6 . 0 ) and diluted to obtain a protein concentration of 1 concentration of 10 mM . Finally a 40 mM aqueous sodium mg/ ml . A 50 - fold molar excess of aminooxy - PSA reagent periodate solution is added to give a concentration of 400 with a MW of 20 kD (described above ) is added followed by UM . m - toluidine as a nucleophilic catalyst ( 10 mM final concen [ 0452 ] The reaction mixture is incubated for 120 + / - 10 tration ) and NaI04 ( final concentration : 400 M ). The cou min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under pling reaction is performed for 2 hours in the dark under gentle shaking . Then the reaction is stopped by the addition gentle shaking at room temperature . Subsequently , the reac of an aqueous L - cysteine solution ( 1 M ) to give a final tion is quenched with cysteine for 60 min at RT ( cysteine concentration of 10 mM in the reaction mixture and incu concentration : 10 mM ) . Then the conductivity of the reac bation for 60 + / - 5 min . tion mixture is adjusted by adding a buffer containing [0453 ]. The obtained insulin conjugate is purified by ion ammonium acetate ( 50 mM Hepes , 350 mM sodium chlo exchange chromatography . The PSA - insulin containing ride, 5 mM calcium chloride , 8 M ammonium acetate , pH fractions of the eluate are collected and concentrated by 6 . 9 ) and loaded onto a column filled with Phenyl Sepharose ultra - / diafiltration (UF /DF ) using a membrane made of FF GE( Healthcare, Fairfield , Conn .) pre - equilibrated with regenerated cellulose (Millipore ) . 50 mM Hepes , 2 . 5 M ammonium acetate , 350 mM sodium [0454 ] The conjugates prepared by use of this procedure chloride , 5 mM calcium chloride , 0 .01 % Tween 80 , pH 6 . 9 . are analytically characterized by measuring total protein , Subsequently the conjugate is eluted with 50 mM Hepes , 5 biological activity according to methods known in the art , mM calcium chloride , pH 7 . 5 . Finally, the PSA - insulin and determination of the polysialyation degree by measuring containing fractions are collected and subjected to UF /DF by the PSA content ( resorcinol assay ). use of a membrane made of regenerated cellulose (Milli pore ). The preparation is analytically characterized by mea Example 26 suring total protein (Bradford ) and biological activity Polysialylation of Interferon - Alpha Using according to methods known in the art. Aminooxy -PSA and m - Toluidine as a Nucleophilic [0447 ] In an alternative embodiment, Method 3 is carried Catalyst out as follows. As described herein , the amino acid sequence of insulin is first modified to incorporate at least one glycosylation site . Following purification , insulin is glyco Method 1 : sylated in vitro according to methods known in the art . [ 0455 ] A starting concentration of interferon - alpha is transferred into a reaction buffer ( e . g . , 50 mM Hepes , 350 [0448 ] Insulin is transferred into reaction buffer ( e . g . 50 mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and mM Hepes , 350 mM sodium chloride, 5 mM calcium diluted to obtain a protein concentration of 1 mg/ml . To this chloride , pH 6 . 0 ) and diluted to obtain a protein concentra solution , NaI04 is added to give a final concentration of 200 tion of 1 mg/ ml . A 50 - fold molar excess of aminooxy -PSA UM . The oxidation is carried at RT for 30 min in the dark reagent with a MW of 20 kD (described above ) is added under gentle shaking . The reaction is then quenched with followed by m - toluidine as a nucleophilic catalyst ( 10 mM cysteine ( final concentration : 10 mM ) for 60 min at RT. final concentration ) and NalO4 ( final concentration : 400 10456 ] The solution is next subjected to UF /DF employing UM ) . The coupling reaction is performed for 2 hours in the Vivaspin centrifugal filtrators to remove excess periodate , dark under gentle shaking at room temperature . Subse quencher and the byproducts thereof or, in the alternative , to quently , the reaction is quenched with cysteine for 60 min at an IEX column with a volume of 20 ml (Merck EMD TMAE RT ( cysteine concentration : 10 mm ) and the conjugate is ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes , 5 purified by ion exchange chromatography . PSA - insulin con mM CaC12 , pH 7 . 0 ) . The column is equilibrated with 5 CV taining fractions of the eluate are collected and subjected to Buffer A . The oxidized interferon -alpha is eluted with Buffer UF/ DF by use of a membrane made of regenerated cellulose B (20 mM Hepes, 5 mM CaCl2 , 1M NaCl, pH 7 . 0 ) . The (Millipore ) . The preparation is analytically characterized by interferon - alpha containing fractions are collected . The pro measuring total protein (Bradford ) and biological activity tein content is determined (Coomassie , Bradford ) and according to methods known in the art. adjusted to 1 mg/ ml with reaction buffer and adjusted to pH 6 . 0 by dropwise addition of 0 . 5 M HCl. Method 4 : [0457 ] A 50 - fold molar excess of aminooxy - PSA reagent with a MW of 20 kD (described above ) is added followed by [0449 ] As described herein , the amino acid sequence of m - toluidine as a nucleophilic catalyst ( final concentration : insulin is first modified to incorporate at least one glycosy 10 mM ). The coupling reaction is performed for 2 hours in lation site . Following purification , insulin is glycosylated in the dark under gentle shaking at room temperature . The vitro according to methods known in the art . excess of aminooxy reagent is removed by means of HIC . [0450 ] Insulin is dissolved in or transferred to a reaction 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 US 2017 /0240590 A1 Aug . 24 , 2017 60

M ammonium acetate , pH 6 .9 ) and loaded onto a column [0464 ] The obtained PSA - interferon -alpha conjugate is filled with 80 ml Phenyl Sepharose FF (GE Healthcare further purified by ion exchange chromatography. The PSA Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes, 2 . 5 M interferon - alpha conjugate containing fractions are collected ammonium acetate , 350 mM sodium chloride , 5 mM cal and concentrated by ultra - /diafiltration (UF /DF ) using a cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted membrane made of regenerated cellulose with an appropri with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . ate molecular weight cut off (Millipore ) . Finally the PSA - interferon - alpha containing fractions are collected and subjected to UF /DF by use of a membrane Method 3 : made of regenerated cellulose (Millipore ). The preparation is next analytically characterized by measuring total protein [0465 ] Interferon - alpha is transferred into reaction buffer (Coomassie , Bradford ) and biological activity according to (50 mM Hepes , 350 mM sodium chloride , 5 mM calcium methods known in the art . chloride , pH 6 . 0 ) and diluted to obtain a protein concentra [0458 ] In an alternative embodiment, Method 1 is carried tion of 1 mg/ ml . A 50 - fold molar excess of a PSA aminooxy out as follows. Interferon - alpha is transferred into a reaction reagent with a MW of 20 kD (described above ) is added buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride, 5 mM followed by m - toluidine as a nucleophilic catalyst ( 10 mM calcium chloride , pH 6 . 0 ) and diluted to obtain a protein final concentration ) and NalO4 ( final concentration : 400 concentration of 1 mg/ ml . To this solution , NalO4 is added UM ). The coupling reaction is performed for 2 hours in the to give a final concentration of 200 UM . The oxidation is dark under gentle shaking at room temperature . Subse carried at RT for 30 min in the dark under gentle shaking . quently , the reaction is quenched with cysteine for 60 min at The reaction is then quenched with cysteine ( final concen RT (cysteine concentration : 10 mM ) . Then the conductivity tration : 10 mM ) for 60 min at RT. of the reaction mixture is adjusted by adding a buffer [0459 ] The solution is next subjected to UF /DF employing containing ammonium acetate (50 mM Hepes, 350 mm Vivaspin centrifugal filtrators to remove excess periodate , sodium chloride , 5 mM calcium chloride , 8 M ammonium quencher and the byproducts thereof. acetate , pH 6 . 9) and loaded onto a column filled with Phenyl 10460 ) A 50 - fold molar excess of aminooxy - PSA reagent Sepharose FF (GE Healthcare , Fairfield , Conn . ) pre - equili with a MW of 20 kD (described above ) is added followed by brated with 50 mM Hepes, 2 . 5 M ammonium acetate , 350 m - toluidine as a nucleophilic catalyst ( final concentration : mM sodium chloride , 5 mM calcium chloride , 0 . 01 % Tween 10 mM ) . The coupling reaction is performed for 2 hours in 80 , pH 6 . 9 . Subsequently the conjugate is eluted with 50 the dark under gentle shaking at room temperature . The mM Hepes , 5 mM calcium chloride , pH 7 .5 . Finally , the excess of aminooxy reagent is removed by means of ion PSA - interferon - alpha containing fractions are collected and exchange chromatography. The PSA - interferon - alpha con subjected to UF/ DF by use of a membrane made of regen taining fractions of the eluate are collected and subjected to erated cellulose (Millipore ). The preparation is analytically UF /DF by use of a membrane made of regenerated cellulose characterized by measuring total protein (Bradford ) and (Millipore ) . The preparation is next analytically character biological activity according to methods known in the art. ized by measuring total protein (Coomassie , Bradford ) and [0466 ]. In an alternative embodiment, Method 3 is carried biological activity according to methods known in the art . out as follows. Interferon - alpha is transferred into reaction buffer (e . g. 50 mM Hepes, 350 mM sodium chloride, 5 mM Method 2 : calcium chloride , pH 6 . 0 ) and diluted to obtain a protein concentration of 1 mg/ml . A 50 - fold molar excess of ami [ 0461] Interferon -alpha is transferred or dissolved in reac nooxy - PSA reagent with a MW of 20 kD (described above ) tion buffer ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 is added followed by m - toluidine as a nucleophilic catalyst mM calcium chloride , pH 6 . 0 ) to get a final protein con ( 10 mM final concentration ) and NalO4 ( final concentration : centration of 1 . 0 + / - 0 . 25 mg/ml . Then the pH of the solution 400 uM ) . The coupling reaction is performed for 2 hours in is corrected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous the dark under gentle shaking at room temperature . Subse HCl solution . Subsequently, a 40 mM aqueous sodium quently , the reaction is quenched with cysteine for 60 min at periodate solution is added within 10 minutes to give a RT ( cysteine concentration : 10 mM ) and the conjugate is concentration of 200 UM . The oxidation reaction is carried purified by ion exchange chromatography. The PSA - inter out for 30 + / - 5 min at a temperature ( T ) of T = + 22 + / - 2° C . feron - alpha containing fractions of the eluate are collected Then the reaction is stopped by addition of an aqueous and subjected to UF /DF by use of a membrane made of L - cysteine solution ( 1 M ) within 15 minutes at T = + 22 + / - 2° regenerated cellulose (Millipore ). The preparation is ana C . to give a final concentration of 10 mM in the reaction lytically characterized by measuring total protein (Bradford ) mixture and incubation for 60 + / - 5 min . and biological activity according to methods known in the [0462 ] The oxidized interferon - alpha is further purified by art. ion exchange chromatography. The oxidized interferon alpha containing fractions of the eluate are collected and Method 4 : used for the conjugation reaction . [0463 ] The aminooxy -polysialic acid (PSA -ONH2 ) [0467 ] Interferon - alpha is dissolved in or transferred to a reagent is added in a 50 - fold molar excess to the eluate reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium chlo containing the purified oxidized interferon - gamma within a ride , 5 mM calcium chloride , pH 6 . 0 ) to get a final protein maximum time period ( t ) of 15 minutes under gentle stirring . concentration of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the Then an aqueous m - toluidine solution (50 mM ) is added solution is corrected to 6 . 0 by drop wise addition of a 0 .5 N within 15 minutes to get a final concentration of 10 mM . The aqueous HCl solution . reaction mixture is incubated for 120 + / – 10 min . at pH 6 .0 in [0468 ] Subsequently , the aminooxy -polysialic acid (PSA the dark at a temperature ( T ) of T = + 22 + / - 2° C . under gentle ONH2) reagent is added in a 50 - fold molar excess to this shaking (protein concentration : 1 mg/ml ) . interferon -alpha solution within a maximum time period ( t ) US 2017 /0240590 A1 Aug . 24 , 2017 of 15 minutes under gentle stirring . Then an aqueous Millipore ). The final diafiltration step is performed against m - toluidine solution (50 mM ) is added within 15 minutes to histidine buffer , pH 6 . 9 containing 150 mM NaCl. The get a final concentration of 10 mM . Finally , a 40 mm preparation is analytically characterized by measuring total aqueous sodium periodate solution is added to give a con protein (Bradford ) and biological activity according to meth centration of 400 uM . ods known in the art . For the PSA - Interferon -gamma con [0469 ] The reaction mixture is incubated for 120 + / - 10 jugate a specific activity of > 50 % in comparison to native min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under Interferon - gamma is determined . The conjugate is addition gentle shaking . Then the reaction is stopped by the addition ally analytically characterized by Size Exclusion HPLC of an aqueous L - cysteine solution ( 1 M ) to give a final using a Agilent 1200 HPLC system equipped with a Shodex concentration of 10 mM in the reaction mixture and incu KW 803 column under conditions as previously described bation for 60 + / - 5 min . (Kolarich et al , Transfusion 2006 ; 46 : 1959 - 77 ) . It is shown [0470 ] The obtained interferon -alpha conjugate is purified that the preparation contains no free Interferon gamma. by ion -exchange chromatography . The PSA - interferon -al pha containing fractions of the eluate are collected and Method 2 : concentrated by ultra - /diafiltration (UF /DF ) using a mem brane made of regenerated cellulose (Millipore ). [0475 ] 10 mg interferon - gamma is dissolved in 8 ml [ 0471] The conjugates prepared by use of this procedure histidine buffer, pH 6 . 0 ( 20 mM L - histidine , 150 mM NaCl) . are analytically characterized by measuring total protein , 200 ul of an aqueous sodium periodate solution ( 5 mM ) and biological activity according to methods known in the art, 2 ml of an aqueous m - toluidine solution ( 50 mM ) are then and determination of the polysialyation degree by measuring added . Subsequently the aminooxy -PSA reagent with a MW of 20 kD (described above ) is added to give a 5 - fold molar the PSA content (resorcinol assay ) . reagent excess . The mixture is incubated for 2 h in the dark Example 27 at room temperature under gentle stirring and quenched for 15 min at room temperature by the addition of 100 ul of 1 Polysialylation of Interferon -Gamma Using M aqueous cysteine solution . Aminooxy - PSA and m - Toluidine as a Nucleophilic [0476 ] The free interferon gamma is removed by means of Catalyst cation exchange chromatography (CEC ) . The reaction mix ture is diluted with 20 ml Buffer A ( 50 mM Hepes , pH 6 . 5 ) Method 1: and loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE Healthcare, Fairfield , Conn . ) pre -equilibrated with Buffer A . [ 0472 ] 10 mg interferon - gamma is dissolved in 5 ml Then the column is eluted with Buffer B (50 mM Hepes, 1 histidine buffer , pH 6 .0 ( 20 mM L - histidine, 150 mM NaCl) . M NaCl, pH 6 . 5 ) . Free interferon - gamma is eluted by 100 ul of an aqueous sodium periodate solution (5 mM ) is washing the column with 25 % Buffer B and the conjugate at then added and the reaction mixture is incubated for 1 h in 50 % Buffer B . The conductivity of the conjugate containing the dark at 4° C . under gentle stirring and quenched for 15 fractions is subsequently raised to ~ 190 mS/ cm with Buffer min at room temperature by the addition of 50 ul of a 1 M C ( 50 mM Hepes , 5 M NaC1, pH 6 . 9 ) and loaded onto a 20 aqueous cysteine solution . The mixture is subsequently ml HiPrep Butyl FF 16 / 10 column (GE Healthcare, Fairfield , subjected to UF /DF employing Vivaspin 15R 10 kD cen Conn . ) pre - equilibrated with Buffer D ( 50 mM Hepes , 3 M trifugal filtrators to remove excess periodate, quencher and NaCl, pH 6 .9 ) . Free PSA - reagent is washed out within 5 CV the byproducts thereof . Buffer D . Subsequently , the conjugate is eluted with 100 % [0473 ] The retentate ( approx . 7 ml) , containing oxidized Buffer E ( 50 mM Hepes , pH 6 . 9 ) . The conjugate containing interferon - gamma, is mixed with 2 ml of an aqueous fractions are concentrated by UF /DF using a 10 kD mem m - toluidine solution (50 mM ) and incubated for 30 min at brane made of regenerated cellulose (88 cm2, cutoff 10 room temperature . Then aminooxy - PSA reagentwith a MW kD Millipore/ ) . The final diafiltration step is performed of 20 kD (described above ) is added to give a 5 - fold molar against histidine buffer , pH 6 . 9 containing 150 mM NaCl. reagent excess. This mixture is incubated for 2 .5 h at RT in The preparation is analytically characterized by measuring the dark under gentle stirring . total protein (Bradford ) and biological activity according to [0474 ] The free Interferon - gamma is removed by means of methods known in the art. For the PSAinterferon - gamma cation exchange chromatography (CEC ) . The reaction mix conjugate a specific activity of > 50 % in comparison to ture is diluted with 20 mlBuffer A (50 mM Hepes, pH 6 . 5 ) native interferon - gamma is determined . The conjugate is and loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE additionally analytically characterized by Size Exclusion Healthcare, Fairfield , Conn . ) pre -equilibrated with Buffer A . HPLC using a Agilent 1200 HPLC system equipped with a Then the column is eluted with Buffer B (50 mM Hepes , 1 Shodex KW 803 column under conditions as previously M NaCl, pH 6 . 5 ) . Free interferon - gamma is eluted by described (Kolarich et al, Transfusion 2006 ; 46 : 1959- 77 ) . It washing the column with 25 % Buffer B and the conjugate at is shown that the preparation contains no free interferon 50 % Buffer B . The conductivity of the conjugate containing gamma. fractions is subsequently raised to - 190 mS/ cm with Buffer C (50 mM Hepes, 5 M NaCl, pH 6 . 9 ) and loaded onto a 20 Method 3 : ml HiPrep Butyl FF 16 / 10 column (GE Healthcare , Fairfield , Conn . ) pre -equilibrated with Buffer D (50 mM Hepes , 3 M [0477 ] 10 mg interferon - gamma is dissolved in 8 ml NaCl, pH 6 . 9 ) . Free PSA - reagent is washed outwithin 5 CV histidine buffer, pH 6 . 0 (20 mM L -histidine , 150 mM NaCl) . Buffer D . Subsequently , the conjugate is eluted with 100 % 200 ul of an aqueous sodium periodate solution ( 5 mM ) and Buffer E (50 mM Hepes , pH 6 .9 ) . The conjugate containing 2 ml of an aqueous m - toluidine solution ( 50 mM ) are then fractions are concentrated by UF /DF using a 10 kD mem added . Subsequently the aminooxy - PSA reagent with a MW brane made of regenerated cellulose ( 88 cm2, cutoff 10 kD , of 20 kD (described above ) is added to give a 5 -fold molar US 2017 /0240590 A1 Aug . 24 , 2017 reagent excess. The mixture is incubated for 2 h in the dark biological activity according to methods known in the art, at room temperature under gentle stirring and quenched for and determination of the polysialyation degree by measuring 15 min at room temperature by the addition of 100 ul of 1 the PSA content ( resorcinol assay ) . M aqueous cysteine solution . [0478 ] The free interferon gamma is removed by means of Example 28 cation exchange chromatography (CEC ) . The reaction mix ture is diluted with 20 ml Buffer A (50 mM Hepes , pH 6 . 5 ) Polysialylation of G -CSF Using Aminooxy -PSA and loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE and m - Toluidine as a Nucleophilic Catalyst Healthcare , Fairfield , Conn . ) pre -equilibrated with Buffer A . Then the column is eluted with Buffer B (50 mM Hepes , 1 Method 1: M NaCl, pH 6 . 5 ) . Free interferon - gamma is eluted by [0484 ] A starting concentration of granulocyte -colony washing the column with 25 % Buffer B and the conjugate at stimulating factor (G - CSF) is transferred into a reaction 50 % Buffer B . The conductivity of the conjugate containing buffer ( e . g . , 50 mM Hepes , 350 mM sodium chloride , 5 mM fractions is subsequently raised to - 190 mS/ cm with Buffer calcium chloride , pH 6 .0 ) and diluted to obtain a protein C (50 mM Hepes, 5 M NaCl, pH 6 . 9 ) and loaded onto a 20 concentration of 1 mg/ ml . To this solution , NalO4 is added ml HiPrep Butyl FF 16 / 10 column (GE Healthcare , Fairfield , to give a final concentration of 200 uM . The oxidation is Conn . ) pre - equilibrated with Buffer D (50 mM Hepes , 3 M carried at RT for 30 min in the dark under gentle shaking. NaCl, pH 6 . 9 ) . Free PSA - reagent is washed out within 5 CV The reaction is then quenched with cysteine ( final concen Buffer D . Subsequently the conjugate is eluted with 100 % tration : 10 mM ) for 60 min at RT. Buffer E (50 mM Hepes , pH 6 .9 ) . The conjugate containing [ 0485 ] The solution is next subjected to UF /DF employing fractions are concentrated by UF /DF using a 10 kD mem Vivaspin centrifugal filtrators to remove excess periodate , brane made of regenerated cellulose ( 88 cm2 , cut - off 10 quencher and the byproducts thereof or, in the alternative , to kD /Millipore ). The final diafiltration step is performed an IEX column with a volume of 20 ml (Merck EMD TMAE against histidine buffer, pH 6 . 9 containing 150 mM NaCl. ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes, 5 The preparation is analytically characterized by measuring mM CaCl2 , pH 7 . 0 ) . The column is equilibrated with 5 CV total protein (Bradford ) and biological activity according to Buffer A . The oxidized G -CSF is eluted with Buffer B ( 20 methods known in the art . For the PSAinterferon - gamma mM Hepes, 5 mM CaCl2 , 1 M NaCl, pH 7. 0 ). The G - CSF conjugate a specific activity of > 50 % in comparison to containing fractions are collected . The protein content is native interferon - gamma is determined . The conjugate is determined (Coomassie , Bradford ) and adjusted to 1 mg/ ml additionally analytically characterized by Size Exclusion with reaction buffer and adjusted to pH 6 . 0 by dropwise HPLC using a Agilent 1200 HPLC system equipped with a addition of 0 . 5 M HCl. Shodex KW 803 column under conditions as previously [0486 ] A 50 - fold molar excess of aminooxy - PSA reagent described (Kolarich et al, Transfusion 2006 ; 46 : 1959 -77 ). It with a MW of 20 kD ( described above ) is added followed by is shown that the preparation contains no free interferon m -toluidine as a nucleophilic catalyst ( final concentration : gamma. 10 mM ) . The coupling reaction is performed for 2 hours in the dark under gentle shaking at room temperature . The Method 4 : excess of aminooxy reagent is removed by means of HIC . [ 0479 ] Interferon - gamma is dissolved in or transferred to The conductivity of the reaction mixture is adjusted by a reaction buffer ( e . g . 50 mM Hepes, 350 mM sodium adding a buffer containing ammonium acetate (50 mM chloride, 5 mM calcium chloride , pH 6 . 0 ) to get a final Hepes , 350 mM sodium chloride, 5 mM calcium chloride, 8 protein concentration of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of M ammonium acetate , pH 6 . 9 ) and loaded onto a column the solution is corrected to 6 . 0 by drop wise addition of a 0 . 5 filled with 80 ml Phenyl Sepharose FF (GE Healthcare , N aqueous HC1 solution . Fairfield , Conn . ) pre -equilibrated with 50 mM Hepes, 2 . 5 M [0480 ] Subsequently , the aminooxy - polysialic acid (PSA ammonium acetate , 350 mM sodium chloride , 5 mM cal ONH2) reagent is added in a 50 - fold molar excess to this cium chloride , pH 6 . 9 . Subsequently , the conjugate is eluted interferon - gamma solution within a maximum time period with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . ( t ) of 15 minutes under gentle stirring . Then an aqueous Finally the PSA - G - CSF - containing fractions are collected m - toluidine solution (50 mM ) is added within 15 minutes to and subjected to UF /DF by use of a membrane made of get a final concentration of 10 mM . Finally , a 40 mm regenerated cellulose (Millipore ) . The preparation is next aqueous sodium periodate solution is added to give a con - analytically characterized by measuring total protein (Coo centration of 400 uM . massie , Bradford ) and biological activity according to meth [0481 ] The reaction mixture is incubated for 120 + /- 10 ods known in the art . min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under [0487 ] In an alternative embodiment, Method 1 is carried gentle shaking . Then the reaction is stopped by the addition out as follows. Granulocyte - colony stimulating factor of an aqueous L - cysteine solution ( 1 M ) to give a final ( G - CSF ) is transferred into a reaction buffer ( e . g . , 50 mM concentration of 10 mM in the reaction mixture and incu Hepes , 350 mM sodium chloride , 5 mM calcium chloride, bation for 60 + / - 5 min . pH 6 . 0 ) and diluted to obtain a protein concentration of 1 [ 0482] The obtained interferon - gamma conjugate is puri mg/ ml . To this solution , NalO4 is added to give a final fied by ion - exchange chromatography . The PSA - interferon concentration of 200 uM . The oxidation is carried at RT for gamma containing fractions of the eluate are collected and 30 min in the dark under gentle shaking . The reaction is then concentrated by ultra - /diafiltration (UF /DF ) using a mem quenched with cysteine ( final concentration : 10 mM ) for 60 brane made of regenerated cellulose (Millipore ). min at RT. [ 0483] The conjugates prepared by use of this procedure [0488 ] The solution is next subjected to UF /DF employing are analytically characterized by measuring total protein , Vivaspin centrifugal filtrators to remove excess periodate , US 2017 /0240590 A1 Aug . 24 , 2017 63

quencher and the byproducts thereof. A 50 - fold molar excess quenched with cysteine for 60 min at RT ( cysteine concen of aminooxy - PSA reagent with a MW of 20 kD ( described tration : 10 mM ) . Then the conductivity of the reaction above ) is added followed by m - toluidine as a nucleophilic mixture is adjusted by adding a buffer containing ammo catalyst ( final concentration : 10 mM ) . The coupling reaction nium acetate ( 50 mM Hepes, 350 mM sodium chloride , 5 is performed for 2 hours in the dark under gentle shaking at mM calcium chloride , 8 M ammonium acetate , pH 6 . 9 ) and room temperature . The excess of aminooxy reagent is loaded onto a column filled with Phenyl Sepharose FF (GE removed by means of ion exchange chromatography. The Healthcare , Fairfield , Conn . ) pre - equilibrated with 50 mM PSA - G - CSF containing fractions of the eluate are collected Hepes , 2 . 5 M ammonium acetate , 350 mM sodium chloride , and subjected to UF /DF by use of a membrane made of 5 mM calcium chloride, 0 .01 % Tween 80 , pH 6 . 9 . Subse regenerated cellulose (Millipore ) . The preparation is next quently the conjugate is eluted with 50 mM Hepes , 5 mM analytically characterized by measuring total protein (Coo calcium chloride , pH 7 . 5 . Finally, the PSA - G - CSF - contain massie , Bradford ) and biological activity according to meth ing fractions are collected and subjected to UF /DF by use of ods known in the art. a membrane made of regenerated cellulose (Millipore ) . The preparation is analytically characterized by measuring total Method 2 : protein (Bradford ) and biological activity according to meth ods known in the art . [0489 ] G - CSF is transferred or dissolved in reaction buffer [ 0495 ] In an alternative embodiment, Method 3 is carried ( e . g . 50 mM Hepes, 350 mM sodium chloride, 5 mM out as follows . Granulocyte - colony stimulating factor calcium chloride , pH 6 . 0 ) to get a final protein concentration ( G -CSF ) is transferred into reaction buffer ( e . g . 50 mM of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor Hepes , 350 mM sodium chloride , 5 mM calcium chloride, rected to 6 . 0 by drop wise addition of a 0 .5 N aqueous HC1 pH 6 .0 ) and diluted to obtain a protein concentration of 1 solution . Subsequently , a 40 mM aqueous sodium periodate mg/ml . A 50 - fold molar excess of aminooxy - PSA reagent solution is added within 10 minutes to give a concentration with a MW of 20 kD (described above ) is added followed by of 200 uM . The oxidation reaction is carried out for 30 + / - 5 m -toluidine as a nucleophilic catalyst ( 10 mM final concen min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction tration ) and NalO4 ( final concentration : 400 uM ) . The is stopped by addition of an aqueous L -cysteine solution (1 coupling reaction is performed for 2 hours in the dark under M ) within 15 minutes at T = + 22 + / - 2° C . to give a final gentle shaking at room temperature . Subsequently , the reac concentration of 10 mM in the reaction mixture and incu tion is quenched with cysteine for 60 min at RT ( cysteine bation for 60 + / - 5 min . concentration : 10 mM ) and the conjugate is purified by ion [0490 ] The oxidized G -CSF is further purified by ion exchange chromatography. The PSA - G - CSF containing exchange chromatography. The oxidized G -CSF containing fractions of the eluate are collected and subjected to UF /DF fractions of the eluate are collected and used for the conju by use of a membrane made of regenerated cellulose (Mil gation reaction . lipore ) . The preparation is analytically characterized by [0491 ] The aminooxy -polysialic acid (PSA - ONH2) measuring total protein (Bradford ) and biological activity reagent is added in a 50 - fold molar excess to the eluate according to methods known in the art . containing the purified oxidized G - CSF within a maximum time period ( t ) of 15 minutes under gentle stirring . Then an Method 4 : aqueous m - toluidine solution (50 mM ) is added within 15 minutes to get a final concentration of 10 mM . The reaction [0496 ] G - CSF is dissolved in or transferred to a reaction mixture is incubated for 120 + / – 10 min . at pH 6 . 0 in the dark buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride , 5 mM at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking calcium chloride , pH 6 . 0 ) to get a final protein concentration of 1 . 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is cor (protein concentration : 1 mg/ml ) . rected to 6 . 0 by drop wise addition of a 0 .5 N aqueous HCI [0492 ] The obtained PSA - G - CSF conjugate is further solution . purified by ion exchange chromatography . The PSA - G -CSF [0497 ] Subsequently , the aminooxy -polysialic acid (PSA conjugate containing fractions are collected and concen ONH2) reagent is added in a 50 - fold molar excess to this trated by ultra - /diafiltration (UF /DF ) using a membrane G - CSF solution within a maximum time period ( t ) of 15 made of regenerated cellulose with an appropriate molecular minutes under gentle stirring. Then an aqueous m - toluidine weight cut off (Millipore ) . solution (50 mm ) is added within 15 minutes to get a final [0493 ] The conjugate prepared by use of this procedure is concentration of 10 mM . Finally , a 40 mM aqueous sodium analytically characterized by measuring total protein , bio periodate solution is added to give a concentration of 400 logical activity , and determination of the polysialyation UM . degree by measuring the PSA content ( resorcinol assay ) . [0498 ] The reaction mixture is incubated for 120 + / - 10 min . in the dark at a temperature ( T ) of T = + 22 +/ - 2° C . under Method 3 : gentle shaking . Then the reaction is stopped by the addition [ 0494 ] Granulocyte -colony stimulating factor (G - CSF ) is of an aqueous L -cysteine solution (1 M ) to give a final transferred into reaction buffer ( 50 mM Hepes , 350 mm concentration of 10 mM in the reaction mixture and incu sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and bation for 60 + / - 5 min . diluted to obtain a protein concentration of 1 mg/ ml . A [04991 . The obtained G -CSF conjugate is purified by ion 50 - fold molar excess of aminooxy -PSA reagent with a MW exchange chromatography . The PSA - G - CSF containing of 20 kD (described above) is added followed by m - tolui fractions of the eluate are collected and concentrated by dine as a nucleophilic catalyst (10 mM final concentration ) ultra - /diafiltration (UF / DF ) using a membrane made of and NalO4 (final concentration : 400 uM ). The coupling regenerated cellulose (Millipore ) . reaction is performed for 2 hours in the dark under gentle (0500 ] The conjugates prepared by use of this procedure shaking at room temperature . Subsequently , the reaction is are analytically characterized by measuring total protein , US 2017 /0240590 A1 Aug . 24 , 2017 64 biological activity according to methods known in the art , above ) is added followed by m - toluidine as a nucleophilic and determination of the polysialyation degree by measuring catalyst ( final concentration : 10 mM ) . The coupling reaction the PSA content (resorcinol assay ) . is performed for 2 hours in the dark under gentle shaking at room temperature. The excess of aminooxy reagent is Example 29 removed by means of ion exchange chromatography The PSA -Humira containing fractions of the elutae are collected Polysialylation of Humira Using Aminooxy - PSA and subjected to UF /DF by use of a membrane made of and m - Toluidine as a Nucleophilic Catalyst regenerated cellulose (Millipore ) . The preparation is next analytically characterized by measuring total protein (Coo Method 1 : massie , Bradford ) and biological activity according to meth [0501 ] A starting concentration of Humira is transferred ods known in the art . into a reaction buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and diluted to Method 2 : obtain a protein concentration of 1 mg /ml . To this solution , [0506 ] Humira is transferred or dissolved in reaction buf NalO4 is added to give a final concentration of 200 uM . The fer ( e .g . 50 mM Hepes, 350 mM sodium chloride, 5 mM oxidation is carried at RT for 30 min in the dark under gentle calcium chloride , pH 6 . 0 ) to get a final protein concentration shaking. The reaction is then quenched with cysteine ( final of 1. 0 + / - 0 . 25 mg/ ml . Then the pH of the solution is cor concentration : 10 mM ) for 60 min at RT . rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 [0502 ] The solution is next subjected to UF /DF employing solution . Subsequently , a 40 mM aqueous sodium periodate Vivaspin centrifugal filtrators to remove excess periodate , solution is added within 10 minutes to give a concentration quencher and the byproducts thereof or, in the alternative , to of 200 uM . The oxidation reaction is carried out for 30 + / - 5 an IEX column with a volume of 20 ml (Merck EMD TMAE min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction ( M ) ) which is equilibrated with Buffer A ( 20 mM Hepes, 5 is stopped by addition of an aqueous L - cysteine solution ( 1 mM CaCl2 , pH 7 .0 ) . The column is equilibrated with 5 CV M ) within 15 minutes at T = + 22 + / - 2° C . to give a final Buffer A . The oxidized Humira is eluted with Buffer B ( 20 concentration of 10 mM in the reaction mixture and incu mM Hepes , 5 mM CaC12 , 1M NaCl, pH 7 . 0 ) . The Humira bation for 60 + / - 5 min . containing fractions are collected . The protein content is [ 0507] The oxidized Humira is further purified by ion determined (Coomassie , Bradford ) and adjusted to 1 mg/ml exchange chromatography. The oxidized Humira containing with reaction buffer and adjusted to pH 6 .0 by dropwise fractions of the eluate are collected and used for the conju addition of 0 .5M HC1. gation reaction . ( 0503 A 50 - fold molar excess of aminooxy - PSA reagent [ 0508 ] The aminooxy - polysialic acid (PSA - ONH2) with a MW of 20 kD (described above ) is added followed by reagent is added in a 50 - fold molar excess to the eluate m - toluidine as a nucleophilic catalyst ( final concentration : containing the purified oxidized Humira within a maximum 10 mM ) . The coupling reaction is performed for 2 hours in time period ( t) of 15 minutes under gentle stirring . Then an the dark under gentle shaking at room temperature . The aqueous m - toluidine solution ( 50 mM ) is added within 15 excess of aminooxy reagent is removed by means of HIC . minutes to get a final concentration of 10 mM . The reaction The conductivity of the reaction mixture is adjusted by mixture is incubated for 120 + / – 10 min . at pH 6 . 0 in the dark adding a buffer containing ammonium acetate (50 mM at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking Hepes, 350 mM sodium chloride , 5 mM calcium chloride , 8 (protein concentration : 1 mg/ ml ) . M ammonium acetate , pH 6 . 9 ) and loaded onto a column [ 0509 ] The obtained PSA -Humira conjugate is further filled with 80 ml Phenyl Sepharose FF (GE Healthcare , purified by ion exchange chromatography. The PSA -Humira Fairfield , Conn . ) pre - equilibrated with 50 mM Hepes, 2 . 5 M conjugate containing fractions are collected and concen ammonium acetate , 350 mM sodium chloride , 5 mM cal cium chloride , pH 6 . 9 . Subsequently the conjugate is eluted trated by ultra - /diafiltration (UF /DF ) using a membrane with 50 mM Hepes buffer pH 7 . 5 containing 5 mM CaC12 . made of regenerated cellulose with an appropriate molecular Finally , the PSA - Humira containing fractions are collected weight cut off (Millipore ). and subjected to UF /DF by use of a membrane made of [0510 ] The conjugate prepared by use of this procedure is regenerated cellulose (Millipore ) . The preparation is next analytically characterized by measuring total protein , bio analytically characterized by measuring total protein (Coo logical activity , and determination of the polysialyation massie , Bradford ) and biological activity according to meth degree by measuring the PSA content ( resorcinol assay ). ods known in the art. [0504 ] In an alternative embodiment, Method 1 is carried Method 3 : out as follows. Humira is transferred into a reaction buffer [0511 ] Humira is transferred into reaction buffer (50 mM ( e .g . 50 mM Hepes, 350 mM sodium chloride, 5 mM Hepes , 350 mM sodium chloride, 5 mM calcium chloride , calcium chloride , pH 6 . 0 ) and diluted to obtain a protein pH 6 . 0 ) and diluted to obtain a protein concentration of 1 concentration of 1 mg/ ml . To this solution , NalO4 is added mg/ ml . A 50 - fold molar excess of aminooxy - PSA reagent to give a final concentration of 200 UM . The oxidation is with a MW of 20 kD (described above ) is added followed by carried at RT for 30 min in the dark under gentle shaking . m - toluidine as a nucleophilic catalyst ( 10 mM final concen The reaction is then quenched with cysteine ( final concen tration ) and NalO4 ( final concentration : 400 uM ) . The tration : 10 mM ) for 60 min at RT. coupling reaction is performed for 2 hours in the dark under (0505 ] The solution is next subjected to UF/ DF employing gentle shaking at room temperature . Subsequently , the reac Vivaspin centrifugal filtrators to remove excess periodate , tion is quenched with cysteine for 60 min at RT ( cysteine quencher and the byproducts thereof. A 50 - fold molar excess concentration : 10 mM ) . Then the conductivity of the reac of aminooxy -PSA reagent with a MW of 20 kD (described tion mixture is adjusted by adding a buffer containing US 2017 /0240590 A1 Aug . 24 , 2017 65 ammonium acetate (50 mM Hepes , 350 mM sodium chlo Example 30 ride , 5 mM calcium chloride , 8 M ammonium acetate , pH 6 . 9 ) and loaded onto a column filled with Phenyl Sepharose Polysialylation of Prolia Using Aminooxy -PSA and FF (GE Healthcare, Fairfield , Conn . ) pre - equilibrated with m - Toluidine as a Nucleophilic Catalyst 50 mM Hepes, 2 . 5 M ammonium acetate , 350 mM sodium chloride, 5 mM calcium chloride , 0 .01 % Tween 80 , pH 6 . 9 . Subsequently the conjugate is eluted with 50 mM Hepes , 5 Method 1: mM calcium chloride , pH 7 .5 . Finally the PSA -Humira [0518 ] A starting concentration of Prolia is transferred into containing fractions are collected and subjected to UF /DF by a reaction buffer ( e . g . 50 mM Hepes , 350 mM sodium use of a membrane made of regenerated cellulose (Milli chloride, 5 mM calcium chloride, pH 6 . 0 ) and diluted to pore ). The preparation is analytically characterized by mea obtain a protein concentration of 1 mg/ ml . To this solution , suring total protein (Bradford ) and biological activity NalO4 is added to give a final concentration of 200 uM . The according to methods known in the art. oxidation is carried at RT for 30 min in the dark under gentle [ 0512 ] In an alternative embodiment, Method 3 is carried shaking . The reaction is then quenched with cysteine ( final out as follows. Humira is transferred into reaction buffer concentration : 10 mM ) for 60 min at RT. ( e . g . 50 mM Hepes , 350 mM sodium chloride , 5 mM [ 0519 ] The solution is next subjected to UF /DF employing calcium chloride , pH 6 . 0 ) and diluted to obtain a protein Vivaspin centrifugal filtrators to remove excess periodate , concentration of 1 mg/ ml . A 50 -fold molar excess of ami quencher and the byproducts thereof or, in the alternative , to nooxy - PSA reagent with a MW of 20 kD (described above ) an IEX column with a volume of 20 ml (Merck EMD TMAE is added followed by m - toluidine as a nucleophilic catalyst ( M )) which is equilibrated with Buffer A (20 mM Hepes, 5 ( 10 mM final concentration ) and NaI04 ( final concentration : 400 uM ). The coupling reaction is performed for 2 hours in mM CaC12 , pH 7 . 0 ) . The column is equilibrated with 5 CV the dark under gentle shaking at room temperature . Subse Buffer A . The oxidized Prolia is eluted with Buffer B ( 20 quently , the reaction is quenched with cysteine for 60 min at mM Hepes, 5 mM CaCl2 , 1M NaCl, pH 7 . 0 ). The Prolia RT ( cysteine concentration : 10 mM ) and the conjugate is containing fractions are collected . The protein content is purified by ion exchange chromatography. The PSA -Humira determined (Coomassie , Bradford ) and adjusted to 1 mg/ ml containing fractions of the eluate are collected and subjected with reaction buffer and adjusted to pH 6 .0 by dropwise to UF /DF by use of a membrane made of regenerated addition of 0 . 5 M HCl. cellulose (Millipore ) . The preparation is analytically char [0520 ] A 50 - fold molar excess of aminooxy -PSA reagent acterized by measuring total protein (Bradford ) and biologi with a MW of 20 kD ( described above ) is added followed by cal activity according to methods known in the art . m -toluidine as a nucleophilic catalyst ( final concentration : 10 mM ) . The coupling reaction is performed for 2 hours in the dark under gentle shaking at room temperature . The Method 4 : excess of aminooxy reagent is removed by means of HIC . [0513 ] Humira is dissolved in or transferred to a reaction 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 . Fairfield , Conn . ) pre - equilibrated with 50 mM Hepes, 2 . 5 M [0514 ] Subsequently , the aminooxy - polysialic acid (PSA ammonium acetate , 350 mM sodium chloride , 5 mM cal ONH2) reagent is added in a 50 - fold molar excess to this cium chloride, pH 6 . 9 . Subsequently the conjugate is eluted Humira 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 -Prolia containing fractions are collected solution ( 50 mM ) is added within 15 minutes to get a final 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 [0515 ] The reaction mixture is incubated for 120 + / – 10 ods known in the art . min . in the dark at a temperature ( T ) ofT = + 22 + / - 2° C . under [0521 ] In an alternative embodiment, Method 1 is carried gentle shaking . Then the reaction is stopped by the addition out as follows. 10 mg Prolia is dissolved in 5 ml histidine of an aqueous L - cysteine solution ( 1 M ) to give a final buffer , pH 6 . 0 (20 mM L -histidine , 150 mM NaCl) . 100 ul concentration of 10 mM in the reaction mixture and incu of an aqueous sodium periodate solution ( 5 mm ) is then bation for 60 + / - 5 min . added and the reaction mixture is incubated for 1 h in the [0516 ] The obtained Humira -conjugate is purified by ion dark at 4° C . under gentle stirring and quenched for 15 min exchange chromatography. The PSA -Humira containing at room temperature by the addition of 50 ul of a 1 M fractions of the eluate are collected and concentrated by aqueous cysteine solution . The mixture is subsequently ultra - / diafiltration (UF /DF ) using a membrane made of subjected to UF /DF employing Vivaspin 15R 10 kD cen regenerated cellulose (Millipore ) . trifugal filtrators to remove excess periodate , quencher and [ 0517 ] The conjugates prepared by use of this procedure the byproducts thereof. are analytically characterized by measuring total protein , [ 0522 ] The retentate ( approx . 7 ml) , containing oxidized biological activity according to methods known in the art , Prolia , is mixed with 2 ml of an aqueous m - toluidine and determination of the polysialyation degree by measuring solution ( 50 mM ) and incubated for 30 min at room tem the PSA content ( resorcinol assay ) . perature . Then aminooxy -PSA reagent with a MW of 20 kD US 2017 /0240590 A1 Aug . 24 , 2017

(described above ) is added to give a 5 - fold molar reagent diafiltration (UF /DF ) using a membrane made of regener excess . This mixture is incubated for 2 . 5 h at RT in the dark ated cellulose with an appropriate molecular weight cut off under gentle stirring . (Millipore ) . [0523 ] The free Prolia is removed by means of cation [ 0528 ] The conjugate prepared by use of this procedure is exchange chromatography (CEC ) . The reaction mixture is analytically characterized by measuring total protein , bio diluted with 20 ml Buffer A (50 mM Hepes , pH 6 . 5 ) and logical activity , and determination of the polysialyation loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE degree by measuring the PSA content ( resorcinol assay ) . Healthcare , Fairfield , Conn . ) pre - equilibrated with Buffer A . Then the column is eluted with Buffer B ( 50 mM Hepes , 1 Method 3 : M NaCl, pH 6 . 5 ). Free Prolia is eluted by washing the [0529 ] Prolia is transferred into reaction buffer ( 50 mM column with 25 % Buffer B and the conjugate at 50 % Buffer Hepes, 350 mM sodium chloride , 5 mM calcium chloride, B . The conductivity of the conjugate containing fractions is pH 6 . 0 ) and diluted to obtain a protein concentration of 1 subsequently raised to ~ 190 mS/ cm with Buffer C (50 mM mg/ ml . A 50 - fold molar excess of aminooxy - PSA reagent Hepes, 5 M NaCl, pH 6 . 9 ) and loaded onto a 20 ml HiPrep with a MW of 20 kD ( described above ) is added followed by Butyl FF 16 / 10 column (GE Healthcare , Fairfield , Conn . ) m - toluidine as a nucleophilic catalyst ( 10 mM final concen pre -equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, tration ) and NalO . ( final concentration : 400 uM ) . The cou pH 6 . 9 ) . Free PSA - reagent is washed out within 5 CV Buffer pling reaction is performed for 2 hours in the dark under D . Subsequently , the conjugate is eluted with 100 % Buffer gentle shaking at room temperature . Subsequently , the reac E ( 50 mM Hepes , pH 6 . 9 ). The conjugate containing frac tion is quenched with cysteine for 60 min at RT ( cysteine tions are concentrated by UF /DF using a 10 kD membrane concentration : 10 mM ). Then the conductivity of the reac made of regenerated cellulose ( 88 cm2, cut -off 10 kD , tion mixture is adjusted by adding a buffer containing Millipore ) . The final diafiltration step is performed against ammonium acetate (50 mM Hepes, 350 mM sodium chlo histidine buffer, pH 6 . 9 containing 150 ml NaCl. The ride , 5 mM calcium chloride, 8 M ammonium acetate , pH preparation is analytically characterized by measuring total 6 . 9 ) and loaded onto a column filled with Phenyl Sepharose protein (Bradford ) and biological activity according to meth FF (GE Healthcare , Fairfield , Conn .) pre - equilibrated with ods known in the art . For the PSA -Prolia conjugate a specific 50 mM Hepes , 2 . 5 M ammonium acetate , 350 mM sodium activity of > 50 % in comparison to native Prolia is deter chloride , 5 mM calcium chloride , 0 .01 % Tween 80 , pH 6 . 9 . mined . The conjugate is additionally analytically character Subsequently the conjugate is eluted with 50 mM Hepes , 5 ized by Size Exclusion HPLC using a Agilent 1200 HPLC mM calcium chloride, pH 7 .5 . Finally the PSA Prolia system equipped with a Shodex KW 803 column under containing fractions are collected and subjected to UF /DF by conditions as previously described (Kolarich et al , Transfu use of a membrane made of regenerated cellulose (Milli sion 2006 ; 46 : 1959 - 77 ) . It is shown that the preparation pore ). The preparation is analytically characterized by mea contains no free Prolia . suring total protein (Bradford ) and biological activity according to methods known in the art . Method 2 : [0530 ] In an alternative embodiment, Method 3 is carried 10524 ] Prolia is transferred or dissolved in reaction buffer out as follows. 10 mg Prolia is dissolved in 8 ml histidine (e .g . 50 mM Hepes, 350 mM sodium chloride, 5 mM buffer , pH 6 . 0 ( 20 mM L - histidine , 150 mM NaCl) . 200 ul calcium chloride , pH 6 . 0 ) to get a final protein concentration of an aqueous sodium periodate solution (5 mM ) and 2 ml of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor of an aqueous m - toluidine solution (50 mM ) are then added . rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 Subsequently the aminooxy - PSA reagent with a MW of 20 solution . Subsequently , a 40 mM aqueous sodium periodate kD (described above ) is added to give a 5 fold molar reagent solution is added within 10 minutes to give a concentration excess . The mixture is incubated for 2 h in the dark at room of 200 uM . The oxidation reaction is carried out for 30 + / - 5 temperature under gentle stirring and quenched for 15 min min at a temperature ( T ) ofT = + 22 + / - 2° C . Then the reaction at room temperature by the addition of 100 ul of 1 M is stopped by addition of an aqueous L - cysteine solution ( 1 aqueous cysteine solution . M ) within 15 minutes at T = + 22 + / - 2° C . to give a final 10531] The free Prolia is removed by means of cation concentration of 10 mM in the reaction mixture and incu exchange chromatography (CEC ). The reaction mixture is bation for 60 + / – 5 min . diluted with 20 ml Buffer A (50 mM Hepes, pH 6 . 5 ) and [ 0525 ] The oxidized Prolia is further purified by ion loaded onto a 20 ml HiPrep SPFF 16 / 10 column (GE exchange chromatography. The oxidized Prolia containing Healthcare , Fairfield , Conn . ) pre - equilibrated with Buffer A . fractions of the eluate are collected and used for the conju Then the column is eluted with Buffer B (50 mM Hepes , 1 gation reaction . M NaCl, pH 6 . 5 ) . Free Prolia is eluted by washing the [ 0526 ] The aminooxy - polysialic acid (PSA - ONH2) column with 25 % Buffer B and the conjugate at 50 % Buffer reagent is added in a 50 - fold molar excess to the eluate B . The conductivity of the conjugate containing fractions is containing the purified oxidized Prolia within a maximum subsequently raised to - 190 mS/ cm with Buffer C (50 mM time period ( t ) of 15 minutes under gentle stirring . Then an Hepes , 5 M NaC1, pH 6 . 9 ) and loaded onto a 20 ml HiPrep aqueous m -toluidine solution (50 mM ) is added within 15 Butyl FF 16 / 10 column (GE Healthcare, Fairfield , Conn . ) minutes to get a final concentration of 10 mM . The reaction pre - equilibrated with Buffer D ( 50 mM Hepes , 3 M NaCl, mixture is incubated for 120 + / - 10 min . at pH 6 . 0 in the dark pH 6 . 9 ). Free PSA -reagent is washed out within 5 CV Buffer at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking D . Subsequently the conjugate is eluted with 100 % Buffer E (protein concentration : 1 mg/ml ) . (50 mM Hepes , pH 6 . 9 ) . The conjugate containing fractions [ 0527 ] The obtained Prolia conjugate is further purified by are concentrated by UF /DF using a 10 kD membrane made ion exchange chromatography . The Prolia conjugate con of regenerated cellulose (88 cm², cut -off 10 kD Millipore/ ) . taining fractions are collected and concentrated by ultra- / The final diafiltration step is performed against histidine US 2017 /0240590 A1 Aug . 24 , 2017 buffer, pH 6 .9 containing 150 mM NaCl. The preparation is group . An example of this type of reagent is the Sunbright® analytically characterized by measuring total protein ( Brad CA series from NOF (NOF Corp ., Tokyo , Japan ) . EPO is ford ) and biological activity according to methods known in dissolved in 7 . 0 mlhistidine buffer, pH 6 . 0 ( 20 mM L -his the art . For the PSA -Prolia conjugate a specific activity of tidine, 150 mM NaCl, 5 mM CaCl2 ). An aqueous sodium > 50 % in comparison to native Prolia is determined . The periodate solution ( 5 mM ) is then added and the reaction conjugate is additionally analytically characterized by Size mixture is incubated for 1 h in the dark at 4° C . under gentle Exclusion HPLC using a Agilent 1200 HPLC system stirring and quenched for 15 min at room temperature by the equipped with a Shodex KW 803 column under conditions addition of 7 . 5 al of a 1 M aqueous cysteine solution . The as previously described (Kolarich et al, Transfusion 2006 ; mixture is subsequently subjected to UF /DF employing 46 : 1959 - 77 ) . It is shown that the preparation contains no Vivaspin centrifugal filtrators to remove excess periodate , free Prolia . quencher and the byproducts thereof . [0539 ] The retentate containing oxidized EPO is next Method 4 : mixed with an aqueous m - toluidine solution (50 mM ) and [0532 ] Prolia is dissolved in or transferred to a reaction incubated for 30 min at room temperature . aminooxy -PEG buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride , 5 mM reagent with a MW of 20 kD is then added to give a 5 - fold calcium chloride , pH 6 . 0 ) to get a final protein concentration molar reagent excess . This mixture is incubated for 2 . 5 h at of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor room temperature in the dark under gentle stirring . rected to 6 . 0 by drop wise addition of a 0 .5 N aqueous HC1 [0540 ] Finally , the PEG - EPO conjugate is purified by solution . ion -exchange chromatography ( e . g . on Q Sepharose FF ) . [0533 ] Subsequently the aminooxy -polysialic acid (PSA For example , 1. 5 mg protein /ml gel is loaded on the column ONH , ) reagent is added in a 50 - fold molar excess to this equilibrated with 50 mM Hepes buffer, pH 7 . 4 containing 5 Prolia — solution within a maximum time period ( t ) of 15 mM CaCl2 . The conjugate is eluted with 50 mM Hepes minutes under gentle stirring . Then an aqueous m -toluidine buffer containing 5 mM CaCl2 and 500 mM sodium chlo solution (50 mM ) is added within 15 minutes to get a final ride , pH 7 . 4 and is then subjected to UF /DF using an concentration of 10 mM . Finally a 40 mM aqueous sodium appropriate MW cutoff membrane. 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 [0534 ] The reaction mixture is incubated for 120 + / - 10 ods known in the art . min . in the dark at a temperature ( T ) of T = + 22 + / - 2° C . under 0541] In an alternative embodiment, Method 1 is carried gentle shaking . Then the reaction is stopped by the addition out as follows. EPO is PEGylated by use of a linear 20 kD of an aqueous L -cysteine solution ( 1 M ) to give a final PEGylation reagent containing an aminooxy group . An concentration of 10 mM in the reaction mixture and incu example of this type of reagent is the Sunbright® CA series bation for 60 + / – 5 min . from NOF (NOF Corp ., Tokyo , Japan ). 10 mg EPO is [0535 ] The obtained Prolia conjugate is purified by ion dissolved in 5 ml histidine buffer, pH 6 . 0 (20 mM L -histi exchange chromatography. The PSA -Prolia containing frac dine , 150 mM NaCl) . 100 ul of an aqueous sodium periodate tions of the eluate are collected and concentrated by ultra - / solution (5 mM ) is then added and the reaction mixture is diafiltration (UF /DF ) using a membrane made of incubated for 1 h in the dark at 4° C . under gentle stirring regenerated cellulose (Millipore ). and quenched for 15 min at room temperature by the [ 0536 ] The conjugates prepared by use of this procedure addition of 50 ul of a 1 M aqueous cysteine solution . The are analytically characterized by measuring total protein , mixture is subsequently subjected to UF /DF employing biological activity according to methods known in the art , Vivaspin 15R 10 kD centrifugal filtrators to remove excess and determination of the polysialyation degree by measuring periodate, quencher and the byproducts thereof. the PSA content ( resorcinol assay ) . 10542 ]. The retentate (approx . 7 ml) , containing oxidized EPO , is mixed with 2 ml of an aqueous m -toluidine solution Example 31 (50 mM ) and incubated for 30 min at room temperature . Polysialylation of Other Therapeutic Proteins Then aminooxy - PEG reagent with a MW of 20 kD (de scribed above ) is added to give a 5 - fold molar reagent [0537 ] Polysialylation reactions performed in the presence excess . This mixture is incubated for 2 . 5 h at RT in the dark of alternative nucleophilic catalysts like m -toluidine or under gentle stirring . 0 - aminobenzoic acid as described herein may be extended to [0543 ] Finally , the PEG -EPO conjugate is purified by other therapeutic proteins. For example , in various aspects ion - exchange chromatography on Sepharose FF . The of the invention , the above polysialylation or PEGylation reaction mixture is diluted with 20 ml Buffer A ( 50 mM reactions as described herein with PSA aminooxy or PEG Hepes , pH 7 . 5 ) and loaded onto a 20 ml HiPrep QFF 16 / 10 aminooxy reagents is repeated with therapeutic proteins column (GE Healthcare, Fairfield , Conn . ) pre - equilibrated such as those proteins described herein . with Buffer A . Then the column is eluted with Buffer B ( 50 Example 32 mM Hepes , 1 M NaCl, pH 7 . 5 ) . Free EPO is eluted by washing the column with 25 % Buffer B and the conjugate at PEGylation of EPO Using an Aminooxy -PEG 50 % Buffer B . The conjugate containing fractions are con Reagent and m - Toluidine as a Nucleophilic centrated by UF /DF using a 10 kD membrane made of Catalyst regenerated cellulose (88 cm2, cutoff 10 kD /Millipore ) . The final diafiltration step is performed against histidine buffer, Method 1: pH 7 . 2 containing 150 mM NaCl. The preparation is ana [ 0538 ] Erythropoietin ( EPO ) is PEGylated by use of a lytically characterized by measuring total protein (Bradford ) linear 20 kD PEGylation reagent containing an aminooxy and biological activity biological activity according to meth US 2017 /0240590 A1 Aug . 24 , 2017 ods known in the art. For the PEG -EPO conjugate a specific [0551 ] Finally , the PEG - EPO conjugate is purified by activity of > 50 % in comparison to native EPO is determined . ion - exchange chromatography on Q Sepharose FF . 1 . 5 mg The conjugate is additionally analytically characterized by protein /ml gel is loaded on the column pre equilibrated with Size Exclusion HPLC using a Agilent 1200 HPLC system 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The equipped with a Shodex KW 803 column under conditions conjugate is eluted with 50 mM Hepes buffer containing 5 as previously described (Kolarich et al, Transfusion 2006 ; mM CaCl2 and 500 mM sodium chloride , pH 7 . 4 and is then 46 : 1959 - 77 ) . It is shown that the preparation contains no subjected to UF /DF using a membrane . The preparation is free EPO . analytically characterized by measuring total protein (Brad ford ) and biological activity according to methods known in Method 2 : the art . 10552 ] In an alternative embodiment, Method 3 is carried [0544 ] EPO is PEGylated by use of a linear 20 kD out as follows . EPO is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An 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 ). from NOF (NOF Corp . , Tokyo , Japan ) . 10 mg EPO is [0545 ] EPO is transferred or dissolved in reaction buffer dissolved in ~ 8 ml histidine buffer , pH 6 . 0 (20 mM L -his ( e .g . 50 mM Hepes, 350 mM sodium chloride, 5 mM tidine , 150 mM NaCl) . 200 ul of an aqueous sodium calcium chloride , pH 6 . 0 ) to get a final protein concentration periodate solution ( 5 mM ) and 2 ml of an aqueous m - tolui of 1 . 0 + / - 0 .25 mg/ ml . Then the pH of the solution is cor dine solution (50 mm ) are then added . Subsequently , the rected to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 aminooxy -PEG reagent with a MW of 20 kD (described solution . Subsequently a 40 mM aqueous sodium periodate above ) is added to give a 5 - fold molar reagent excess . The solution is added within 10 minutes to give a concentration mixture is incubated for 2 h in the dark at room temperature of 200 uM . The oxidation reaction is carried out for 30 + / - 5 under gentle stirring and quenched for 15 min at room min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction temperature by the addition of 100 ul of 1 M aqueous is stopped by addition of an aqueous L -cysteine solution (1 cysteine solution . M ) within 15 minutes at T = + 22 + / - 2° C . to give a final [0553 ] Finally , the PEG - EPO conjugate is purified by concentration of 10 mM in the reaction mixture and incu ion -exchange chromatography on Q Sepharose FF . The bation for 60 + / - 5 min . reaction mixture is diluted with 20 ml Buffer A (50 mM [0546 ] The oxidized EPO is further purified by ion Hepes , pH 7 . 5 ) and loaded onto a 20 mlHiPrep QFF 16 / 10 exchange chromatography. The oxidized EPO containing column (GE Healthcare , Fairfield , Conn . ) pre - equilibrated fractions of the eluate are collected and used for the conju with Buffer A . Then the column is eluted with Buffer B (50 gation reaction . mM Hepes , 1 M NaCl, pH 7. 5 ). Free EPO is eluted by [ 0547 ] The aminooxy -PEG reagent with a MW of 20 kD washing the column with 25 % Buffer B and the conjugate at reagent is added in a 50 - fold molar excess to the eluate 50 % Buffer B . The conjugate containing fractions are con containing the purified oxidized EPO within a maximum centrated by UF /DF using a 10 kD membrane made of time period ( t ) of 15 minutes under gentle stirring . Then an regenerated cellulose ( 88 cm2, cut -off 10 kD /Millipore ) . The aqueous m - toluidine solution (50 mM ) is added within 15 final diafiltration step is performed against histidine buffer , minutes to get a final concentration of 10 mM . The reaction pH 7 . 2 containing 150 mM NaCl. The preparation is ana mixture is incubated for 120 + / – 10 min . in the dark at a lytically characterized by measuring total protein (Bradford ) temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . and biological activity according to methods known in the [0548 ] The obtained PEG -EPO conjugate is further puri art . For the PEG -EPO conjugate a specific activity of > 50 % fied by ion exchange chromatography. The PEG - EPO con in comparison to native EPO is determined . The conjugate jugate containing fractions are collected and concentrated by is additionally analytically characterized by Size Exclusion ultra - / diafiltration (UF /DF ) using a membrane made of HPLC using a Agilent 1200 HPLC system equipped with a regenerated cellulose with an appropriate molecular weight Shodex KW 803 column under conditions as previously cut off (Millipore ). described (Kolarich et al, Transfusion 2006 ; 46 : 1959 -77 ) . It [0549 ] The conjugate prepared by use of this procedure is shown that the preparation contains no free EPO . are analytically characterized by measuring total protein and biological activity according to methods known in the art . Method 4 : [0554 ] EPO is PEGylated by use of a linear 20 kD Method 3 : PEGylation reagent containing an aminooxy group . An [0550 ] EPO is PEGylated by use of a linear 20 kD example of this type of reagent is the Sunbright® CA series PEGylation reagent containing an aminooxy group . An from NOF (NOF Corp . , Tokyo , Japan ). An initial concen example of this type of reagent is the Sunbright® CA series tration or weight of EPO is transferred or dissolved in Hepes from NOF (NOF Corp ., Tokyo , Japan ) . EPO is dissolved in buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM Hepes buffer ( 50 mM Hepes , 150 mM sodium chloride , 5 calcium chloride, pH 6 . 0 ) to get a final protein concentration mM calcium chloride , pH 6 .0 ) and mixed with an aqueous of 2 mg EPO /ml . Subsequently an 5 mM aqueous sodium sodium periodate solution ( 10 mm ) , and an aqueous periodate solution is added within 15 minutes to give a final m - toluidine solution (50 mM ). Subsequently , the aminooxy concentration of 100 uM , followed by addition of an 50 mM reagent is added to give a 20 -fold molar reagent excess. The aqueous m -toluidine solution to get a final concentration of mixture is incubated for 2 h in the dark at room temperature 10 mM within a time period of 30 minutes . Then the under gentle stirring and quenched for 15 min at room aminooxy - PEG reagent with a MW of 20 kD (described temperature by the addition of 8 ul of aqueous cysteine above ) is added to give a 20 - fold molar reagent excess . After solution ( 1 M ) . correction of the pH to 6 . 0 the mixture is incubated for 2 h US 2017 /0240590 A1 Aug . 24 , 2017 60 in the dark at room temperature under gentle stirring and Vivaspin centrifugal filtrators to remove excess periodate , quenched for 15 min at room temperature by the addition of quencher and the byproducts thereof. a 1 M aqueous L - cysteine solution to give a final concen 10561 ] The retentate containing oxidized Ang - 2 is next tration of 10 mM . mixed with an aqueous m - toluidine solution (50 mm ) and [0555 ] The PEG -EPO conjugate is purified by means of incubated for 30 min at room temperature . Aminooxy - PEG ion exchange chromatography ( IEC ) . The conjugate con reagent with a MW of 20 kD is then added to give a 5 - fold taining fractions of the eluate are concentrated by UF /DF molar reagent excess . This mixture is incubated for 2 . 5 h at using a 10 kD membrane made of regenerated cellulose ( 88 room temperature in the dark under gentle stirring . cm², cutoff 10 kD /Millipore ) . The final diafiltration step is 10562 ]. Finally , the PEG -Ang - 2 conjugate is purified by performed against Hepes buffer ( 50 mM Hepes , 5 mM ion -exchange chromatography . The conjugate containing CaCl2 , pH 7 . 5 ) . fraction of the eluate are collected and then subjected to [0556 ] The preparation is analytically characterized by UF /DF using an appropriate MW cutoff membrane. The measuring total protein (Bradford and BCA procedure ) and preparation is next analytically characterized by measuring biological activity according to known methods. total protein (Coomassie , Bradford ) and biological activity according to methods known in the art . Example 33 Method 2 : PEGylation of Ang - 2 Using an Aminooxy - PEG Reagent and m - Toluidine as a Nucleophilic [ 0563 ] Ang - 2 is PEGylated by use of a linear 20 kD Catalyst PEGylation reagent containing an aminooxy group . An example of this type of reagent is the Sunbright® CA series Method 1: from NOF (NOF Corp ., Tokyo , Japan ). [ 0564 ) Ang - 2 is transferred or dissolved in reaction buffer [0557 ] Ang - 2 is PEGylated by use of a linear 20 kD ( e .g . 50 mM Hepes, 350 mM sodium chloride , 5 mM PEGylation reagent containing an aminooxy group . An calcium chloride , pH 6 . 0 ) to get a final protein concentration example of this type of reagent is the Sunbright® CA series of 1 . 0 + / - 0 . 25 mg /ml . Then the pH of the solution is cor from NOF (NOF Corp ., Tokyo , Japan ). Ang - 2 is dissolved rected to 6 . 0 by drop wise addition of a 0 .5N aqueous HC1 in 7 .0 mlhistidine buffer , pH 6 .0 ( 20 mM L -histidine , 150 solution . Subsequently a 40 mM aqueous sodium periodate mM NaCl, 5 mM CaCl2 ). An aqueous sodium periodate solution is added within 10 minutes to give a concentration solution ( 5 mm ) is then added and the reaction mixture is of 200 uM . The oxidation reaction is carried out for 30 + / - 5 incubated for 1 h in the dark at 4° C . under gentle stirring min at a temperature ( T ) of T = + 22 + / - 2° C . Then the reaction and quenched for 15 min at room temperature by the is stopped by addition of an aqueous L - cysteine solution ( 1 addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The M ) within 15 minutes at T = + 22 + / - 2° C . to give a final mixture is subsequently subjected to UF /DF employing concentration of 10 mM in the reaction mixture and incu Vivaspin centrifugal filtrators to remove excess periodate , bation for 60 + / - 5 min . quencher and the byproducts thereof. [0565 ] The oxidized Ang - 2 is further purified by ion 10558 ]. The retentate containing oxidized Ang - 2 is next exchange chromatography. The oxidized Ang - 2 containing mixed with an aqueous m -toluidine solution (50 mM ) and fractions of the eluate are collected and used for the conju incubated for 30 min at room temperature . Aminooxy - PEG gation reaction . reagent with a MW of 20 kD is then added to give a 5 - fold [0566 ] The aminooxy -PEG reagent with a MW of 20 kD molar reagent excess . This mixture is incubated for 2 . 5 h at reagent is added in a 50 - fold molar excess to the eluate room temperature in the dark under gentle stirring . containing the purified oxidized Ang - 2 within a maximum [0559 ] Finally , the PEG -Ang - 2 conjugate is purified by time period (t ) of 15 minutes under gentle stirring. Then an ion -exchange chromatography (e . g. on Q Sepharose FF ). aqueous m - toluidine solution (50 mM ) is added within 15 For example , 1 . 5 mg protein /ml gel is loaded on the column minutes to get a final concentration of 10 mM . The reaction equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 mixture is incubated for 120 + / – 10 min . in the dark at a mM CaC12 . The conjugate is eluted with 50 mM Hepes temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . buffer containing 5 mM CaCl2 and 500 mM sodium chlo [ 0567 ] The obtained PEG - Ang - 2 conjugate is further puri ride, pH 7 . 4 and is then subjected to UF /DF using an fied by ion exchange chromatography . The PEG - Ang - 2 appropriate MW cutoff membrane . The preparation is next conjugate containing fractions are collected and concen analytically characterized by measuring total protein (Coo trated by ultra - /diafiltration (UF /DF ) using a membrane massie , Bradford ) and biological activity according to meth made of regenerated cellulose with an appropriate molecular ods known in the art. weight cut off (Millipore ) . [ 0560] In an alternative embodiment, Method 1 is carried out as follows. Ang - 2 is PEGylated by use of a linear 20 kD [0568 ] The conjugate prepared by use of this procedure PEGylation reagent containing an aminooxy group . An are analytically characterized by measuring total protein and example of this type of reagent is the Sunbright® CA series biological activity according to methods known in the art. from NOF (NOF Corp . , Tokyo , Japan ) . Ang- 2 is dissolved in 7 .0 ml histidine buffer, pH 6 .0 (20 mM L - histidine , 150 Method 3 : mM NaC1, 5 mM CaC12 ). An aqueous sodium periodate [0569 ] Ang - 2 is PEGylated by use of a linear 20 kD solution (5 mm ) is then added and the reaction mixture is PEGylation reagent containing an aminooxy group . An incubated for 1 h in the dark at 4° C . under gentle stirring example of this type of reagent is the Sunbright® CA series and quenched for 15 min at room temperature by the from NOF (NOF Corp . , Tokyo , Japan ) . Ang- 2 is dissolved addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , mixture is subsequently subjected to UF /DF employing 5 mM calcium chloride , pH 6 .0 ) and mixed with an aqueous US 2017 /0240590 A1 Aug . 24 , 2017 70

sodium periodate solution ( 10 mm ) , and an aqueous cm2, cutoff 10 kD /Millipore ). The final diafiltration step is m - toluidine solution ( 50 mM ) . Subsequently the aminooxy performed against Hepes buffer (50 mM Hepes, 5 mM reagent is added to give a 20 - fold molar reagent excess . The CaCl2 , pH 7 . 5 ). mixture is incubated for 2 h in the dark at room temperature [ 0575 ] The preparation is analytically characterized by under gentle stirring and quenched for 15 min at room measuring total protein (Bradford and BCA procedure ) and temperature by the addition of 8 ul of aqueous cysteine biological activity according to known methods. solution ( 1 M ). [0576 ] Subsequently , the free Ang -2 is removed by means [0570 ] Finally , the PEG - Ang -2 conjugate is purified by of ion exchange chromatography ( IEC ) . The conjugate con ion -exchange chromatography on Q Sepharose FF. 1. 5 mg taining fractions of the eluate are concentrated by UF /DF . protein /ml gel is loaded on the column pre equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The Example 34 conjugate is eluted with 50 mM Hepes buffer containing 5 mM CaC12 and 500 mM sodium chloride, pH 7 . 4 and is then PEGylation of VEGF Using an Aminooxy - PEG subjected to UF/ DF using a membrane . The preparation is Reagent and m - Toluidine as a Nucleophilic analytically characterized by measuring total protein (Brad Catalyst ford ) and biological activity according to methods known in the art . Method 1 : [0571 ] In an alternative embodiment, Method 3 is carried [0577 ] VEGF is PEGylated by use of a linear 20 kD out as follows. Ang - 2 is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An 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 ) . VEGF is dissolved from NOF (NOF Corp . , Tokyo , Japan ) . Ang - 2 is dissolved in 7 .0 ml histidine buffer , pH 6 .0 (20 mM L -histidine , 150 in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , mM NaCl, 5 mM CaC12 ) . An aqueous sodium periodate 5 mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous solution ( 5 mM ) is then added and the reaction mixture is sodium periodate solution ( 10 mM ) , and an aqueous incubated for 1 h in the dark at 4° C . under gentle stirring m - toluidine solution (50 mM ) . Subsequently the aminooxy and quenched for 15 min at room temperature by the reagent is added to give a 20 - fold molar reagent excess . The addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The mixture is incubated for 2 h in the dark at room temperature mixture is subsequently subjected to UF /DF employing under gentle stirring and quenched for 15 min at room Vivaspin centrifugal filtrators to remove excess periodate , temperature by the addition of 8 ul of aqueous cysteine quencher and the byproducts thereof. [0578 ] The retentate containing oxidized VEGF is next solution ( 1 M ). mixed with an aqueous m - toluidine solution ( 50 mM ) and [0572 ] Finally the PEG - Ang - 2 conjugate is purified by incubated for 30 min at room temperature . Aminooxy -PEG ion - exchange chromatography The conjugate containg fre reagent with a MW of 20 kD is then added to give a 5 - fold actions of the eluate are collected and then subjected to molar reagent excess . This mixture is incubated for 2 . 5 h at UF/ DF . The preparation is analytically characterized by room temperature in the dark under gentle stirring . measuring total protein ( Bradford ) and biological activity [ 0579 ] Finally , the PEG - VEGF conjugate is purified by according to methods known in the art. ion -exchange chromatography ( e . g . , on Q Sepharose FF ) . For example , 1 . 5 mg protein /ml gel is loaded on the column Method 4 : equilibrated with 50 mM Hepes buffer , pH 7 .4 containing 5 mM CaC12 . The conjugate is eluted with 50 mM Hepes [ 0573] Ang - 2 is PEGylated by use of a linear 20 kD buffer containing 5 mM CaC12 and 500 mM sodium chlo PEGylation reagent containing an aminooxy group . An ride, pH 7 . 4 and is then subjected to UF /DF using an example of this type of reagent is the Sunbright® CA series appropriate MW cutoff membrane . The preparation is next from NOF (NOF Corp ., Tokyo , Japan ) . An initial concen analytically characterized by measuring total protein ( Coo tration or weight of Ang - 2 is transferred or dissolved in massie , Bradford ) and biological activity according to meth Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 ods known in the art . mM calcium chloride, pH 6 . 0 ) to get a final protein con [0580 ] In an alternative embodiment, Method 1 is carried centration of 2 mg Ang - 2 /ml . Subsequently an 5 mM aque out as follows. VEGF is PEGylated by use of a linear 20 kD ous sodium periodate solution is added within 15 minutes to PEGylation reagent containing an aminooxy group . An give a final concentration of 100 UM , followed by addition example of this type of reagent is the Sunbright® CA series of an 50 mM aqueous m - toluidine solution to get a final from NOF (NOF Corp . , Tokyo , Japan ) . VEGF is dissolved concentration of 10 mM within a time period of 30 minutes. in 7 . 0 ml histidine buffer, pH 6 . 0 ( 20 mM L -histidine , 150 Then the aminooxy - PEG reagent with a MW of 20 kD mM NaCl, 5 mM CaC12 ) . An aqueous sodium periodate ( described above ) is added to give a 20 - fold molar reagent solution ( 5 mM ) is then added and the reaction mixture is excess . After correction of the pH to 6 . 0 the mixture is incubated for 1 h in the dark at 4° C . under gentle stirring 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 an 1 M aqueous L -cysteine solution to mixture is subsequently subjected to UF /DF employing give a final concentration of 10 mM . Vivaspin centrifugal filtrators to remove excess periodate , [0574 ] The PEG -Ang - 2 conjugate is purified by means of quencher and the byproducts thereof. ion exchange chromatography ( IEC ) . The conjugate con [0581 ] The retentate containing oxidized VEGF is next taining fractions of the eluate are concentrated by UF /DF mixed with an aqueous m - toluidine solution (50 mM ) and using a 10 kD membrane made of regenerated cellulose (88 incubated for 30 min at room temperature . Aminooxy - PEG US 2017 /0240590 A1 Aug . 24 , 2017 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 ) . [ 0582 ] Finally , the PEG - VEGF conjugate is purified by [0589 ] Finally , the PEG -VEGF conjugate is purified by ion -exchange chromatography The conjugate containg frac ion -exchange chromatography on Q Sepharose FF . 1. 5 mg tions of the eluate are collected and then subjected to UF /DF protein /ml gel is loaded on the column pre equilibrated with using an appropriate MW cutoff membrane . The preparation 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The is next analytically characterized by measuring total protein conjugate is eluted with 50 mM Hepes buffer containing 5 (Coomassie , Bradford ) and biological activity according to mM CaC12 and 500 mM sodium chloride , pH 7 . 4 and is then methods known in the art . subjected to UF /DF using a membrane . The preparation is analytically characterized by measuring total protein (Brad ford ) and biological activity according to methods known in Method 2 : the art. [ 0583] VEGF is PEGylated by use of a linear 20 kD [0590 ] In an alternative embodiment, Method 3 is carried PEGylation reagent containing an aminooxy group . An out as follows. VEGF is PEGylated by use of a linear 20 kD example of this type of reagent is the Sunbright® CA series PEGylation reagent containing an aminooxy group . An from NOF (NOF Corp . , Tokyo , Japan ) . VEGF is transferred example of this type of reagent is the Sunbright® CA series or dissolved in reaction buffer (e . g . 50 mM Hepes, 350 mM from NOF (NOF Corp . , Tokyo , Japan ) . VEGF is dissolved sodium chloride , 5 mM calcium chloride , pH 6 .0 ) to get a in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , final protein concentration of 1. 0 + / - 0 .25 mg/ml . Then the 5 mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous pH of the solution is corrected to 6 . 0 by drop wise addition sodium periodate solution ( 10 mM ) , and an aqueous of a 0 . 5 N aqueous HCl solution . Subsequently , a 40 mm m -toluidine solution (50 mM ) . Subsequently , the aminooxy aqueous sodium periodate solution is added within 10 min reagent is added to give a 20 - fold molar reagent excess. The mixture is incubated for 2 h in the dark at room temperature utes to give a concentration of 200 uM . The oxidation under gentle stirring and quenched for 15 min at room reaction is carried out for 30 + / - 5 min at a temperature ( T ) temperature by the addition of 8 ul of aqueous cysteine of T = + 22 + / - 2° C . Then the reaction is stopped by addition solution ( 1 M ). of an aqueous L - cysteine solution ( 1 M ) within 15 minutes [0591 ] Finally, the PEG - VEGF conjugate is purified by at T = + 22 + / - 2° C . to give a final concentration of 10 mM in ion -exchange chromatography. The conjugate fractions of the reaction mixture and incubation for 60 + / - 5 min . the eluate are collected and then subjected to UF /DF . The [ 0584 ] The oxidized VEGF is further purified by ion preparation is analytically characterized by measuring total exchange chromatography. The oxidized VEGF containing protein (Bradford ) and biological activity according to meth fractions of the eluate are collected and used for the conju gation reaction . ods known in the art . [0585 ] The aminooxy - PEG reagent with a MW of 20 kD Method 4 : reagent is added in a 50 - fold molar excess to the eluate [0592 ] VEGF is PEGylated by use of a linear 20 kD containing the purified oxidized VEGF within a maximum PEGylation reagent containing an aminooxy group . An time period ( t ) of 15 minutes under gentle stirring . Then an example of this type of reagent is the Sunbright® CA series aqueous m - toluidine solution ( 50 mM ) is added within 15 from NOF (NOF Corp . , Tokyo , Japan ) . An initial concen minutes to get a final concentration of 10 mM . The reaction tration or weight of VEGF is transferred or dissolved in mixture is incubated for 120 + / – 10 min . in the dark at a Hepes buffer ( 50 mM Hepes, 150 mM sodium chloride , 5 temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . mM calcium chloride , pH 6 .0 ) to get a final protein con [0586 ] The obtained PEG - VEGF conjugate is further puri centration of 2 mg VEGF /ml . Subsequently , an 5 mM fied by ion exchange chromatography . The PEG - VEGF aqueous sodium periodate solution is added within 15 min conjugate containing fractions are collected and concen utes to give a final concentration of 100 UM , followed by trated by ultra - /diafiltration (UF /DF ) using a membrane addition of an 50 mM aqueous m - toluidine solution to get a made of regenerated cellulose with an appropriate molecular final concentration of 10 mM within a time period of 30 weight cut off (Millipore ) . minutes . Then the aminooxy - PEG reagent with a MW of 20 [ 0587 ] The conjugate prepared by use of this procedure kD (described above ) is added to give a 20 - fold molar are analytically characterized by measuring total protein and reagent excess . After correction of the pH to 6 . 0 the mixture biological activity according to methods known in the art . is incubated for 2 h in the dark at room temperature under gentle stirring and quenched for 15 min at room temperature Method 3 : by the addition of an 1 M aqueous L -cysteine solution to give a final concentration of 10 mM . [0588 ] VEGF is PEGylated by use of a linear 20 kD [ 0593] The PEG -VEGF conjugate is purified by means of PEGylation reagent containing an aminooxy group . An ion exchange chromatography ( IEC ) . The conjugate con example of this type of reagent is the Sunbright® CA series taining fractions of the eluate are concentrated by UF /DF from NOF (NOF Corp . , Tokyo , Japan ) . VEGF is dissolved using a 10 kD membrane made of regenerated cellulose ( 88 in Hepes buffer (50 mM Hepes, 150 mM sodium chloride , cm2, cut -off 10 kD /Millipore ) . The final diafiltration step is 5 mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous performed against Hepes buffer (50 mM Hepes, 5 mM sodium periodate solution ( 10 mM ) , and an aqueous CaCl2, pH 7. 5) . m -toluidine solution (50 mm ). Subsequently , the aminooxy [0594 ] The preparation is analytically characterized by reagent is added to give a 20 - fold molar reagent excess . The measuring total protein ( Bradford and BCA procedure ) and mixture is incubated for 2 h in the dark at room temperature biological activity according to known methods. US 2017 /0240590 A1 Aug . 24 , 2017

Example 35 Method 2 : PEGylation of EGF Using an Aminooxy - PEG [0601 ] EGF is PEGylated by use of a linear 20 kD Reagent and m - Toluidine as a Nucleophilic PEGylation reagent containing an aminooxy group . An Catalyst example of this type of reagent is the Sunbright® CA series from NOF (NOF Corp . , Tokyo , Japan ) . EGF is transferred or Method 1: dissolved in reaction buffer ( e . g . 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6 . 0 ) to get a [0595 ] EGF is PEGylated by use of a linear 20 kD final protein concentration of 1 . 0 + / - 0 .25 mg/ ml . Then the PEGylation reagent containing an aminooxy group . An pH of the solution is corrected to 6 . 0 by drop wise addition example of this type of reagent is the Sunbright® CA series of a 0 . 5 N aqueous HC1 solution . Subsequently , a 40 mM from NOF (NOF Corp . , Tokyo , Japan ) . EGF is dissolved in aqueous sodium periodate solution is added within 10 min 7 .0 ml histidine buffer , pH 6 .0 (20 mM L -histidine , 150 mM utes to give a concentration of 200 uM . The oxidation NaC1, 5 mM CaC12 ) . An aqueous sodium periodate solution reaction is carried out for 30 + / - 5 min at a temperature ( T ) ( 5 mM ) is then added and the reaction mixture is incubated of T = + 22 +/ - 2° C . Then the reaction is stopped by addition for 1 h in the dark at 4° C . under gentle stirring and quenched of an aqueous L -cysteine solution ( 1 M ) within 15 minutes for 15 min at room temperature by the addition of 7 . 5 ul of at T = + 22 + / - 2° C . to give a final concentration of 10 mM in a 1 M aqueous cysteine solution . The mixture is subse the reaction mixture and incubation for 60 + / - 5 min . quently subjected to UF /DF employing Vivaspin centrifugal [0602 ] The oxidized EGF is further purified by ion filtrators to remove excess periodate , quencher and the exchange chromatography . The oxidized EGF containing byproducts thereof. fractions of the eluate are collected and used for the conju [ 0596 ] The retentate containing oxidized EGF is next gation reaction . mixed with an aqueous m - toluidine solution ( 50 mM ) and [0603 ] The aminooxy -PEG reagent with a MW of 20 kD incubated for 30 min at room temperature . Aminooxy -PEG reagent is added in a 50 - fold molar excess to the eluate reagent with a MW of 20 kD is then added to give a 5 - fold containing the purified oxidized NGF within a maximum molar reagent excess . This mixture is incubated for 2 .5 h at time period ( t ) of 15 minutes under gentle stirring . Then an room temperature in the dark under gentle stirring . aqueous m - toluidine solution (50 mM ) is added within 15 [ 0597 ] Finally , the PEG -EGF conjugate is purified by minutes to get a final concentration of 10 mM . The reaction ion - exchange chromatography ( e . g . , on Q Sepharose FF ) . mixture is incubated for 120 +/ – 10 min . in the dark at a For example , 1 . 5 mg protein /ml gel is loaded on the column temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 [0604 ] The obtained PEG - EGF conjugate is further puri mM CaC12 . The conjugate is eluted with 50 mM Hepes fied by ion exchange chromatography . The PEG - EGF con buffer containing 5 mM CaC12 and 500 mM sodium chlo jugate containing fractions are collected and concentrated by ride , pH 7 . 4 and is then subjected to UF /DF using an ultra - / diafiltration (UF /DF ) using a membrane made of appropriate MW cutoff membrane . The preparation is next regenerated cellulose with an appropriate molecular weight analytically characterized by measuring total protein (Coo cut off (Millipore ). massie , Bradford ) and biological activity according to meth [0605 ] The conjugate prepared by use of this procedure ods known in the art . are analytically characterized by measuring total protein and [ 0598 ] In an alternative embodiment, Method 1 is carried out as follows. EGF is PEGylated by use of a linear 20 kD biological activity according to methods known in the art. PEGylation reagent containing an aminooxy group . An example of this type of reagent is the Sunbright® CA series Method 3: from NOF (NOF Corp . , Tokyo , Japan ) . EGF is dissolved in [0606 ] EGF is PEGylated by use of a linear 20 kD 7 . 0 ml histidine buffer , pH 6 . 0 ( 20 mM L - histidine, 150 mM PEGylation reagent containing an aminooxy group . An NaC1, 5 mM CaCl2 ) . An aqueous sodium periodate solution example of this type of reagent is the Sunbright® CA series ( 5 mM ) is then added and the reaction mixture is incubated from NOF (NOF Corp ., Tokyo , Japan ). EGF is dissolved in for 1 h in the dark at 4° C . under gentle stirring and quenched Hepes buffer ( 50 mM Hepes , 150 mM sodium chloride , 5 for 15 min at room temperature by the addition of 7 . 5 ul of mM calcium chloride, pH 6 .0 ) and mixed with an aqueous a 1 M aqueous cysteine solution . The mixture is subse sodium periodate solution ( 10 mm ) , and an aqueous quently subjected to UF/ DF employing Vivaspin centrifugal m - toluidine solution ( 50 mM ). Subsequently the aminooxy filtrators to remove excess periodate , quencher and the reagent is added to give a 20 -fold molar reagent excess . The byproducts thereof. mixture is incubated for 2 h in the dark at room temperature [ 0599 ] The retentate containing oxidized EGF is next under gentle stirring and quenched for 15 min at room mixed with an aqueous m - toluidine solution ( 50 mM ) and temperature by the addition of 8 ul of aqueous cysteine incubated for 30 min at room temperature . Aminooxy -PEG solution ( 1 M ) . reagent with a MW of 20 kD is then added to give a 5 - fold [ 0607 ] Finally , the PEG - EGF conjugate is purified by molar reagent excess . This mixture is incubated for 2 . 5 h at ion - exchange chromatography on Q - Sepharose FF . 1 . 5 mg room temperature in the dark under gentle stirring . protein /ml gel is loaded on the column pre equilibrated with [0600 ] Finally , the PEG - EGF conjugate is purified by 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The ion - exchange chromatography . The conjugate containg frac conjugate is eluted with 50 mM Hepes buffer containing 5 tions of the eluate are collected and then subjected to UF/ DF mM CaCl2 and 500 mM sodium chloride, pH 7 . 4 and is then using an appropriate MW cutoff membrane . The preparation subjected to UF /DF using a membrane . The preparation is is next analytically characterized by measuring total protein analytically characterized by measuring total protein (Brad (Coomassie , Bradford ) and biological activity according to ford ) and biological activity according to methods known in methods known in the art. the art . US 2017 /0240590 A1 Aug . 24 , 2017

[0608 ] In an alternative embodiment, Method 3 is carried NaCl, 5 mM CaCl2 ) . An aqueous sodium periodate solution out as follows. EGF is PEGylated by use of a linear 20 kD ( 5 mM ) is then added and the reaction mixture is incubated PEGylation reagent containing an aminooxy group . An for 1 h in the dark at 4° C . under gentle stirring and quenched example of this type of reagent is the Sunbright® CA series for 15 min at room temperature by the addition of 7 . 5 ul of from NOF (NOF Corp ., Tokyo , Japan ). EGF is dissolved in a 1 M aqueous cysteine solution . The mixture is subse Hepes buffer (50 mM Hepes, 150 mM sodium chloride , 5 quently subjected to UF /DF employing Vivaspin centrifugal mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous filtrators to remove excess periodate , quencher and the sodium periodate solution ( 10 mM ) , and an aqueous byproducts thereof . m -toluidine solution (50 mM ) . Subsequently the aminooxy [ 0614 ] The retentate containing oxidized NGF is next reagent is added to give a 20 - fold molar reagent excess . The mixed with an aqueous m - toluidine solution (50 mM ) and mixture is incubated for 2 h in the dark at room temperature incubated for 30 min at room temperature . Aminooxy -PEG under gentle stirring and quenched for 15 min at room reagent with a MW of 20 kD is then added to give a 5 - fold temperature by the addition of 8 ul of aqueous cysteine molar reagent excess. This mixture is incubated for 2 .5 h at solution ( 1 M ) . room temperature in the dark under gentle stirring . [ 0609 ] Finally , the PEG - EGF conjugate is purified by [ 0615 ] Finally , the PEG -NGF conjugate is purified by ion - exchange chromatography . The conjugate containing ion -exchange chromatography ( e . g ., on Q - Sepharose FF ) . fractions of the eluate are collected and then subjected to For example , 1 . 5 mg protein /ml gel is loaded on the column UF /DF . The preparation is analytically characterized by equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 measuring total protein (Bradford ) and biological activity mM CaC12 . The conjugate is eluted with 50 mM Hepes according to methods known in the art. buffer containing 5 mM CaCl2 and 500 mM sodium chlo ride, pH 7 . 4 and is then subjected to UF /DF using an Method 4 : appropriate MW cutoff membrane . The preparation is next [ 0610 ] EGF is PEGylated by use of a linear 20 kD analytically characterized by measuring total protein ( Coo PEGylation reagent containing an aminooxy group . An massie , Bradford ) and biological activity according to meth example of this type of reagent is the Sunbright® CA series ods known in the art . from NOF (NOF Corp . , Tokyo , Japan ) . An initial concen [0616 ] In an alternative embodiment, Method 1 is carried tration or weight of EGF is transferred or dissolved in Hepes out as follows . NGF is PEGylated by use of a linear 20 kD buffer (50 mM Hepes , 150 mM sodium chloride, 5 mM PEGylation reagent containing an aminooxy group . An calcium chloride, pH 6 . 0 ) to get a final protein concentration example of this type of reagent is the Sunbright® CA series of 2 mg EGF/ ml . Subsequently an 5 mM aqueous sodium from NOF ( NOF Corp ., Tokyo , Japan ). NGF is dissolved in periodate solution is added within 15 minutes to give a final 7 . 0 ml histidine buffer , pH 6 . 0 ( 20 mM L -histidine , 150 mM concentration of 100 uM , followed by addition of an 50 mM NaCl, 5 mM CaCl2 ) . An aqueous sodium periodate solution aqueous m - toluidine solution to get a final concentration of ( 5 mM ) is then added and the reaction mixture is incubated 10 mM within a time period of 30 minutes . Then the for 1 h in the dark at 4° C . under gentle stirring and quenched aminooxy -PEG reagent with a MW of 20 kD ( described for 15 min at room temperature by the addition of 7 . 5 ul of above ) is added to give a 20 - fold molar reagent excess . After a 1 M aqueous cysteine solution . The mixture is subse correction of the pH to 6 . 0 the mixture is incubated for 2 h quently subjected to UF /DF employing Vivaspin centrifugal in the dark at room temperature under gentle stirring and filtrators to remove excess periodate , quencher and the quenched for 15 min at room temperature by the addition of byproducts thereof. an 1 M aqueous L - cysteine solution to give a final concen [0617 ] The retentate containing oxidized NGF is next tration of 10 mM . mixed with an aqueous m - toluidine solution (50 mM ) and [0611 ] The PEG - EGF conjugate is purified by means of incubated for 30 min at room temperature . Aminooxy - PEG ion exchange chromatography ( IEC ) . The conjugate con reagent with a MW of 20 kD is then added to give a 5 - fold taining fractions of the eluate are concentrated by UF /DF molar reagent excess . This mixture is incubated for 2. 5 h at using a 10 kD membrane made of regenerated cellulose (88 room temperature in the dark under gentle stirring . cm2 , cutoff 10 kD /Millipore ) . The final diafiltration step is [ 0618 ] Finally , the PEG -NGF conjugate is purified by performed against Hepes buffer (50 mM Hepes, 5 mM ion - exchange chromatography ( The conjugate containing CaC12 , pH 7 . 5 ) . fractions of the eluate are collected and then subjected to [0612 ] The preparation is analytically characterized by UF /DF using an appropriate MW cutoff membrane. The measuring total protein (Bradford and BCA procedure ) and preparation is next analytically characterized by measuring biological activity according to known methods . total protein (Coomassie , Bradford ) and biological activity according to methods known in the art. Example 36 Method 2 : PEGylation of NGF Using an Aminooxy - PEG [0619 ] NGF is PEGylated by use of a linear 20 kD Reagent and m - Toluidine as a Nucleophilic PEGylation reagent containing an aminooxy group . An Catalyst example of this type of reagent is the Sunbright® CA series from NOF (NOF Corp ., Tokyo , Japan ). NGF is transferred Method 1 : or dissolved in reaction buffer ( e . g . 50 mM Hepes, 350 mm [0613 ] NGF is PEGylated by use of a linear 20 kD sodium chloride , 5 mM calcium chloride , pH 6 .0 ) to get a PEGylation reagent containing an aminooxy group . An final protein concentration of 1 . 0 + / - 0 .25 mg/ ml . Then the example of this type of reagent is the Sunbright® CA series pH of the solution is corrected to 6 . 0 by drop wise addition from NOF (NOF Corp . , Tokyo , Japan ) . NGF is dissolved in of a 0 .5N aqueous HCl solution . Subsequently a 40 mm 7 . 0 ml histidine buffer, pH 6 .0 ( 20 mM L -histidine , 150 mM aqueous sodium periodate solution is added within 10 min US 2017 /0240590 A1 Aug . 24 , 2017 utes to give a concentration of 200 UM . The oxidation under gentle stirring and quenched for 15 min at room reaction is carried out for 30 + / - 5 min at a temperature ( T ) temperature by the addition of 8 ul of aqueous cysteine of T = + 22 + / - 2° C . Then the reaction is stopped by addition solution ( 1 M ) . of an aqueous L - cysteine solution ( 1 M ) within 15 minutes [ 0627 ] Finally , the PEG - NGF conjugate is purified by at T = + 22 + / - 2° C . to give a final concentration of 10 mM in ion -exchange chromatography . The conjugate containg frac the reaction mixture and incubation for 60 + /- 5 min . tions are collected and then subjected to UF /DF . The prepa [ 0620 ] The oxidized NGF is further purified by ion ration is analytically characterized by measuring total pro exchange chromatography. The oxidized NGF containing tein (Bradford ) and biological activity according to methods fractions of the eluate are collected and used for the conju known in the art . gation reaction . [ 0621] The aminooxy - PEG reagent with a MW of 20 kD Method 4 : reagent is added in a 50 - fold molar excess to the eluate [0628 ] NGF is PEGylated by use of a linear 20 kD containing the purified oxidized NGF within a maximum PEGylation reagent containing an aminooxy group . An time period (t ) of 15 minutes under gentle stirring. Then an example of this type of reagent is the Sunbright® CA series aqueous m - toluidine solution ( 50 mM ) is added within 15 from NOF (NOF Corp . , Tokyo , Japan ) . An initial concen minutes to get a final concentration of 10 mM . The reaction tration or weight of NGF is transferred or dissolved in Hepes mixture is incubated for 120 + / – 10 min . in the dark at a buffer (50 mM Hepes , 150 mM sodium chloride , 5 mM temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . calcium chloride, pH 6 . 0 ) to get a final protein concentration [0622 ] The obtained PEG -NGF conjugate is further puri of 2 mg NGF/ ml . Subsequently an 5 mM aqueous sodium fied by ion exchange chromatography. The PEG -NGF con periodate solution is added within 15 minutes to give a final jugate containing fractions are collected and concentrated by concentration of 100 uM , followed by addition of an 50 mM ultra - / diafiltration (UF /DF ) using a membrane made of aqueous m - toluidine solution to get a final concentration of regenerated cellulose with an appropriate molecular weight 10 mM within a time period of 30 minutes . Then the cut off (Millipore ) . aminooxy -PEG reagent with a MW of 20 kD (described [ 0623 ] The conjugate prepared by use of this procedure above ) is added to give a 20 -fold molar reagent excess . After are analytically characterized by measuring total protein and correction of the pH to 6 . 0 the mixture is incubated for 2 h biological activity according to methods known in the art . in the dark at room temperature under gentle stirring and quenched for 15 min at room temperature by the addition of Method 3 : an 1 M aqueous L - cysteine solution to give a final concen [ 0624 ] NGF is PEGylated by use of a linear 20 kD tration of 10 mM . PEGylation reagent containing an aminooxy group . An [0629 ] The PEG -NGF conjugate is purified by means of example of this type of reagent is the Sunbright® CA series ion exchange chromatography ( IEC ) . The conjugate con from NOF (NOF Corp ., Tokyo , Japan ). NGF is dissolved in taining fractions of the eluate are concentrated by UF /DF Hepes buffer (50 mM Hepes, 150 mM sodium chloride , 5 using a 10 kD membrane made of regenerated cellulose ( 88 mM calcium chloride , pH 6 .0 ) and mixed with an aqueous cm2, cutoff 10 kD /Millipore ) . The final diafiltration step is sodium periodate solution ( 10 mm ), and an aqueous performed against Hepes buffer ( 50 mM Hepes, 5 mM m - toluidine solution (50 mM ) . Subsequently the aminooxy CaCl2 , pH 7 .5 ). reagent is added to give a 20 - fold molar reagent excess . The [ 0630 ] The preparation is analytically characterized by mixture is incubated for 2 h in the dark at room temperature measuring total protein (Bradford and BCA procedure ) and under gentle stirring and quenched for 15 min at room biological activity according to known methods. temperature by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . Example 37 [ 0625 ] Finally , the PEG - NGF conjugate is purified by ion - exchange chromatography on Q Sepharose FF. 1 . 5 mg PEGylation of HGH Using an Aminooxy - PEG protein /ml gel is loaded on the column pre equilibrated with Reagent and m - Toluidine as a Nucleophilic 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The Catalyst conjugate is eluted with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7. 4 and is then Method 1 : subjected to UF /DF using a membrane . The preparation is [ 0631 ] As described herein , the amino acid sequence of analytically characterized by measuring total protein (Brad human growth hormone (HGH ) is first modified to incor ford ) and biological activity according to methods known in porate at least one glycosylation site . Following purification , the art . HGH is glycosylated in vitro according to methods known 10626 ] In an alternative embodiment , Method 3 is carried in the art. out as follows. NGF is PEGylated by use of a linear 20 kD [0632 ] HGH is PEGylated by use of a linear 20 kD PEGylation reagent containing an aminooxy group . An 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 ). NGF is dissolved in from NOF (NOF Corp ., Tokyo , Japan ). HGH is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 7 . 0 mlhistidine buffer , pH 6 .0 ( 20 mM L -histidine , 150 mm mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous NaCl, 5 mM CaC12 ) . An aqueous sodium periodate solution sodium periodate solution ( 10 mm ) , and an aqueous ( 5 mM ) is then added and the reaction mixture is incubated m -toluidine solution (50 mM ). Subsequently the aminooxy for 1 h in the dark at 4° C . under gentle stirring and quenched reagent is added to give a 20 - fold molar reagent excess . The for 15 min at room temperature by the addition of 7 . 5 ul of mixture is incubated for 2 h in the dark at room temperature a 1 M aqueous cysteine solution . The mixture is subse US 2017 /0240590 A1 Aug . 24 , 2017 75 quently subjected to UF/ DF employing Vivaspin centrifugal sodium chloride , 5 mM calcium chloride, pH 6 .0 ) to get a filtrators to remove excess periodate , quencher and the final protein concentration of 1 . 0 + / - 0 . 25 mg /ml . Then the byproducts thereof. pH of the solution is corrected to 6 . 0 by drop wise addition [ 0633 ] The retentate containing oxidized HGH is next of a 0 .5N aqueous HCl solution . Subsequently , a 40 mM mixed with an aqueous m - toluidine solution (50 mM ) and aqueous sodium periodate solution is added within 10 min incubated for 30 min at room temperature . Aminooxy -PEG utes to give a concentration of 200 UM . The oxidation reagent with a MW of 20 kD is then added to give a 5 - fold reaction is carried out for 30 + / - 5 min at a temperature ( T ) molar reagent excess . This mixture is incubated for 2 . 5 h at of T = + 22 + / - 2° C . Then the reaction is stopped by addition room temperature in the dark under gentle stirring . of an aqueous L - cysteine solution ( 1 M ) within 15 minutes [0634 ) Finally, the PEG -HGH conjugate is purified by at T = + 22 + / - 2° C . to give a final concentration of 10 mM in ion - exchange chromatography ( e . g . , on Q Sepharose FF ) . the reaction mixture and incubation for 60 + / – 5 min . For example, 1 . 5 mg protein /ml gel is loaded on the column 0641 ] The oxidized HGH is further purified by ion equilibrated with 50 mM Hepes buffer, pH 7 . 4 containing 5 exchange chromatography. The oxidized HGH containing mM CaC12 . The conjugate is eluted with 50 mM Hepes fractions of the eluate are collected and used for the conju buffer containing 5 mM CaCl2 and 500 mM sodium chlo gation reaction . ride, pH 7 .4 and is then subjected to UF /DF using an ( 0642 ] The aminooxy -PEG reagent with a MW of 20 kD appropriate MW cutoff membrane . The preparation is next reagent is added in a 50 - fold molar excess to the eluate analytically characterized by measuring total protein (Coo containing the purified oxidized HGH within a maximum massie , Bradford ) and biological activity according to meth time period ( t ) of 15 minutes under gentle stirring . Then an ods known in the art. aqueous m - toluidine solution (50 mM ) is added within 15 [0635 ] In n alternative embodiment, Method 1 is carried minutes to get a final concentration of 10 mM . The reaction out as follows . As described herein , the amino acid sequence mixture is incubated for 120 + / - 10 min . in the dark at a of human growth hormone (HGH ) is first modified to temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . incorporate at least one glycosylation site . Following puri 10643 ] The obtained PEG -HGH conjugate is further puri fication , HGH is glycosylated in vitro according to methods fied by ion exchange chromatography . The PEG - NGF con known in the art. jugate containing fractions are collected and concentrated by [ 0636 ] HGH is PEGylated by use of a linear 20 kD ultra - /diafiltration (UF /DF ) using a membrane made of PEGylation reagent containing an aminooxy group . An regenerated cellulose with an appropriate molecular weight example of this type of reagent is the Sunbright® CA series cut off (Millipore ) . from NOF (NOF Corp . , Tokyo , Japan ) . HGH is dissolved in [ 0644 ] The conjugate prepared by use of this procedure 7 . 0 ml histidine buffer , pH 6 . 0 ( 20 mM L - histidine, 150 mM are analytically characterized by measuring total protein and NaCl, 5 mM CaC12 ) . An aqueous sodium periodate solution biological activity according to methods known in the art . ( 5 mM ) is then added and the reaction mixture is incubated for 1 h in the dark at 4° C . under gentle stirring and quenched Method 3 : for 15 min at room temperature by the addition of 7 . 5 ul of [ 0645 ] As described herein , the amino acid sequence of a 1 M aqueous cysteine solution . The mixture is subse human growth hormone (HGH ) is first modified to incor quently subjected to UF/ DF employing Vivaspin centrifugal porate at least one glycosylation site . Following purification , filtrators to remove excess periodate , quencher and the HGH is glycosylated in vitro according to methods known byproducts thereof. in the art . [0637 ] The retentate containing oxidized HGH is next [0646 ] HGH is PEGylated by use of a linear 20 kD mixed with an aqueous m - toluidine solution (50 mM ) and PEGylation reagent containing an aminooxy group . An incubated for 30 min at room temperature . Aminooxy - PEG example of this type of reagent is the Sunbright® CA series reagent with a MW of 20 kD is then added to give a 5 - fold from NOF (NOF Corp . , Tokyo , Japan ) . HGH is dissolved in molar reagent excess . This mixture is incubated for 2 .5 h at Hepes buffer (50 mM Hepes , 150 mM sodium chloride , 5 room temperature in the dark under gentle stirring . mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous [ 0638 ] Finally , the PEG -HGH conjugate is purified by sodium periodate solution ( 10 mM ), and an aqueous ion - exchange chromatography ( The conjugate containg m - toluidine solution (50 mm ) . Subsequently the aminooxy fractions of the eluate are collected and then subjected to reagent is added to give a 20 - fold molar reagent excess. The UF /DF using an appropriate MW cutoff membrane. The mixture is incubated for 2 h in the dark at room temperature preparation is next analytically characterized by measuring under gentle stirring and quenched for 15 min at room total protein (Coomassie , Bradford ) and biological activity temperature by the addition of 8 ul of aqueous cysteine according to methods known in the art . solution ( 1 M ) . 0647 ] Finally , the PEG - HGH conjugate is purified by Method 2 : ion - exchange chromatography on Q - Sepharose FF . 1 . 5 mg [ 0639] As described herein , the amino acid sequence of protein /ml gel is loaded on the column pre equilibrated with human growth hormone (HGH ) is first modified to incor 50 mM Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The porate at least one glycosylation site . Following purification , conjugate is eluted with 50 mM Hepes buffer containing 5 HGH is glycosylated in vitro according to methods known mM CaCl2 and 500 mM sodium chloride , pH 7 . 4 and is then in the art. subjected to UF /DF using a membrane . The preparation is [0640 ] HGH is PEGylated by use of a linear 20 kD analytically characterized by measuring total protein ( Brad PEGylation reagent containing an aminooxy group . An ford ) and biological activity according to methods known in example of this type of reagent is the Sunbright® CA series the art. from NOF (NOF Corp . , Tokyo , Japan ) . HGH is transferred [ 0648 ] In an alternative embodiment, Method 3 is carried or dissolved in reaction buffer (e . g . 50 mM Hepes , 350 mM out as follows. As described herein , the amino acid sequence US 2017 /0240590 A1 Aug . 24 , 2017 76 of human growth hormone (HGH ) is first modified to example of this type of reagent is the Sunbright® CA series incorporate at least one glycosylation site . Following puri from NOF (NOF Corp . , Tokyo , Japan ). TNF - alpha is dis fication , HGH is glycosylated in vitro according to methods solved in 7 . 0 mlhistidine buffer, pH 6 .0 (20 mM L -histidine , known in the art . HGH is PEGylated by use of a linear 20 150 mM NaC1, 5 mM CaCl2 ). An aqueous sodium periodate KD PEGylation reagent containing an aminooxy group . An solution ( 5 mM ) is then added and the reaction mixture is example of this type of reagent is the Sunbright® CA series from NOF (NOF Corp . , Tokyo , Japan ) . HGH is dissolved in incubated for 1 h in the dark at 4° C . under gentle stirring Hepes buffer ( 50 mM Hepes, 150 mM sodium chloride , 5 and quenched for 15 min at room temperature by the mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The sodium periodate solution ( 10 mm ) , and an aqueous mixture is subsequently subjected to UF /DF employing m - toluidine solution (50 mm ) . Subsequently the aminooxy Vivaspin centrifugal filtrators to remove excess periodate , reagent is added to give a 20 - fold molar reagent excess . The quencher and the byproducts thereof. mixture is incubated for 2 h in the dark at room temperature [0655 ) The retentate containing oxidized TNF - alpha is under gentle stirring and quenched for 15 min at room next mixed with an aqueous m - toluidine solution ( 50 mM ) temperature by the addition of 8 ul of aqueous cysteine and incubated for 30 min at room temperature . Aminooxy solution ( 1 M ) . PEG reagent with a MW of 20 kD is then added to give a [ 0649 Finally, the PEG -HGH conjugate is purified by 5 - fold molar reagent excess . This mixture is incubated for ion - exchange chromatography. The conjugate containg frac 2 . 5 h at room temperature in the dark under gentle stirring . tions are collected and then subjected to UF /DF . The prepa (0656 ] Finally , the PEG - TNF - alpha conjugate is purified ration is analytically characterized by measuring total pro by ion -exchange chromatography ( e . g . , on Q - Sepharose tein (Bradford ) and biological activity according to methods FF ) . For example , 1 . 5 mg protein /ml gel is loaded on the known in the art . column equilibrated with 50 mM Hepes buffer , pH 7 . 4 Method 4 : containing 5 mM CaC12 . The conjugate is eluted with 50 [0650 ] As described herein , the amino acid sequence of mM Hepes buffer containing 5 mM CaCl2 and 500 mm human growth hormone (HGH ) is first modified to incor sodium chloride , pH 7 . 4 and is then subjected to UF /DF porate at least one glycosylation site . Following purification , using an appropriate MW cutoff membrane . The preparation HGH is glycosylated in vitro according to methods known is next analytically characterized by measuring total protein in the art. (Coomassie , Bradford ) and biological activity according to [0651 ] HGH is PEGylated by use of a linear 20 kD methods known in the art . PEGylation reagent containing an aminooxy group . An [0657 ] In an alternative embodiment, Method 1 is carried example of this type of reagent is the Sunbright® CA series out as follows. TNF - alpha is PEGylated by use of a linear 20 from NOF (NOF Corp ., Tokyo , Japan ) . An initial concen KD PEGylation reagent containing an aminooxy group . An tration or weight of HGH is transferred or dissolved in example of this type of reagent is the Sunbright® CA series Hepes buffer (50 mM Hepes, 150 mM sodium chloride , 5 from NOF (NOF Corp . , Tokyo , Japan ) . TNF - alpha is dis mM calcium chloride , pH 6 . 0 ) to get a final protein con solved in 7 . 0 mlhistidine buffer , pH 6 . 0 ( 20 mM L -histidine , centration of 2 mgHGH /ml . Subsequently an 5 mM aqueous 150 mM NaC1, 5 mM CaC12 ). An aqueous sodium periodate sodium periodate solution is added within 15 minutes to give solution ( 5 mM ) is then added and the reaction mixture is a final concentration of 100 uM , followed by addition of an incubated for 1 h in the dark at 4° C . under gentle stirring 50 mM aqueous m - toluidine solution to get a final concen and quenched for 15 min at room temperature by the tration of 10 mM within a time period of 30 minutes . Then addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The the aminooxy - PEG reagent with a MW of 20 kD ( described mixture is subsequently subjected to UF /DF employing above ) is added to give a 20 - fold molar reagent excess . After Vivaspin centrifugal filtrators to remove excess periodate , correction of the pH to 6 . 0 the mixture is incubated for 2 h quencher and the byproducts thereof. in the dark at room temperature under gentle stirring and [0658 ] The retentate containing oxidized TNF - alpha is quenched for 15 min at room temperature by the addition of next mixed with an aqueous m -toluidine solution (50 mM ) a 1 M aqueous L - cysteine solution to give a final concen and incubated for 30 min at room temperature . Aminooxy tration of 10 mM . PEG reagent with a MW of 20 kD is then added to give a [ 0652 ] The PEG - HGH conjugate is purified by means of 5 - fold molar reagent excess . This mixture is incubated for ion exchange chromatography ( IEC ). The conjugate con 2 . 5 h at room temperature in the dark under gentle stirring . taining fractions of the eluate are concentrated by UF /DF 106591. Finally , the PEG - TNF -alpha conjugate is purified using a 10 kD membrane made of regenerated cellulose (88 by ion -exchange chromatography . The conjugate containg cm2 , cutoff 10 kD /Millipore ) . The final diafiltration step is fractions of the eluate are collected and then subjected to performed against Hepes buffer ( 50 mM Hepes , 5 mM UF /DF using an appropriate MW cutoff membrane. The CaCl2 , PH 7 . 5 ) . preparation is next analytically characterized by measuring [ 0653] The preparation is analytically characterized by total protein (Coomassie , Bradford ) and biological activity measuring total protein (Bradford and BCA procedure ) and according to methods known in the art . biological activity according to known methods. Example 38 Method 2 : PEGylation of TNF - Alpha Using an [ 0660 ] TNF - alpha is PEGylated by use of a linear 20 kD Aminooxy -PEG Reagent and m - Toluidine as a PEGylation reagent containing an aminooxy group . An Nucleophilic Catalyst example of this type of reagent is the Sunbright® CA series from NOF (NOF Corp ., Tokyo , Japan ) . TNF - alpha is trans Method 1 : ferred or dissolved in reaction buffer (e . g. 50 mM Hepes, [0654 ] TNF -alpha is PEGylated by use of a linear 20 kD 350 mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) PEGylation reagent containing an aminooxy group . An to get a final protein concentration of 1 . 0 + / - 0 . 25 mg/ml . US 2017 /0240590 A1 Aug . 24 , 2017

Then the pH of the solution is corrected to 6 . 0 by drop wise nooxy reagent is added to give a 20 - fold molar reagent addition of a 0 .5N aqueous HC1 solution . Subsequently a 40 excess . The mixture is incubated for 2 h in the dark at room mM aqueous sodium periodate solution is added within 10 temperature under gentle stirring and quenched for 15 min minutes to give a concentration of 200 UM . The oxidation at room temperature by the addition of 8 ul of aqueous reaction is carried out for 30 + / – 5 min at a temperature ( T ) cysteine solution ( 1 M ) . of T = + 22 + / - 2° C . Then the reaction is stopped by addition [0668 ] Finally , the PEG - TNF - alpha conjugate is purified of an aqueous L - cysteine solution ( 1 M ) within 15 minutes by ion - exchange chromatography . The conjugate containing at T = + 22 + / - 2° C . to give a final concentration of 10 mM in fractions are collected and then subjected to UF /DF . The the reaction mixture and incubation for 60 + / - 5 min . preparation is analytically characterized by measuring total [ 0661 ] The oxidized TNF - alpha is further purified by ion protein (Bradford ) and biological activity according to meth exchange chromatography. The oxidized TNF - alpha con ods known in the art. taining fractions of the eluate are collected and used for the conjugation reaction . Method 4 : [ 0662 ] The aminooxy - PEG reagent with a MW of 20 kD [0669 ] TNF- alpha is PEGylated by use of a linear 20 kD reagent is added in a 50 - fold molar excess to the eluate PEGylation reagent containing an aminooxy group . An containing the purified oxidized TNF alpha within a maxi example of this type of reagent is the Sunbright® CA series mum time period (t ) of 15 minutes under gentle stirring . from NOF (NOF Corp ., Tokyo , Japan ). An initial concen Then an aqueous m - toluidine solution ( 50 mM ) is added tration or weight of TNF - alpha is transferred or dissolved in within 15 minutes to get a final concentration of 10 mM . The Hepes buffer (50 mM Hepes , 150 mM sodium chloride , 5 reaction mixture is incubated for 120 + / – 10 min . in the dark mM calcium chloride , pH 6 .0 ) to get a final protein con at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . centration of 2 mg TNF -alpha /ml . Subsequently , an 5 mM 10663 ) The obtained PEG - TNF - alpha conjugate is further aqueous sodium periodate solution is added within 15 min purified by ion exchange chromatography. The PEG - TNF utes to give a final concentration of 100 UM , followed by alpha conjugate containing fractions are collected and con addition of an 50 mM aqueous m - toluidine solution to get a centrated by ultra - /diafiltration (UF /DF ) using a membrane final concentration of 10 mM within a time period of 30 made of regenerated cellulose with an appropriate molecular minutes . Then the aminooxy - PEG reagent with a MW of 20 weight cut off (Millipore ) . kD (described above ) is added to give a 20 - fold molar [ 0664 ] The conjugate prepared by use of this procedure reagent excess . After correction of the pH to 6 . 0 the mixture are analytically characterized by measuring total protein and is incubated for 2 h in the dark at room temperature under biological activity according to methods known in the art . gentle stirring and quenched for 15 min at room temperature by the addition of an 1 M aqueous L -cysteine solution to Method 3 : give a final concentration of 10 mM . [ 0665 ) TNF -alpha is PEGylated by use of a linear 20 kD [0670 ] The PEG - TNF - alpha conjugate is purified by PEGylation reagent containing an aminooxy group . An means of ion exchange chromatography (IEC ) . The conju example of this type of reagent is the Sunbright® CA series gate containing fractions of the eluate are concentrated by from NOF (NOF Corp ., Tokyo , Japan ) . TNF - alpha is dis UF /DF using a 10 kD membrane made of regenerated solved in Hepes buffer (50 mM Hepes , 150 mM sodium cellulose (88 cm2, cutoff 10 kD /Millipore ) . The final dia chloride, 5 mM calcium chloride , pH 6 . 0 ) and mixed with an filtration step is performed against Hepes buffer (50 mM aqueous sodium periodate solution ( 10 mM ), and an aque Hepes, 5 mM CaC12 , PH 7 . 5 ) . ous m - toluidine solution (50 mm ) . Subsequently the ami [0671 ] The preparation is analytically characterized by nooxy reagent is added to give a 20 - fold molar reagent measuring total protein (Bradford and BCA procedure ) and excess . The mixture is incubated for 2 h in the dark at room biological activity according to known methods. temperature under gentle stirring and quenched for 15 min at room temperature by the addition of 8 ul of aqueous Example 39 cysteine solution ( 1 M ) . [0666 ] Finally , the PEG - TNF - alpha conjugate is purified PEGylation of Insulin Using an Aminooxy -PEG by ion -exchange chromatography on Q - Sepharose FF . 1 . 5 Reagent and m - Toluidine as a Nucleophilic mg protein /ml gel is loaded on the column pre equilibrated Catalyst with 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The conjugate is eluted with 50 mM Hepes buffer containing Method 1 : 5 mM CaC12 and 500 mM sodium chloride , pH 7 . 4 and is [0672 ] As described herein , the amino acid sequence of then subjected to UF /DF using a membrane . The preparation insulin is first modified to incorporate at least one glycosy is analytically characterized by measuring total protein lation site . Following purification , insulin is glycosylated in (Bradford ) and biological activity according to methods vitro according to methods known in the art. Insulin is known in the art . PEGylated by use of a linear 20 kD PEGylation reagent [0667 ] In an alternative embodiment, Method 3 is carried containing an aminooxy group . An example of this type of out as follows . TNF -alpha is PEGylated by use of a linear 20 reagent is the Sunbright® CA series from NOF (NOF Corp . , kD PEGylation reagent containing an aminooxy group . An Tokyo , Japan ) . Insulin is dissolved in 7 . 0 ml histidine buffer , example of this type of reagent is the Sunbright® CA series pH 6 . 0 ( 20 mM L -histidine , 150 mM NaC1, 5 mM CaC12 ) . from NOF (NOF Corp . , Tokyo , Japan ). TNF -alpha is dis An aqueous sodium periodate solution ( 5 mm ) is then added solved in Hepes buffer ( 50 mM Hepes , 150 mM sodium and the reaction mixture is incubated for 1 h in the dark at chloride , 5 mM calcium chloride, pH 6 . 0 ) and mixed with an 4° C . under gentle stirring and quenched for 15 min at room aqueous sodium periodate solution ( 10 mM ) , and an aque - temperature by the addition of 7 . 5 ul of a 1 M aqueous ous m - toluidine solution (50 mm ). Subsequently , the ami cysteine solution . The mixture is subsequently subjected to US 2017 /0240590 A1 Aug . 24 , 2017 78

UF/ DF employing Vivaspin centrifugal filtrators to remove of a 0 . 5 N aqueous HCl solution . Subsequently , a 40 mM excess periodate, quencher and the byproducts thereof. aqueous sodium periodate solution is added within 10 min [ 0673] The retentate containing oxidized insulin is next utes to give a concentration of 200 UM . The oxidation mixed with an aqueous m - toluidine solution (50 mM ) and reaction is carried out for 30 + / – 5 min at a temperature ( T ) incubated for 30 min at room temperature . Aminooxy - PEG of T = + 22 + / - 2° C . Then the reaction is stopped by addition reagent with a MW of 20 kD is then added to give a 5 - fold of an aqueous L -cysteine solution ( 1 M ) within 15 minutes molar reagent excess. This mixture is incubated for 2 . 5 h at at T = + 22 + / - 2° C . to give a final concentration of 10 mM in room temperature in the dark under gentle stirring . the reaction mixture and incubation for 60 + / - 5 min . 106741 Finally , the PEG - insulin conjugate is purified by [0680 ] The oxidized insulin is further purified by ion ion - exchange chromatography ( e . g . , on Q - Sepharose FF ) . exchange chromatography . The oxidized insulin containing For example , 1 . 5 mg protein /ml gel is loaded on the column fractions of the eluate are collected and used for the conju equilibrated with 50 mM Hepes buffer, pH 7 .4 containing 5 gation reaction . mM CaC12 . The conjugate is eluted with 50 mM Hepes [0681 ] The aminooxy -PEG reagent with a MW of 20 kD buffer containing 5 mM CaC12 and 500 mM sodium chlo reagent is added in a 50 - fold molar excess to the eluate ride , pH 7 . 4 and is then subjected to UF /DF using an containing the purified oxidized insulin within a maximum appropriate MW cutoff membrane . The preparation is next time period ( t ) of 15 minutes under gentle stirring . Then an analytically characterized by measuring total protein (Coo aqueous m - toluidine solution (50 mM ) is added within 15 massie , Bradford ) and biological activity according to meth minutes to get a final concentration of 10 mM . The reaction ods known in the art . mixture is incubated for 120 + / - 10 min . in the dark at a [0675 ] In an alternative embodiment, Method 1 is carried temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . out as follows. As described herein , the amino acid sequence [0682 ] The obtained PEG - insulin conjugate is further of insulin is first modified to incorporate at least one purified by ion exchange chromatography. The PEG - insulin glycosylation site . Following purification , insulin is glyco conjugate containing fractions are collected and concen sylated in vitro according to methods known in the art . trated by ultra - / diafiltration (UF /DF ) using a membrane Insulin is PEGylated by use of a linear 20 kD PEGylation made of regenerated cellulose with an appropriate molecular reagent containing an aminooxy group . An example of this weight cut off (Millipore ) . type of reagent is the Sunbright® CA series from NOF (NOF [ 0683] The conjugate prepared by use of this procedure Corp . , Tokyo , Japan ). Insulin is dissolved in 7 . 0 mlhistidine are analytically characterized by measuring total protein and buffer, pH 6 . 0 ( 20 mM L -histidine , 150 mM NaCl, 5 mM biological activity according to methods known in the art . CaCl2 ). An aqueous sodium periodate solution ( 5 mm ) is then added and the reaction mixture is incubated for 1 h in Method 3: the dark at 4° C . under gentle stirring and quenched for 15 [ 0684 ] As described herein , the amino acid sequence of min at room temperature by the addition of 7 . 5 al of a 1 M insulin is first modified to incorporate at least one glycosy aqueous cysteine solution . The mixture is subsequently lation site . Following purification , insulin is glycosylated in subjected to UF /DF employing Vivaspin centrifugal filtra vitro according to methods known in the art . tors to remove excess periodate , quencher and the byprod [0685 ] Insulin is PEGylated by use of a linear 20 kD ucts thereof. PEGylation reagent containing an aminooxy group . An [ 0676 ] The retentate containing oxidized insulin is next example of this type of reagent is the Sunbright® CA series mixed with an aqueous m - toluidine solution (50 mM ) and from NOF (NOF Corp ., Tokyo , Japan ) . Insulin is dissolved incubated for 30 min at room temperature . Aminooxy -PEG in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , reagent with a MW of 20 kD is then added to give a 5 - fold 5 mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous molar reagent excess . This mixture is incubated for 2 . 5 h at sodium periodate solution ( 10 mM ), and an aqueous room temperature in the dark under gentle stirring . m - toluidine solution ( 50 mm ) . Subsequently, the aminooxy [ 0677 ] Finally, the PEG -insulin conjugate is purified by reagent is added to give a 20 -fold molar reagent excess. The ion -exchange chromatography. The conjugate containg frac mixture is incubated for 2 h in the dark at room temperature tions of the eluate are collected and then subjected to UF /DF under gentle stirring and quenched for 15 min at room using an appropriate MW cutoff membrane . The preparation temperature by the addition of 8 ul of aqueous cysteine is next analytically characterized by measuring total protein solution ( 1 M ) . ( Coomassie , Bradford ) and biological activity according to [0686 ] Finally, the PEG - insulin conjugate is purified by methods known in the art. ion - exchange chromatography on Q Sepharose FF . 1 . 5 mg protein /ml gel is loaded on the column pre equilibrated with Method 2 : 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The [0678 ] As described herein , the amino acid sequence of conjugate is eluted with 50 mM Hepes buffer containing 5 insulin is first modified to incorporate at least one glycosy mM CaCl2 and 500 mM sodium chloride , pH 7 . 4 and is then lation site . Following purification , insulin is glycosylated in subjected to UF /DF using a membrane . The preparation is vitro according to methods known in the art. analytically characterized by measuring total protein (Brad [0679 ] Insulin is PEGylated by use of a linear 20 kD ford ) and biological activity according to methods known in PEGylation reagent containing an aminooxy group . An the art . example of this type of reagent is the Sunbright® CA series 0687 ] In an alternative embodiment, Method 3 is carried from NOF (NOF Corp ., Tokyo , Japan ). Insulin is transferred out as follows. As described herein , the amino acid sequence or dissolved in reaction buffer (e . g. 50 mM Hepes , 350 mM of insulin is first modified to incorporate at least one sodium chloride, 5 mM calcium chloride, pH 6 . 0 ) to get a glycosylation site . Following purification , insulin is glyco final protein concentration of 1 . 0 + / - 0 . 25 mg/ ml . Then the sylated in vitro according to methods known in the art. pH of the solution is corrected to 6 . 0 by drop wise addition Insulin is PEGylated by use of a linear 20 kD PEGylation US 2017 /0240590 A1 Aug . 24 , 2017

reagent containing an aminooxy group . An example of this dissolved in 7 . 0 ml histidine buffer , pH 6 . 0 ( 20 mM L - his type of reagent is the Sunbright® CA series from NOF (NOF tidine , 150 mM NaC1, 5 mM CaC12 ) . An aqueous sodium Corp ., Tokyo , Japan ) . Insulin is dissolved in Hepes buffer periodate solution ( 5 mm ) is then added and the reaction (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium mixture is incubated for 1 h in the dark at 4° C . under gentle chloride , pH 6 . 0 ) and mixed with an aqueous sodium stirring and quenched for 15 min at room temperature by the periodate solution ( 10 mM ), and an aqueous m -toluidine addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The solution ( 50 mm ) . Subsequently the aminooxy reagent is mixture is subsequently subjected to UF /DF employing added to give a 20 - fold molar reagent excess . The mixture Vivaspin centrifugal filtrators to remove excess periodate , is incubated for 2 h in the dark at room temperature under quencher and the byproducts thereof . gentle stirring and quenched for 15 min at room temperature [0694 ] The retentate containing oxidized interferon -alpha by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . is next mixed with an aqueous m - toluidine solution (50 mm ) [ 0688 ) Finally , the insulin - conjugate is purified by ion - and incubated for 30 min at room temperature . Aminooxy exchange chromatography. The conjugate containing frac PEG reagent with a MW of 20 kD is then added to give a tions are collected and then subjected to UF /DF . The prepa 5 - fold molar reagent excess . This mixture is incubated for ration is analytically characterized by measuring total 2 . 5 h at room temperature in the dark under gentle stirring. protein (Bradford ) and biological activity according to meth [0695 ] Finally , the PEG - interferon - alpha conjugate is ods known in the art . purified by ion - exchange chromatography ( e . g . , on Q - Sep harose FF ) . For example , 1 . 5 mg protein /ml gel is loaded on Method 4 : the column equilibrated with 50 mM Hepes buffer, pH 7 . 4 [ 0689 ] As described herein , the amino acid sequence of containing 5 mM CaC12 . The conjugate is eluted with 50 insulin is first modified to incorporate at least one glycosy mM Hepes buffer containing 5 mM CaC12 and 500 mm lation site . Following purification , insulin is glycosylated in sodium chloride , pH 7 . 4 and is then subjected to UF /DF vitro according to methods known in the art . using an appropriate MW cutoff membrane . The preparation [ 0690 ] Insulin is PEGylated by use of a linear 20 kD is next analytically characterized by measuring total protein PEGylation reagent containing an aminooxy group . An (Coomassie , Bradford ) and biological activity according to example of this type of reagent is the Sunbright® CA series methods known in the art. from NOF (NOF Corp . , Tokyo , Japan ) . An initial concen [0696 ] In an alternative embodiment, Method 1 is carried tration or weight of insulin is transferred or dissolved in out as follows. Interferon - alpha is PEGylated by use of a Hepes buffer ( 50 mM Hepes , 150 mM sodium chloride , 5 linear 20 kD PEGylation reagent containing an aminooxy mM calcium chloride , pH 6 . 0 ) to get a final protein con group . An example of this type of reagent is the Sunbright® centration of 2 mg insulin /ml . Subsequently an 5 mM CA series from NOF (NOF Corp . , Tokyo , Japan ) . Interferon aqueous sodium periodate solution is added within 15 min alpha is dissolved in 7 . 0 ml histidine buffer, pH 6 . 0 ( 20 mM utes to give a final concentration of 100 UM , followed by L -histidine , 150 mM NaCl, 5 mM CaC12 ) . An aqueous addition of an 50 mM aqueous m - toluidine solution to get a sodium periodate solution (5 mm ) is then added and the final concentration of 10 mM within a time period of 30 reaction mixture is incubated for 1 h in the dark at 4° C . minutes . Then the aminooxy - PEG reagentwith a MW of 20 under gentle stirring and quenched for 15 min at room kD ( described above ) is added to give a 20 - fold molar temperature by the addition of 7 . 5 ul of a 1 M aqueous reagent excess . After correction of the pH to 6 . 0 the mixture cysteine solution . The mixture is subsequently subjected to is incubated for 2 h in the dark at room temperature under UF /DF employing Vivaspin centrifugal filtrators to remove gentle stirring and quenched for 15 min at room temperature excess periodate , quencher and the byproducts thereof. by the addition of a 1 M aqueous L -cysteine solution to give 0697 ] The retentate containing oxidized interferon -alpha a final concentration of 10 mM . is next mixed with an aqueous m - toluidine solution (50 mM ) [0691 ] The PEG - insulin conjugate is purified by means of and incubated for 30 min at room temperature . Aminooxy ion exchange chromatography ( IEC ). The conjugate con PEG reagent with a MW of 20 kD is then added to give a taining fractions of the eluate are concentrated by UF /DF 5 - fold molar reagent excess . This mixture is incubated for using a 10 kD membrane made of regenerated cellulose (88 2 . 5 h at room temperature in the dark under gentle stirring . cm2, cut - off 10 kD /Millipore ). The final diafiltration step is [0698 ] Finally , the PEG - interferon - alpha conjugate is performed against Hepes buffer ( 50 mM Hepes, 5 mM purified by ion - exchange chromatography The conjugate CaC12 , pH 7. 5 ). containing freactions are collected and then subjected to [ 0692 ] The preparation is analytically characterized by UF /DF using an appropriate MW cutoff membrane . The measuring total protein (Bradford and BCA procedure ) and preparation is next analytically characterized by measuring biological activity according to known methods. total protein ( Coomassie , Bradford ) and biological activity according to methods known in the art. Example 40 Method 2 : PEGylation of Interferon -Alpha Using an [0699 ] Interferon - alpha is PEGylated by use of a linear 20 Aminooxy -PEG Reagent and m - Toluidine as a kD PEGylation reagent containing an aminooxy group . An Nucleophilic Catalyst example of this type of reagent is the Sunbright® CA series from NOF (NOF Corp ., Tokyo , Japan ) . Interferon - alpha is Method 1: transferred or dissolved in reaction buffer ( e. g . 50 mM [ 0693 ] Interferon -alpha is PEGylated by use of a linear 20 Hepes , 350 mM sodium chloride , 5 mM calcium chloride , kD PEGylation reagent containing an aminooxy group . An pH 6 . 0 ) to get a final protein concentration of 1 .0 + / - 0 .25 example of this type of reagent is the Sunbright® CA series mg/ ml . Then the pH of the solution is corrected to 6 . 0 by from NOF (NOF Corp ., Tokyo , Japan ). Interferon -alpha is drop wise addition of a 0 . 5 N aqueous HCl solution . Sub US 2017 /0240590 A1 Aug . 24 , 2017 80

sequently , a 40 mM aqueous sodium periodate solution is excess . The mixture is incubated for 2 h in the dark at room added within 10 minutes to give a concentration of 200 uM . temperature under gentle stirring and quenched for 15 min The oxidation reaction is carried out for 30 + / - 5 min at a at room temperature by the addition of 8 ul of aqueous temperature ( T ) of T = + 22 + / - 2° C . Then the reaction is cysteine solution ( 1 M ) . stopped by addition of an aqueous L - cysteine solution ( 1 M ) [ 0707 ] Finally , the PEG - interferon - alpha conjugate is within 15 minutes at T = + 22 +/ - 2° C . to give a final concen purified by ion - exchange chromatography . The conjugate tration of 10 mM in the reaction mixture and incubation for containg fractions are collected and then subjected to UF /DF 60 + / - 5 min . using a membrane . The preparation is analytically charac [ 0700 ] The oxidized interferon - alpha is further purified by terized by measuring total protein ( Bradford ) and biological ion exchange chromatography. The oxidized interferon activity according to methods known in the art. alpha containing fractions of the eluate are collected and used for the conjugation reaction . Method 4 : [ 0701 ] The aminooxy - PEG reagent with a MW of 20 kD reagent is added in a 50 - fold molar excess to the eluate [0708 ] Interferon - alpha is PEGylated by use of a linear 20 containing the purified oxidized interferon - alpha within a KD PEGylation reagent containing an aminooxy group . An maximum time period ( t ) of 15 minutes under gentle stirring . example of this type of reagent is the Sunbright® CA series Then an aqueous m - toluidine solution ( 50 mM ) is added from NOF (NOF Corp ., Tokyo , Japan ). An initial concen within 15 minutes to get a final concentration of 10 mM . The tration or weight of interferon -alpha is transferred or dis reaction mixture is incubated for 120 + / – 10 min . in the dark solved in Hepes buffer (50 mM Hepes , 150 mM sodium at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . chloride, 5 mM calcium chloride , pH 6 . 0 ) to get a final [0702 ] The obtained PEG -interferon - alpha conjugate is protein concentration of 2 mg interferon - alpha /ml . Subse further purified by ion exchange chromatography. The PEG quently , an 5 mM aqueous sodium periodate solution is interferon alpha conjugate containing fractions are collected added within 15 minutes to give a final concentration of 100 and concentrated by ultra - / diafiltration (UF /DF ) using a UM , followed by addition of an 50 mM aqueous m - toluidine membrane made of regenerated cellulose with an appropri solution to get a final concentration of 10 mM within a time ate molecular weight cut off (Millipore ) . period of 30 minutes . Then the aminooxy - PEG reagent with [ 0703 ] The conjugate prepared by use of this procedure a MW of 20 kD (described above ) is added to give a 20 - fold are analytically characterized by measuring total protein and molar reagent excess . After correction of the pH to 6 . 0 the mixture is incubated for 2 h in the dark at room temperature biological activity according to methods known in the art. under gentle stirring and quenched for 15 min at room Method 3 : temperature by the addition of an 1 M aqueous L - cysteine solution to give a final concentration of 10 mM . [0704 ] Interferon - alpha is PEGylated by use of a linear 20 [0709 ] The PEG -interferon - alpha conjugate is purified by kD PEGylation reagent containing an aminooxy group . An means of ion exchange chromatography ( IEC ) . The conju example of this type of reagent is the Sunbright® CA series gate containing fractions of the eluate are concentrated by from NOF (NOF Corp . , Tokyo , Japan ) . Interferon -alpha is UF/ DF using a 10 kD membrane made of regenerated dissolved in Hepes buffer ( 50 mM Hepes, 150 mM sodium cellulose ( 88 cm2 , cutoff 10 kD /Millipore ) . The final dia chloride , 5 mM calcium chloride, pH 6 . 0 ) and mixed with an filtration step is performed against Hepes buffer (50 mM aqueous sodium periodate solution ( 10 mM ) , and an aque Hepes, 5 mM CaCl2 , pH 7 . 5 ) . ous m - toluidine solution (50 mm ) . Subsequently the ami [ 0710 ] The preparation is analytically characterized by nooxy reagent is added to give a 20 -fold molar reagent measuring total protein (Bradford and BCA procedure ) and excess . The mixture is incubated for 2 h in the dark at room temperature under gentle stirring and quenched for 15 min biological activity according to known methods. at room temperature by the addition of 8 ul of aqueous cysteine solution ( 1 M ) . Example 41 [ 0705 ] Finally , the PEG - interferon -alpha conjugate is PEGylation of Interferon -Gamma Using an purified by ion -exchange chromatography on Q -Sepharose Aminooxy - PEG Reagent and m - Toluidine as a FF . 1 . 5 mg protein /ml gel is loaded on the column pre Nucleophilic Catalyst equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The conjugate is eluted with 50 mM Hepes buffer containing 5 mM CaC12 and 500 mM sodium chlo Method 1 : ride , pH 7 . 4 and is then subjected to UF /DF using a [0711 ] Interferon - gamma is PEGylated by use of a linear membrane . The preparation is analytically characterized by 20 kD PEGylation reagent containing an aminooxy group . measuring total protein (Bradford ) and biological activity An example of this type of reagent is the Sunbright® CA according to methods known in the art . series from NOF (NOF Corp ., Tokyo , Japan ). 10 mg Inter [0706 ] In an alternative embodiment, Method 3 is carried feron - gamma is dissolved in 5 ml histidine buffer, pH 6 .0 ( 20 out as follows. Interferon - alpha is PEGylated by use of a mM L -histidine , 150 mM NaCl) . 100 ul of an aqueous linear 20 kD PEGylation reagent containing an aminooxy sodium periodate solution (5 mM ) is then added and the group . An example of this type of reagent is the Sunbright® reaction mixture is incubated for 1 h in the dark at 4° C . CA series from NOF (NOF Corp ., Tokyo , Japan ). Interferon under gentle stirring and quenched for 15 min at room alpha is dissolved in Hepes buffer (50 mM Hepes , 150 mm temperature by the addition of 50 ul of a 1 M aqueous sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) and mixed cysteine solution . The mixture is subsequently subjected to with an aqueous sodium periodate solution ( 10 mM ), and an UF/ DF employing Vivaspin 15R 10 kD centrifugal filtrators aqueous m - toluidine solution ( 50 mm ) . Subsequently the to remove excess periodate , quencher and the byproducts aminooxy reagent is added to give a 20 - fold molar reagent thereof. US 2017 /0240590 A1 Aug . 24 , 2017

[ 0712] The retentate ( approx . 7 ml) , containing oxidized interferon - gamma conjugate containing fractions are col interferon - gamma, is mixed with 2 ml of an aqueous lected and concentrated by ultra - /diafiltration ( UF /DF ) using m - toluidine solution ( 50 mM ) and incubated for 30 min at a membrane made of regenerated cellulose with an appro room temperature. Then aminooxy -PEG reagent with a MW priate molecular weight cut off (Millipore ) . of 20 kD ( described above ) is added to give a 5 - fold molar [0718 ] The conjugate prepared by use of this procedure reagent excess . This mixture is incubated for 2 . 5 h at RT in are analytically characterized by measuring total protein and the dark under gentle stirring . biological activity according to methods known in the art. [0713 ] Finally , the PEG - interferon -gamma conjugate is purified by ion - exchange chromatography on SP Sepharose Method 3 : FF . The reaction mixture is diluted with 20 ml Buffer A (50 mM Hepes , pH 6 . 5 ) and loaded onto a 20 ml HiPrep SPFF [0719 ] Interferon - gamma is PEGylated by use of a linear 16 / 10 column (GE Healthcare , Fairfield , Conn . ) pre -equili 20 kD PEGylation reagent containing an aminooxy group . brated with Buffer A . Then the column is eluted with Buffer An example of this type of reagent is the Sunbright® CA B (50 mM Hepes, 1 M NaCl, pH 6 . 5 ) . Free interferon series from NOF (NOF Corp ., Tokyo , Japan ) . 10 mg inter gamma is eluted by washing the column with 25 % Buffer B feron - gamma is dissolved in ~ 8 ml histidine- buffer , pH 6 . 0 and the conjugate at 50 % Buffer B . The conjugate contain ( 20 mM L -histidine , 150 mM NaCl) . 200 ul of an aqueous ing fractions are concentrated by UF/ DF using a 10 kD sodium periodate solution ( 5 mM ) and 2 ml of an aqueous membrane made of regenerated cellulose ( 88 cm2, cutoff 10 m - toluidine solution (50 mm ) are then added . Subsequently kD /Millipore ) . The final diafiltration step is performed the aminooxy - PEG reagent with a MW of 20 kD (described against histidine buffer , pH 6 . 9 containing 150 mM NaCl. above ) is added to give a 5 - fold molar reagent excess. The The preparation is analytically characterized by measuring mixture is incubated for 2 h in the dark at room temperature total protein (Bradford ) and biological activity according to under gentle stirring and quenched for 15 min at room methods known in the art. For the PEG - interferon - gamma temperature by the addition of 100 ul of 1 M aqueous conjugate a specific activity of > 50 % in comparison to cysteine solution . native Interferon gamma is determined . The conjugate is [0720 ] Finally the PEG - interferon -gamma conjugate is additionally analytically characterized by Size Exclusion purified by ion - exchange chromatography on SP - Sepharose HPLC using a Agilent 1200 HPLC system equipped with a FF . The reaction mixture is diluted with 20 ml Buffer A (50 Shodex KW 803 column under conditions as previously mM Hepes, pH 6 . 5 ) and loaded onto a 20 mlHiPrep SP FF described ( Kolarich et al, Transfusion 2006 ; 46 : 1959 - 77 ) . It 16 / 10 column (GE Healthcare , Fairfield , Conn . ) pre - equili is shown that the preparation contains no free Interferon brated with Buffer A . Then the column is eluted with Buffer gamma . B (50 mM Hepes, 1 M NaC1, pH 6 . 5 ) . Free intergferon gamma is eluted by washing the column with 25 % Buffer B Method 2 : and the conjugate at 50 % Buffer B . The conjugate contain ing fractions are concentrated by UF/ DF using a 10 kD [ 0714 ] Interferon - gamma is PEGylated by use of a linear membrane made of regenerated cellulose ( 88 cm2, cut -off 10 20 kD PEGylation reagent containing an aminooxy group . kD Millipore/ ) . The final diafiltration step is performed An example of this type of reagent is the Sunbright® CA against histidine buffer , pH 6 . 9 containing 150 mM NaCl. series from NOF (NOF Corp . , Tokyo , Japan ) . Interferon The preparation is analytically characterized by measuring gamma is transferred or dissolved in reaction buffer ( e . g . 50 total protein ( Bradford ) and biological activity according to mM Hepes , 350 mM sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) to get a final protein concentration of methods known in the art. For the PEG - interferon - gamma 1 .0 + / - 0 .25 mg /ml . Then the pH of the solution is corrected conjugate a specific activity of > 50 % in comparison to to 6 . 0 by drop wise addition of a 0 . 5 N aqueous HC1 native interferon - gamma is determined . The conjugate is solution . Subsequently a 40 mM aqueous sodium periodate additionally analytically characterized by Size Exclusion solution is added within 10 minutes to give a concentration HPLC using a Agilent 1200 HPLC system equipped with a of 200 uM . The oxidation reaction is carried out for 30 + / - 5 Shodex KW 803 column under conditions as previously min at a temperature ( T ) ofT = + 22 +/ - 2° C . Then the reaction described (Kolarich et al, Transfusion 2006 ; 46 : 1959- 77 ) . It is stopped by addition of an aqueous L - cysteine solution ( 1 is shown that the preparation contains no free interferon M ) within 15 minutes at T = + 22 + / - 2° C . to give a final gamma. concentration of 10 mM in the reaction mixture and incu bation for 60 + / - 5 min . Method 4 : [0715 ] The oxidized interferon - gamma is further purified [0721 ] Interferon - gamma is PEGylated by use of a linear by ion exchange chromatography. The oxidized interferon 20 kD PEGylation reagent containing an aminooxy group . gamma containing fractions of the eluate are collected and An example of this type of reagent is the Sunbright® CA used for the conjugation reaction . series from NOF (NOF Corp . , Tokyo , Japan ). An initial [ 0716 ] The aminooxy -PEG reagent with a MW of 20 kD concentration or weight of interferon - gamma is transferred reagent is added in a 50 -fold molar excess to the eluate or dissolved in Hepes buffer ( 50 mM Hepes , 150 MM containing the purified oxidized interferon - gamma within a sodium chloride , 5 mM calcium chloride, pH 6 .0 ) to get a maximum time period ( t ) of 15 minutes under gentle stirring . final protein concentration of 2 mg interferon - gamma /ml . Then an aqueous m - toluidine solution ( 50 mM ) is added Subsequently an 5 mM aqueous sodium periodate solution is within 15 minutes to get a final concentration of 10 mM . The added within 15 minutes to give a final concentration of 100 reaction mixture is incubated for 120 + / – 10 min . in the dark UM , followed by addition of an 50 mM aqueous m - toluidine at a temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . solution to get a final concentration of 10 mM within a time [0717 ] The obtained PEG - interferon - gamma conjugate is period of 30 minutes . Then the aminooxy -PEG reagent with further purified by ion exchange chromatography. The PEG a MW of 20 kD (described above ) is added to give a 20 - fold US 2017 /0240590 A1 Aug . 24 , 2017 82 molar reagent excess. After correction of the pH to 6 . 0 the mixture is subsequently subjected to UF /DF employing mixture is incubated for 2 h in the dark at room temperature Vivaspin centrifugal filtrators to remove excess periodate , under gentle stirring and quenched for 15 min at room quencher and the byproducts thereof. temperature by the addition of an 1 M aqueous L - cysteine [0728 ] The retentate containing oxidized G -CSF is next solution to give a final concentration of 10 mM . mixed with an aqueous m - toluidine solution (50 mM ) and [0722 ] The PEG - interferon -gamma conjugate is purified incubated for 30 min at room temperature . Aminooxy -PEG by means of ion exchange chromatography ( IEC ) . The reagent with a MW of 20 kD is then added to give a 5 - fold conjugate containing fractions of the eluate are concentrated molar reagent excess . This mixture is incubated for 2 . 5 h at by UF /DF using a 10 kD membrane made of regenerated room temperature in the dark under gentle stirring . cellulose (88 cm2, cutoff 10 kD /Millipore ) . The final dia [0729 ] Finally , the PEG -G -CSF conjugate is purified by filtration step is performed against Hepes buffer (50 mM ion -exchange chromatography ( The conjugate containg Hepes, 5 mM CaCl2 , pH 7 . 5 ) . fractions of the eluate are collected and then subjected to [0723 ] The preparation is analytically characterized by UF/ DF using an appropriate MW cutoff membrane . The measuring total protein (Bradford and BCA procedure ) and preparation is next analytically characterized by measuring biological activity according to known methods. total protein (Coomassie , Bradford ) and biological activity according to methods known in the art . Example 42 Method 2 : PEGylation of G -CSF Using an Aminooxy -PEG Reagent and m - Toluidine as a Nucleophilic [0730 ] G -CSF is PEGylated by use of a linear 20 kD Catalyst PEGylation reagent containing an aminooxy group . An example of this type of reagent is the Sunbright® CA series Method 1 : from NOF (NOF Corp . , Tokyo , Japan ). G - CSF is transferred or dissolved in reaction buffer (e . g . 50 mM Hepes, 350 mM [0724 ] G - CSF is PEGylated by use of a linear 20 kD sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) to get a PEGylation reagent containing an aminooxy group . An final protein concentration of 1 . 0 + / - 0 . 25 mg/ ml . Then the example of this type of reagent is the Sunbright® CA series pH of the solution is corrected to 6 . 0 by drop wise addition from NOF ( NOF Corp . , Tokyo , Japan ). G -CSF is dissolved of a 0 .5N aqueous HCl solution . Subsequently a 40 mM in 7 . 0 ml histidine buffer , pH 6 . 0 (20 mM L - histidine, 150 aqueous sodium periodate solution is added within 10 min mM NaCl, 5 mM CaCl2 ). An aqueous sodium periodate utes to give a concentration of 200 UM . The oxidation solution (5 mm ) is then added and the reaction mixture is reaction is carried out for 30 + / - 5 min at a temperature ( T ) incubated for 1 h in the dark at 4° C . under gentle stirring of T = + 22 + / - 2° C . Then the reaction is stopped by addition and quenched for 15 min at room temperature by the of an aqueous L -cysteine solution ( 1 M ) within 15 minutes addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The at T = + 22 + / - 2° C . to give a final concentration of 10 mM in mixture is subsequently subjected to UF /DF employing the reaction mixture and incubation for 60 + / - 5 min . Vivaspin centrifugal filtrators to remove excess periodate , [0731 ] The oxidized G -CSF is further purified by ion quencher and the byproducts thereof . exchange chromatography . The oxidized G -CSF containing [0725 ] The retentate containing oxidized G - CSF is next fractions of the eluate are collected and used for the conju mixed with an aqueous m -toluidine solution (50 mM ) and gation reaction . incubated for 30 min at room temperature . Aminooxy - PEG reagent with a MW of 20 kD is then added to give a 5 - fold [0732 ] The aminooxy -PEG reagent with a MW of 20 kD molar reagent excess . This mixture is incubated for 2 . 5 h at reagent is added in a 50 - fold molar excess to the eluate room temperature in the dark under gentle stirring . containing the purified oxidized G - CSF within a maximum [0726 ] Finally , the PEG - G -CSF conjugate is purified by time period (t ) of 15 minutes under gentle stirring . Then an ion - exchange chromatography ( e . g . , on Q - Sepharose FF ) . aqueous m - toluidine solution (50 mM ) is added within 15 For example , 1 . 5 mg protein /ml gel is loaded on the column minutes to get a final concentration of 10 mM . The reaction equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 mixture is incubated for 120 + / – 10 min . in the dark at a mM CaC12 . The conjugate is eluted with 50 mM Hepes temperature ( T ) of T = + 22 +/ - 2° C . under gentle shaking . buffer containing 5 mM CaCl2 and 500 mM sodium chlo [0733 ] The obtained PEG -G -CSF conjugate is further ride, pH 7 . 4 and is then subjected to UF /DF using an purified by ion exchange chromatography . The PEG - G - CSF appropriate MW cutoff membrane. The preparation is next conjugate containing fractions are collected and concen analytically characterized by measuring total protein (Coo trated by ultra - /diafiltration (UF / DF ) using a membrane massie , Bradford ) and biological activity according to meth made of regenerated cellulose with an appropriate molecular ods known in the art . weight cut off (Millipore ) . 10727 ]. In an alternative embodiment, Method 1 is carried out as follows. G -CSF is PEGylated by use of a linear 20 kD Method 3 : PEGylation reagent containing an aminooxy group . An [0734 ] G - CSF is PEGylated by use of a linear 20 kD example of this type of reagent is the Sunbright® CA series PEGylation reagent containing an aminooxy group . An from NOF (NOF Corp . , Tokyo , Japan ) . G - CSF is dissolved example of this type of reagent is the Sunbright® CA series in 7 . 0 ml histidine buffer, pH 6 . 0 (20 mM L - histidine , 150 from NOF (NOF Corp . , Tokyo , Japan ) . G -CSF is dissolved mM NaCl, 5 mM CaCl2 ). An aqueous sodium periodate in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , solution ( 5 mM ) is then added and the reaction mixture is 5 mM calcium chloride , pH 6 . 0 ) and mixed with an aqueous incubated for 1 h in the dark at 4° C . under gentle stirring sodium periodate solution (10 mm ), and an aqueous and quenched for 15 min at room temperature by the m - toluidine solution (50 mm ) . Subsequently , the aminooxy addition of 7 .5 ul of a 1 M aqueous cysteine solution . The reagent is added to give a 20 - fold molar reagent excess. The US 2017 /0240590 A1 Aug . 24 , 2017 mixture is incubated for 2 h in the dark at room temperature [0740 ] The preparation is analytically characterized by under gentle stirring and quenched for 15 min at room measuring total protein (Bradford and BCA procedure ) and temperature by the addition of 8 ul of aqueous cysteine biological activity according to known methods . solution (1 M ). [ 0735 ] Finally , the PEG - G -CSF conjugate is purified by Example 43 ion -exchange chromatography on Q -Sepharose FF . 1. 5 mg protein /ml gel is loaded on the column pre equilibrated with PEGylation of Humira Using an Aminooxy -PEG 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The Reagent and m - Toluidine as a Nucleophilic conjugate is eluted with 50 mM Hepes buffer containing 5 Catalyst mM CaC12 and 500 mM sodium chloride, pH 7 . 4 and is then subjected to UF/ DF using a membrane. The preparation is Method 1 : analytically characterized by measuring total protein (Brad [ 0741 ] Humira is PEGylated by use of a linear 20 kD ford ) and biological activity according to methods known in PEGylation reagent containing an aminooxy group . An the art . example of this type of reagent is the Sunbright® CA series [0736 ] In an alternative embodiment, Method 3 is carried from NOF (NOF Corp . , Tokyo , Japan ) . Humira is dissolved out as follows. G - CSF is PEGylated by use of a linear 20 kD in 7 . 0 ml histidine buffer , pH 6 . 0 ( 20 mM L - histidine , 150 PEGylation reagent containing an aminooxy group . An mM NaCl, 5 mM CaCl2 ) . An aqueous sodium periodate example of this type of reagent is the Sunbright® CA series solution (5 mM ) is then added and the reaction mixture is from NOF (NOF Corp ., Tokyo , Japan ). G - CSF is dissolved incubated for 1 h in the dark at 4° C . under gentle stirring in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , and quenched for 15 min at room temperature by the 5 mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The sodium periodate solution ( 10 mm ) , and an aqueous mixture is subsequently subjected to UF /DF employing m - toluidine solution (50 mm ) . Subsequently , the aminooxy Vivaspin centrifugal filtrators to remove excess periodate , reagent is added to give a 20 - fold molar reagent excess . The quencher and the byproducts thereof. mixture is incubated for 2 h in the dark at room temperature [0742 ] The retentate containing oxidized Humira is next under gentle stirring and quenched for 15 min at room mixed with an aqueous m - toluidine solution (50 mM ) and temperature by the addition of 8 ul of aqueous cysteine incubated for 30 min at room temperature . Aminooxy -PEG solution ( 1 M ) . reagent with a MW of 20 kD is then added to give a 5 - fold molar reagent excess. This mixture is incubated for 2 . 5 h at [ 0737 ] Finally , the PEG - G - CSF conjugate is purified by room temperature in the dark under gentle stirring . ion - exchange chromatography. The conjugate containing [0743 ] Finally , the PEG -Humira conjugate is purified by fractions of the eluate are collected and then subjected to ion -exchange chromatography (e .g . , on Q - Sepharose FF ). UF /DF using a membrane . The preparation is analytically For example , 1 . 5 mg protein /ml gel is loaded on the column characterized by measuring total protein (Bradford ) and equilibrated with 50 mM Hepes buffer, pH 7 .4 containing 5 biological activity according to methods known in the art. mM CaC12 . The conjugate is eluted with 50 mM Hepes buffer containing 5 mM CaC12 and 500 mM sodium chlo Method 4 : ride , pH 7 . 4 and is then subjected to UF /DF using an appropriate MW cutoff membrane . The preparation is next [ 0738 ] G - CSF is PEGylated by use of a linear 20 kD analytically characterized by measuring total protein (Coo PEGylation reagent containing an aminooxy group . An massie , Bradford ) and biological activity according to meth example of this type of reagent is the Sunbright® CA series ods known in the art . from NOF (NOF Corp ., Tokyo , Japan ) . An initial concen [0744 ] In an alternative embodiment, Method 1 is carried tration or weight of G -CSF is transferred or dissolved in out as follows. Humira is PEGylated by use of a linear 20 kD Hepes buffer (50 mM Hepes, 150 mM sodium chloride , 5 PEGylation reagent containing an aminooxy group . An mM calcium chloride , pH 6 .0 ) to get a final protein con example of this type of reagent is the Sunbright® CA series centration of 2 mg G -CSF /ml . Subsequently , an 5 mM from NOF (NOF Corp ., Tokyo , Japan ) . Humira is dissolved aqueous sodium periodate solution is added within 15 min in 7 . 0 ml histidine buffer , pH 6 . 0 ( 20 mM L - histidine , 150 utes to give a final concentration of 100 uM , followed by mM NaCl, 5 mM CaC12 ) . An aqueous sodium periodate addition of an 50 mM aqueous m - toluidine solution to get a solution (5 mM ) is then added and the reaction mixture is final concentration of 10 mM within a time period of 30 incubated for 1 h in the dark at 4° C . under gentle stirring minutes. Then the aminooxy - PEG reagent with a MW of 20 and quenched for 15 min at room temperature by the kD ( described above ) is added to give a 20 - fold molar addition of 7 . 5 ul of a 1 M aqueous cysteine solution . The reagent excess . After correction of the pH to 6 . 0 the mixture mixture is subsequently subjected to UF /DF employing is incubated for 2 h in the dark at room temperature under Vivaspin centrifugal filtrators to remove excess periodate , gentle stirring and quenched for 15 min at room temperature quencher and the byproducts thereof. by the addition of an 1 M aqueous L -cysteine solution to [0745 ] The retentate containing oxidized Humira is next give a final concentration of 10 mM . mixed with an aqueous m - toluidine solution (50 mM ) and [0739 ] The G -CSF conjugate is purified by means of ion incubated for 30 min at room temperature . Aminooxy - PEG exchange chromatography (IEC ) . The conjugate containing reagent with a MW of 20 kD is then added to give a 5 - fold fractions of the eluate are concentrated by UF /DF using a 10 molar reagent excess . This mixture is incubated for 2 . 5 h at KD membrane made of regenerated cellulose ( 88 cm2, room temperature in the dark under gentle stirring . cutoff 10 kD /Millipore ). The final diafiltration step is per [0746 ] Finally , the PEG -Humira conjugate is purified by formed against Hepes buffer (50 mM Hepes , 5 mM CaCl2 , ion -exchange chromatography . The conjugate containg frac pH 7 . 5 ) . tions of the eluate are collected and then subjected to UF /DF US 2017 /0240590 A1 Aug . 24 , 2017 84 using an appropriate MW cutoffmembrane . 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 the art . methods known in the art . [0753 ] In an alternative embodiment, Method 3 is carried out as follows. Humira is PEGylated by use of a linear 20 kD Method 2 : PEGylation reagent containing an aminooxy group . An example of this type of reagent is the Sunbright® CA series [ 0747] Humira is PEGylated by use of a linear 20 kD from NOF ( NOF Corp ., Tokyo , Japan ). Humira is dissolved PEGylation reagent containing an aminooxy group . An in Hepes buffer ( 50 mM Hepes , 150 mM sodium chloride, example of this type of reagent is the Sunbright® CA series 5 mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous from NOF (NOF Corp . , Tokyo , Japan ) . Humira is trans sodium periodate solution ( 10 mm ) , and an aqueous ferred or dissolved in reaction buffer ( e . g . 50 mM Hepes, m - toluidine solution (50 mM ) . Subsequently the aminooxy 350 mM sodium chloride , 5 mM calcium chloride , pH 6 .0 ) reagent is added to give a 20 - fold molar reagent excess. The to get a final protein concentration of 1 . 0 + / - 0 . 25 mg/ ml . mixture is incubated for 2 h in the dark at room temperature Then the pH of the solution is corrected to 6 .0 by drop wise under gentle stirring and quenched for 15 min at room addition of a 0 .5N aqueous HC1 solution . Subsequently a 40 temperature by the addition of 8 ul of aqueous cysteine mM aqueous sodium periodate solution is added within 10 solution ( 1 M ) . minutes to give a concentration of 200 uM . The oxidation [ 0754 ] Finally , the PEG - Humira conjugate is purified by reaction is carried out for 30 + / - 5 min at a temperature ( T ) ion -exchange chromatography . The conjugate containing of T = + 22 + / - 2° C . Then the reaction is stopped by addition fractions are collected and then subjected to UF /DF using a of an aqueous L - cysteine solution ( 1 M ) within 15 minutes membrane . The preparation is analytically characterized by at T = + 22 + / - 2° C . to give a final concentration of 10 mM in measuring total protein (Bradford ) and biological activity the reaction mixture and incubation for 60 + / - 5 min . according to methods known in the art. [0748 ] The oxidized Humira is further purified by ion exchange chromatography . The oxidized Humira containing Method 4 : fractions of the eluate are collected and used for the conju [0755 ] Humira is PEGylated by use of a linear 20 kD gation reaction . PEGylation reagent containing an aminooxy group . An [0749 ] The aminooxy - PEG reagent with a MW of 20 kD example of this type of reagent is the Sunbright® CA series reagent is added in a 50 - fold molar excess to the eluate from NOF (NOF Corp ., Tokyo , Japan ). An initial concen containing the purified oxidized Humira within a maximum tration or weight of HJumira is transferred or dissolved in time period (t ) of 15 minutes under gentle stirring . Then an Hepes buffer (50 mM Hepes , 150 mM sodium chloride , 5 aqueous m - toluidine solution ( 50 mM ) is added within 15 mM calcium chloride, pH 6 .0 ) to get a final protein con minutes to get a final concentration of 10 mM . The reaction centration of 2 mg Humira /ml . Subsequently an 5 mM mixture is incubated for 120 + / – 10 min . in the dark at a aqueous sodium periodate solution is added within 15 min temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . utes to give a final concentration of 100 UM , followed by [0750 ] The obtained PEG -Humira conjugate is further addition of an 50 mM aqueous m - toluidine solution to get a purified by ion exchange chromatography . The PEG -Humira final concentration of 10 mM within a time period of 30 conjugate containing fractions are collected and concen minutes . Then the aminooxy -PEG reagent with a MW of 20 trated by ultra - / diafiltration (UF /DF ) using a membrane kD (described above ) is added to give a 20 - fold molar made of regenerated cellulose with an appropriate molecular reagent excess . After correction of the pH to 6 . 0 the mixture weight cut off (Millipore ) . is incubated for 2 h in the dark at room temperature under gentle stirring and quenched for 15 min at room temperature Method 3 : by the addition of a 1 M aqueous L - cysteine solution to give a final concentration of 10 mM . [ 0751 ] Humira is PEGylated by use of a linear 20 kD 10756 ) The Humira conjugate is purified by means of ion PEGylation reagent containing an aminooxy group . An exchange chromatography (IEC ) . The conjugate containing example of this type of reagent is the Sunbright® CA series fractions of the eluate are concentrated by UF / DF using a 10 from NOF (NOF Corp . , Tokyo , Japan ) . Humira is dissolved kD membrane made of regenerated cellulose (88 cm2, in Hepes buffer (50 mM Hepes , 150 mM sodium chloride , cut -off 10 kD /Millipore ) . The final diafiltration step is per 5 mM calcium chloride, pH 6 . 0 ) and mixed with an aqueous formed against Hepes buffer (50 mM Hepes , 5 mM CaCl2 , sodium periodate solution ( 10 mm ) , and an aqueous pH 7 . 5 ). m - toluidine solution (50 mM ) . Subsequently , the aminooxy [0757 ] The preparation is analytically characterized by reagent is added to give a 20 - fold molar reagent excess . The measuring total protein ( Bradford and BCA procedure ) and mixture is incubated for 2 h in the dark at room temperature biological activity according to known methods. under gentle stirring and quenched for 15 min at room temperature by the addition of 8 ul of aqueous cysteine Example 44 solution ( 1 M ). [0752 ] Finally , the PEG -Humira conjugate is purified by PEGylation of Prolia Using an Aminooxy - PEG ion -exchange chromatography on Q - Sepharose FF . 1 . 5 mg Reagent and m - Toluidine as a Nucleophilic protein /ml gel is loaded on the column pre equilibrated with Catalyst 50 mM Hepes buffer , pH 7 . 4 containing 5 mM CaC12 . The conjugate is eluted with 50 mM Hepes buffer containing 5 Method 1 : mM CaCl2 and 500 mM sodium chloride , pH 7 . 4 and is then [0758 ] Prolia is PEGylated by use of a linear 20 kD subjected to UF/ DF using a membrane . The preparation is PEGylation reagent containing an aminooxy group . An US 2017 /0240590 A1 Aug . 24 , 2017 example of this type of reagent is the Sunbright® CA series art . For the PEG -Prolia conjugate a specific activity of > 50 % from NOF (NOF Corp . , Tokyo , Japan ) . Prolia is dissolved in in comparison to native Prolia is determined . The conjugate 7 .0 ml histidine buffer , pH 6 .0 (20 mM L -histidine , 150 mM is additionally analytically characterized by Size Exclusion NaC1, 5 mM CaC12 ) . An aqueous sodium periodate solution HPLC using a Agilent 1200 HPLC system equipped with a ( 5 mm ) is then added and the reaction mixture is incubated Shodex KW 803 column under conditions as previously for 1 h in the dark at 4° C . under gentle stirring and quenched described (Kolarich et al, Transfusion 2006 ; 46 : 1959 -77 ) . It for 15 min at room temperature by the addition of 7 . 5 ul of is shown that the preparation contains no free Prolia . a 1 M aqueous cysteine solution . The mixture is subse quently subjected to UF/ DF employing Vivaspin centrifugal Method 2 : filtrators to remove excess periodate , quencher and the byproducts thereof. [0764 ] Prolia is PEGylated by use of a linear 20 kD [0759 ] The retentate containing oxidized Prolia is next PEGylation reagent containing an aminooxy group . An mixed with an aqueous m - toluidine solution (50 mM ) and example of this type of reagent is the Sunbright® CA series incubated for 30 min at room temperature . Aminooxy -PEG from NOF (NOF Corp . , Tokyo , Japan ) . Prolia is transferred reagent with a MW of 20 kD is then added to give a 5 - fold or dissolved in reaction buffer ( e . g . 50 mM Hepes, 350 mm molar reagent excess . This mixture is incubated for 2 . 5 h at sodium chloride , 5 mM calcium chloride , pH 6 . 0 ) to get a room temperature in the dark under gentle stirring . final protein concentration of 1 . 0 + / - 0 .25 mg/ ml . Then the [0760 ] Finally , the PEG -Prolia conjugate is purified by pH of the solution is corrected to 6 . 0 by drop wise addition ion - exchange chromatography ( e .g ., on Q -Sepharose FF ) . of a 0 .5N aqueous HCl solution . Subsequently , a 40 mm For example , 1 . 5 mg protein /ml gel is loaded on the column aqueous sodium periodate solution is added within 10 min equilibrated with 50 mM Hepes buffer , pH 7 . 4 containing 5 utes to give a concentration of 200 uM . The oxidation mM CaC12 . The conjugate is eluted with 50 mM Hepes reaction is carried out for 30 + / - 5 min at a temperature ( T ) buffer containing 5 mM CaCl2 and 500 mM sodium chlo of T = + 22 + / - 2° C . Then the reaction is stopped by addition ride , pH 7 . 4 and is then subjected to UF /DF using an of an aqueous L -cysteine solution ( 1 M ) within 15 minutes appropriate MW cutoff membrane. The preparation is next at T = + 22 + / - 2° C . to give a final concentration of 10 mM in analytically characterized by measuring total protein (Coo the reaction mixture and incubation for 60 + / - 5 min . massie , Bradford ) and biological activity according to meth [0765 ] The oxidized Prolia is further purified by ion ods known in the art . exchange chromatography . The oxidized Humira containing 10761] In an alternative embodiment, Method 1 is carried fractions of the eluate are collected and used for the conju out as follows. Prolia is PEGylated by use of a linear 20 kD gation reaction . PEGylation reagent containing an aminooxy group . An [0766 ] The aminooxy - PEG reagent with a MW of 20 kD example of this type of reagent is the Sunbright® CA series reagent is added in a 50 - fold molar excess to the eluate from NOF (NOF Corp . , Tokyo , Japan ) . 10 mg rFIX is containing the purified oxidized Prolia within a maximum dissolved in 5 ml histidine - buffer , pH 6 . 0 ( 20 mM L - histi time period ( t ) of 15 minutes under gentle stirring . Then an dine , 150 mM NaCl) . 100 ul of an aqueous sodium periodate aqueous m - toluidine solution (50 mM ) is added within 15 solution ( 5 mM ) is then added and the reaction mixture is minutes to get a final concentration of 10 mM . The reaction incubated for 1 h in the dark at 4° C . under gentle stirring mixture is incubated for 120 + - 10 min . in the dark at a and quenched for 15 min at room temperature by the temperature ( T ) of T = + 22 + / - 2° C . under gentle shaking . addition of 50 ul of a 1 M aqueous cysteine solution . The [0767 ] The obtained PEG - Prolia conjugate is further puri mixture is subsequently subjected to UF /DF employing fied by ion exchange chromatography. The PEG - Prolia Vivaspin 15R 10 kD centrifugal filtrators to remove excess conjugate containing fractions are collected and concen periodate , quencher and the byproducts thereof. trated by ultra - /diafiltration (UF / DF ) using a membrane [ 0762] The retentate (approx . 7 ml) , containing oxidized made of regenerated cellulose with an appropriate molecular Prolia , is mixed with 2 ml of an aqueous m - toluidine weight cut off (Millipore ) . solution ( 50 mM ) and incubated for 30 min at room tem perature . Then aminooxy - PEG reagent with a MW of 20 kD Method 3 : (described above ) is added to give a 5 - fold molar reagent [0768 ] Prolia is PEGylated by use of a linear 20 kD excess . This mixture is incubated for 2 . 5 h at RT in the dark PEGylation reagent containing an aminooxy group . An under gentle stirring . example of this type of reagent is the Sunbright® CA series [0763 ] Finally the PEG - Prolia conjugate is purified by from NOF (NOF Corp . , Tokyo , Japan ) . EPO is dissolved in ion -exchange chromatography on SP Sepharose FF . The Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 reaction mixture is diluted with 20 ml Buffer A ( 50 mM mM calcium chloride, pH 6 .0 ) and mixed with an aqueous Hepes, pH 6 .5 ) and loaded onto a 20 mlHiPrep SP FF 16 / 10 sodium periodate solution ( 10 mm ) , and an aqueous column (GE Healthcare , Fairfield , Conn . ) pre -equilibrated m - toluidine solution (50 mM ) . Subsequently the aminooxy with Buffer A . Then the column is eluted with Buffer B (50 reagent is added to give a 20 - fold molar reagent excess. The mM Hepes, 1 M NaCl, pH 6 . 5 ) . Free Prolia is eluted by mixture is incubated for 2 h in the dark at room temperature washing the column with 25 % Buffer B and the conjugate at under gentle stirring and quenched for 15 min at room 50 % Buffer B . The conjugate containing fractions are con temperature by the addition of 8 ul of aqueous cysteine centrated by UF/ DF using a 10 kD membrane made of solution ( 1 M ). regenerated cellulose (88 cm2, cutoff 10 kD /Millipore ) . The [0769 ] Finally , the PEG -Prolia conjugate is purified by final diafiltration step is performed against histidine buffer , ion -exchange chromatography on Q - Sepharose FF . 1 .5 mg pH 6 . 9 containing 150 mM NaCl. The preparation is ana protein /ml gel is loaded on the column pre equilibrated with lytically characterized by measuring total protein ( Bradford ) 50 mM Hepes buffer, pH 7 . 4 containing 5 mM CaC12 . The and biological activity according to methods known in the conjugate is eluted with 50 mM Hepes buffer containing 5 US 2017 /0240590 A1 Aug . 24 , 2017 86 mM CaCl2 and 500 mM sodium chloride , pH 7 .4 and is then described (Kolarich et al , Transfusion 2006 ; 46 :1959 - 77 ). It subjected to UF/ DF using a membrane . The preparation is is shown that the preparation contains no free Prolia . analytically characterized by measuring total protein (Brad ford ) and biological activity according to methods known in Method 4 : the art . [ 0773 ] Prolia is PEGylated by use of a linear 20 kD [0770 ] In an alternative embodiment, Method 3 is carried PEGylation reagent containing an aminooxy group . An out as follows. example of this type of reagent is the Sunbright® CA series [0771 ] Prolia is PEGylated by use of a linear 20 kD from NOF (NOF Corp . , Tokyo , Japan ). An initial concen PEGylation reagent containing an aminooxy group . An tration or weight of HJumira is transferred or dissolved in Hepes buffer ( 50 mM Hepes , 150 mM sodium chloride , 5 example of this type of reagent is the Sunbright® CA series mM calcium chloride, pH 6 . 0 ) to get a final protein con from NOF (NOF Corp ., Tokyo , Japan ) . 10 mg Prolia is centration of 2 mg Prolia /ml . Subsequently an 5 mM aque dissolved in ~ 8 ml histidine buffer , pH 6 . 0 ( 20 mM L -his ous sodium periodate solution is added within 15 minutes to tidine , 150 mM NaCl) . 200 ul of an aqueous sodium give a final concentration of 100 uM , followed by addition periodate solution ( 5 mM ) and 2 ml of an aqueous m - tolui of an 50 mM aqueous m - toluidine solution to get a final dine solution (50 mm ) are then added . Subsequently , the concentration of 10 mM within a time period of 30 minutes . aminooxy -PEG reagent with a MW of 20 kD (described Then the aminooxy - PEG reagent with a MW of 20 kD above ) is added to give a 5 - fold molar reagent excess . The (described above) is added to give a 20 - fold molar reagent mixture is incubated for 2 h in the dark at room temperature excess . After correction of the pH to 6 . 0 the mixture is under gentle stirring and quenched for 15 min at room incubated for 2 h in the dark at room temperature under temperature by the addition of 100 ul of 1 M aqueous gentle stirring and quenched for 15 min at room temperature cysteine solution . by the addition of an 1 M aqueous L -cysteine solution to [ 0772] Finally the PEG - Prolia conjugate is purified by give a final concentration of 10 mM . ion -exchange chromatography on SP - Sepharose FF . The [0774 ] The Prolia conjugate is purified by means of ion reaction mixture is diluted with 20 ml Buffer A (50 mM exchange chromatography ( IEC ) . The conjugate containing Hepes , pH 6 .5 ) and loaded onto a 20 mlHiPrep SPFF 16 / 10 fractions of the eluate are concentrated by UF /DF using a 10 column (GE Healthcare, Fairfield , Conn .) pre -equilibrated kD membrane made of regenerated cellulose (88 cm2, with Buffer A . Then the column is eluted with Buffer B ( 50 cutoff 10 kD /Millipore ). The final diafiltration step is per mM Hepes, 1 M NaCl, pH 6 . 5 ) . Free Prolia is eluted by formed against Hepes buffer ( 50 mM Hepes , 5 mM CaC12 , washing the column with 25 % Buffer B and the conjugate at pH 7 . 5 ) . 50 % Buffer B . The conjugate containing fractions are con [0775 ] The preparation is analytically characterized by centrated by UF /DF using a 10 kD membrane made of measuring total protein (Bradford and BCA procedure ) and regenerated cellulose (88 cm2, cutoff 10 kD /Millipore ). The biological activity according to known methods . final diafiltration step is performed against histidine buffer , pH 6 . 9 containing 150 mM NaCl. The preparation is ana Example 45 lytically characterized by measuring total protein (Bradford ) and biological activity according to methods known in the PEGylation of a Therapeutic Protein Using art . For the PEG - Prolia conjugate a specific activity of > 50 % Branched PEG in comparison to native Prolia is determined . The conjugate [0776 ] PEGylation of a therapeutic protein of the inven is additionally analytically characterized by Size Exclusion tion may be extended to a branched or linear PEGylation HPLC using a Agilent 1200 HPLC system equipped with a reagent, which is made of an aldehyde and a suitable linker Shodex KW 803 column under conditions as previously containing an active aminooxy group .

SEQUENCE LISTING

< 160 > NUMBER OF SEQ ID NOS : 1 < 210 > SEQ ID NO 1 ? 1 > LENGTH : 422 < 212 > TYPE : PRT < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 1 Leu Asn Arg Pro Lys Arg TyrTyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gin Gly Asn Leu Glu Arg Glu Cys peopleMet Glu Glu Lys Cys Ser Phe Glu 20 in?e 25 za Glu Pro Arg Glu Val Phe Glu Asn Thr Glu Lys Thr Thr Glu Phe Trp Hii 35 40 45 Lys Gin Tyr Val Asp Gly Asp Gin Cys Glu Ser Asn Pro Cys Leu Asn naa 60 Gly Gly Ser Cys Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro US 2017/ 0240590 Al Aug . 24 , 2017 27

- continued Te65 70 | 75 80 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

Val Val Cys Ser ECys ?Thr Glu Gly ETyr Arg Leu Ala Glu Asn Gin Lys 115 120 125

Ser Cys Glu Pro Ala Val Pro Phe Pro EREECys Gly Arg gVal Ser Val Ser & ? 135 Gin Thr Ser Lys Leu Thr Arg Ala Glu Ala Val Phe Pro Asp Val Asp 145 150 160

Tyr Val Asn Pro Thr Glu Ala Glu ?Thr Ile Leu Asp Asn Ile Thr Gln dd3175

Gly Thr Gin Ser Phe Asn Asp Phe ?Thr Arg Val Val Gly Gly Glu Asp

g Ala Lys Pro Gly Gin Phe Pro ?Trp Gin Val Val Leu Asn Gly Lys Val 195 200 205 Asp Ala Phe cys Gly Gly Ser Ile Val Asn Glu Lys Trp Ile Val Thr 215 | g Ala Ala His Cys Val Glu Thr Gly Val Lys Ile Thr Val Val Ala Gly 225 230 235 Glu His Asn Ile Glu Glu Thr Glu His Thr Glu Gin Lys Arg Asn Val 245

Ile Arq Ala Ile Ile Pro His His Asn Tyr Asn Ala Ala Ile Asn Lys 265 270 Tyr Asn His Asp Ile Ala Leu Leu Glu Leu Asp Glu Pro Leu Val Leu 275 280 285 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 305 310 315

Phe His Lys Gly Arg Ser Ala Leu Val Leu 3Gin Tyr Leu Arg Val Pro 325

Leu Val Asp Arg Ala Thr Cys Leu Arg Ser ?Thr Lys Phe Thr Ile Tyr 345 350 mmmm Asn Asn Met LgEPhe Cys Ala Gly Phe His Glu Gly Gly Arg Asp Ser Cys 355 360 365 Gin Gly Asp Ser Gly Gly Pro His Val Thr Glu Val Glu Gly Thr Ser 375 mmm Phe Leu Thr Gly Ile Ile Ser Trp Gly Glu Glu Cys Ala Met Lys Gly 385 390 395 Lys Tyr Gly Ile Tyr Thr Lys Val Ser Arg 1Tyr Val Asn Trp Ile Lys 405 410 415

Glu Lys Thr Lys Leu Thr 420 mmmm US 2017 /0240590 A1 Aug . 24 , 2017 88

1 . A method of conjugating a water soluble polymer to an 13 . The method of claim 1 wherein the conjugating the oxidized carbohydrate moiety of FVIII comprising contact water soluble polymer to the oxidized carbohydrate moiety ing the oxidized carbohydrate moiety with an activated of FVIII is stopped by the addition of a quenching agent water soluble polymer under conditions that allow conjuga selected from the group consisting of L - cysteine , methio tion ; nine, glutathione , glycerol, sodium meta bisulfite said water soluble polymer containing an active aminooxy (Na S ,03 ) , tryptophane , tyrosine , histidine or derivatives group is selected from the group consisting of polyeth thereof, kresol, imidazol, and combinations thereof, ylene glycol (PEG ) , branched PEG , PolyPEG® (War wherein the quenching agent is added in an amount to wick Effect Polymers ; Coventry, UK ), and polysialic result in a final concentration between about 1 mM and acid (PSA ) ; and about 100 mM quenching agent , under conditions said carbohydrate moiety oxidized by incubation with a comprising a time period between about 5 minutes and buffer comprising sodium periodate (NalO . ) ; about 120 minutes ; a temperature between about 2° C . wherein an oxime linkage is formed between the oxidized and about 37° C .; in the presence or absence of light; carbohydrate moiety and the active aminooxy group on and with or without stirring . the water soluble polymer ; and 14 . The method of claim 13 wherein the quenching agent wherein said oxime linkage formation is catalyzed by is L - cysteine . m - toluidine . 15 . The method of claim 14 wherein the L - cysteine is 2 . ( canceled ) added to result in a final concentration of about 10 mM and the conditions comprise a time period of about 60 minutes , 3 . The method according to claim 1 wherein a solution a temperature of about 22° C ., the absence of light and with comprising an initial concentration of FVIII between about stirring . 0 . 3 mg/ ml and about 3 . 0 mg/ ml is adjusted to a pH value 16 . The method of claim 1 comprising : between about 5 . 0 and about 8 . 0 prior to contacting with the a ) a first step comprising adjusting the pH value of a activated water soluble polymer. solution comprising FVIII to a pH value between about 4 . The method of claim 3 wherein the initial concentration 5 . 0 and about 8 . 0 , wherein the concentration of FVIII of FVIII is about 1 . 0 mg/ ml and the pH is about 6 . 0 . is between about 0 . 3 mg/ ml and about 3 . 0 mg/ ml ; 5 . The method of claim 1 wherein FVIII is contacted by b ) a second step comprising oxidizing one or more a desired excess concentration of activated water soluble carbohydrates on FVIII, wherein sodium periodate is polymer , wherein the excess concentration is between about added to the solution in the first step to result in a final 1 -molar and about 300 -molar excess . concentration between about 50 uM and about 1000 6 . The method of claim 5 wherein the excess concentra um , under conditions comprising a time period tion is about 50 - fold molar excess . between about 0 . 1 minutes and about 120 minutes, a 7 . The method of claim 5 wherein FVIII is incubated with temperature between about 2° C . and about 37° C .; in the activated water soluble polymer under conditions com the presence or absence of light, and with or without prising a time period between about 0 . 5 hours and about 24 stirring ; hours ; a temperature between about 2° C . and about 37° C . ; c ) a third step comprising contacting FVIII with a desired in the presence or absence of light ; and with or without excess concentration of activated water soluble poly stirring . mer, wherein the excess concentration is between about 8 . The method according to claim 7 wherein the condi 1 -molar excess and about 300 -molar excess , under tions comprise a time period of about 120 minutes , a conditions comprising a time period between about 0 . 5 temperature of about 22° C ., the absence of light; and with hours and about 24 hours , a temperature between about stirring . 2° C . and about 37° C . ; in the presence or absence of 9 . The method according to claim 1 wherein m -toluidine light; and with or without stirring ; is added in an amount to result in a final concentration d ) a fourth step comprising adding m -toluidine to the between about 1 . 0 mM and about 50 mM , under conditions solution of the third step , wherein m - toluidine is added comprising a time period between about 0 . 1 minutes and to result in a final concentration between about 1 mM about 30 minutes ; a temperature between about 2° C . and and about 50 mM , under conditions comprising a time about 37° C . ; in the presence or absence of light; and with period between about 0 . 1 minutes and about 30 min or without stirring . utes; a temperature between about 2° C . and about 37° 10 . The method of claim 9 wherein the final concentration C . ; in the presence or absence of light, and with or of m - toluidine is about 10 mM , and the conditions comprise without stirring; a time period of up to about 15 minutes, a temperature of e ) a fifth step wherein FVIII is incubated with the acti about 22° C . , the absence of light; and with stirring . vated water soluble polymer and m - toluidine under 11 . The method according to claim 1 wherein sodium conditions that allow conjugation of the activated periodate is added in an amount to result in a final concen water- soluble polymer to one or more oxidized carbo tration between about 50 uM and about 1000 UM , under hydrates on FVIII , said conditions comprising a time conditions comprising a time period between about 0 . 1 period between about 0 .5 hours and about 24 hours , a minutes and 120 minutes ; a temperature between about 2° C . temperature between about 2° C . and about 37° C . ; in and about 37° C . ; in the presence or absence of light; and the presence or absence of light, and with or without with or without stirring . stirring ; and 12 . The method of claim 11 wherein the final concentra f ) a sixth step wherein the conjugating the water soluble tion of sodium periodate is about 400 UM , and the conditions polymer to the one or more oxidized carbohydrates of comprise a time period of about 10 minutes , a temperature FVIII in the fifth step is stopped by the addition of a of about 22° C . , the absence of light and with stirring . quenching agent selected from the group consisting of US 2017 /0240590 A1 Aug . 24 , 2017

L - cysteine ,methionine , glutathione, glycerol, Na S205 28 . The method according to claim 18 wherein the PSA is (sodium meta bisulfite ), tryptophane , tyrosine, histidine prepared by reacting an activated aminooxy linker with or derivatives thereof, kresol, imidazol, and combina oxidized PSA ; tions thereof; wherein the quenching agent is added to wherein the aminooxy linker is selected from the group result in a final concentration of about 1 mM and about consisting of: 100 mm , under conditions comprising a time period between about 5 minutes and about 120 minutes ; a a ) a 3 -oxa -pentane - 1, 3- dioxyamine linker of the formula : temperature between about 2° C . and about 37° C . ; in the presence or absence of light , and with or without stirring . H?NH A NH , 17 . The method of claim 16 wherein the initial concen tration of FVIII in the first step is about 1 mg/ml and the pH is about 6 .0 ; b ) a 3 , 6 , 9 -trioxa - undecane - 1 , 11 - dioxyamine linker of the wherein the final concentration of sodium periodate in the formula : second step is about 400 uM , and the conditions in the fifth step comprise a time period of about 10 minutes, a temperature of about 22° C . , the absence of light and NoH2NOH » NH with stirring ; yoyo NH3 wherein the excess concentration in the third step is about 50 molar excess ; wherein the conditions in the third step comprise a time period of about 15 minutes , a and temperature of about 22° C ., the absence of light and c ) a 3 ,6 , 9 , 12 , 15 - penatoxa -heptadecane - 1, 17 -dioxyamine with stirring ; linker of the formula :

H?N noNºNH- NH2 ,

wherein the final concentration of m -toluidine in the wherein the PSA is oxidized by incubation with sodium fourth step is about 10 mm , and the conditions in the periodate to form a terminal aldehyde group at the fourth step comprise a time period of about 15 minutes , non - reducing end of the PSA . a temperature of about 22° C ., the absence of light and 29 . The method according to claim 28 wherein the ami with stirring ; nooxy linker is 3 - oxa - pentane - 1 , 5 - dioxyamine . 30 . (canceled ) wherein the conditions of incubating FVIII with the 31 . The method according to claim 1 wherein m - toluidine activated water soluble polymer and m - toluidine in the is provided at a concentration between about 1 mM and fifth step comprise a time period of about 2 hours ; a about 50 mM . temperature of about 22° C . , the absence of light; and 32 . (canceled ) with stirring ; and 33 . The method according to claim 1 wherein the m -tolui dine is present in the conjugation reaction at a concentration wherein the quenching agent in the sixth step is L - cyste of about 10 mM . ine ; and wherein the L - cysteine is added to result in a 34 . The method according to claim 1 further comprising final concentration of about 10 mM and the conditions the step of reducing an oxime linkage in the conjugated in the sixth step comprise a time period of about 60 FVIII by incubating the conjugated FVIII in a buffer com minutes, a temperature of about 22° C ., the absence of prising a reducing compound selected from the group con light and with stirring. sisting of sodium cyanoborohydride (NaCNBH2 ), ascorbic acid (vitamin C ) and NaBHz. 18. The method according to claim 1 wherein the water 35 . The method according to claim 34 wherein the reduc soluble polymer is PSA . ing compound is sodium cyanoborohydride (NaCNBH3 ) . 36 . The method according to claim 1 further comprising 19 . The method according to claim 1 wherein the water the step of purifying the conjugated FVIII . soluble polymer is PEG . 37 . The method according to claim 36 wherein the con 20 -21 . ( canceled ) jugated FVIII is purified by a method selected from the group consisting of chromatography , filtration and precipi 22 . The method according to claim 18 wherein the PSA is tation . comprised of about 10 - 300 sialic acid units . 38 . The method according to claim 37 wherein the chro matography is selected from the group consisting ofHydro 23 -26 . (canceled ) phobic Interaction Chromatography (HIC ) , Ion Exchange 27. The method according to claim 1 wherein the oxidized chromatography (IEC ) , Size exclusion chromatography carbohydrate moiety of FVIII is located in the activation (SEC ) , Affinity chromatography, and Reversed - phase chro peptide of FVIII . matography . US 2017 /0240590 A1 Aug . 24 , 2017

39 . The method of claim 38 wherein an anti- chaotropic polyethylene glycol (PEG ), branched PEG , PolyPEG® salt is used in a chromotagraphy loading step and in a (Warwick Effect Polymers ; Coventry , UK ) , and poly chromatography washing step . sialic acid (PSA ) . 40 . The method of claim 38 wherein the chromatography 55 -60 . ( canceled ) takes place in a column . 41 . The method of claim 40 wherein the column com 61 . The method according to claim 1 wherein the water prises a chromatography resin selected from the group soluble polymer containing an active aminooxy group is consisting of Phenyl- Sepharose FF and Butyl- Sepharose FF . prepared by a method comprising : a ) incubating a solution comprising an oxidized water 42 . The method of claim 41 wherein the resin is present soluble polymer with an activated aminooxy linker in the column at a bed height of between about 5 cm and comprising an active aminooxy group under conditions about 20 cm . that allow the formation of a stable oxime linkage 43 . The method according to claim 42 wherein the bed between the oxidized water -soluble polymer and the height is about 10 cm . activated aminooxy linker, said conditions comprising 44 . The method of claim 40 comprising one or more a time period between about 1 minute and about 24 washing steps wherein flow direction is set to up - flow and hours ; a temperature between about 2° C . and about 37° wherein the flow rate is between about 0 . 2 cm /min and about C . ; in the presence or absence of light, and with or 6 . 7 cm /min . without stirring ; thereby forming a water soluble poly 45 . The method according to claim 44 wherein the flow mer containing an active aminooxy group ; rate is about 2 cm /min . b ) incubating a solution comprising the water soluble 46 . The method of claim 1 comprising one or more elution polymer containing an active aminooxy group of step steps wherein flow direction is set to down - flow and wherein a ) with m - toluidine under conditions comprising a time the flow rate is between about 0 . 1 cm /min and about 6 . 7 period between 1 minute and 24 hours; a temperature cm /min . between 2° C . and 37° C .; in the presence or absence 47 . The method according to claim 46 wherein the flow of light; and with or without stirring ; rate is about 1 cm /min . c ) incubating a solution comprising the water soluble 48 . The method of claim 1 further comprising concen polymer containing an active aminooxy group of step trating the conjugated FVII by ultra - / diafiltration (UF /DF ). b ) with a reducing agent under conditions that allow the 49 . The method of claim 1 wherein the final concentration formation of a stable alkoxamine linkage between the of FVIII is between about 0 . 5 and about 3 mg/ ml . oxidized water- soluble polymer and the activated ami 50 . The method according to claim 1 wherein FVIII nooxy linker, said conditions comprising a time period comprises between about 5 and about 11 water soluble between about 1 minute and about 24 hours ; a tem polymer moieties. perature between about 2° C . and about 37° C . ; in the 51 . The method of claim 21 wherein the conjugated FVIII presence or absence of light; and with or without is purified using chromatography ; wherein an anti -chao stirring ; and tropic salt is used for a loading step and for a washing step ; d ) purifying the water soluble polymer containing an the method comprising one or more washing steps wherein active aminooxy group by a method selected from the flow direction is set to up - flow and wherein the flow rate is group consisting of chromatography, filtration and pre between about 0 . 2 cm /min and about 6 . 7 cm /min and one or cipitation more elution steps wherein flow direction is set to down flow and wherein the flow rate is between about 0 . 2 cm /min 62 . The method according to claim 1 wherein said water and about 6 . 7 cm /min ; further comprising concentrating the soluble polymer is oxidized by incubation with sodium conjugated FVIII by ultra - / diafiltration (UF /DF ) . periodate to form a terminal aldehyde group at the non 52. The method of claim 51 wherein the chromatography reducing end of the water -soluble polymer . is hydrophobic interaction chromatography (HIC ) ; wherein 63. The method according to claim 62 wherein the water the one or more washing steps flow rate is about 2 cm /min ; soluble polymer is PSA . and wherein the one or more elution steps flow rate is about 64 . (canceled ) 1 cm /min . 65 . The method according to claim 61 wherein the ami 53 . A modified FVIII produced by the method according nooxy linker is selected from the group consisting of: to claim 1 . 54 . A method of forming an oxime linkage between an a ) a 3 -oxa - pentane - 1, 5 -dioxyamine linker of the formula : oxidized carbohydrate moiety on FVIII and an activated water soluble polymer containing an active aminooxy group HN . NH2, comprising the steps of: H2NO NH2, a ) oxidizing a carbohydrate moiety on FVIII by incubat ing said protein with sodium periodate (NaO2 ) ; and b ) forming an oxime linkage between the oxidized car b ) a 3 , 6 , 9 -trioxa - undecane - 1 , 11 - dioxyamine linker of the bohydrate moiety of FVIII and the activated water formula : soluble polymer containing an active aminooxy group in the presence of m - toluidine under conditions allow HN . NH ing formation of said oxime linkage ; H2No wherein said water soluble polymer containing an active aminooxy group is selected from the group consisting US 2017 /0240590 A1 Aug . 24 , 2017 91

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

H2N NH

66 . The method according to claim 61 wherein the reduc 70 . The method according to claim 61 wherein m - tolui ing agent is selected from the group consisting of sodium dine is added in an amount to result in a final concentration cyanoborohydride (NaCNBHz ) , ascorbic acid ( vitamin C ) between about 1 . 0 mM and about 50 mM . and NaBHz. 71 . The method according to claim 61 further comprising concentrating the conjugated FVIII by ultra - / diafiltration 67 . The method according to claim 66 wherein the reduc (UF /DF ) . ing agent is sodium cyanoborohydride (NaCNBH3 ) . 72 . (canceled ) 68 -69 . ( canceled ) * * * * *