Methods for the purification of polypeptide conjugates

Abstract

The present invention provides processes for the manufacturing of polypeptide conjugates. In particular, the invention provides methods for the purification of polypeptide conjugates, which include at least one polymeric modifying groups, such as a poly(alkylene oxide) moiety. Exemplary poly(alkylene oxide) moieties include poly(ethylene glycol) (PEG) and poly(propylene glycol). In an exemplary process, hydrophobic interaction chromatography (HIC) is used to resolve different glycoforms of glycoPEGylated polypeptides.

1 - 85 . (canceled) 
     
     
         86 . A method of making a composition comprising a first polypeptide conjugate, said first polypeptide conjugate comprising a first number of poly(alkylene oxide) moieties covalently linked to said first polypeptide, said method comprising:
 (a) contacting a mixture comprising said first polypeptide conjugate and a hydrophobic interaction chromatography (HIC) medium; and   (b) eluting said first polypeptide conjugate from said hydrophobic interaction chromatography medium,   thereby making said composition comprising said first polypeptide conjugate, wherein said first polypeptide is a member selected from bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein 15 (BMP-15), neurotrophin-3 (NT-3), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), interferon alpha, interferon beta, interferon gamma, α 1 -antitrypsin (α-1 protease inhibitor), glucocerebrosidase, tissue-type plasminogen activator (TPA), interleukin-2 (IL-2), leptin, hirudin, urokinase, human DNase, insulin, hepatitis B surface protein (HbsAg), chimeric diphtheria toxin-IL-2, human growth hormone (hGH), human chorionic gonadotropin (hCG), thyroid peroxidase (TPO), alpha-galactosidase, alpha-L-iduronidase, beta-glucosidase, alpha-galactosidase A, acid α-glucosidase (acid maltase), anti-thrombin III (AT III), follicle stimulating hormone (FSH), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), fibroblast growth factor 7 (FGF-7), fibroblast growth factor 21 (FGF-21), fibroblast growth factor 23 (FGF-23), prokinetisin, extendin-4, CD4, tumor necrosis factor receptor (TNF-R), α-CD20, P-selectin glycoprotein ligand-1 (PSGL-1), complement, transferrin, glycosylation-dependent cell adhesion molecule (GlyCAM), neural-cell adhesion molecule (N-CAM), TNF receptor-IgG Fc region fusion protein, anti-HER2 monoclonal antibody, monoclonal antibody to respiratory syncytial virus, monoclonal antibody to protein F of respiratory syncytial virus, monoclonal antibody to TNF-α, monoclonal antibody to glycoprotein IIb/IIIa, monoclonal antibody to CD20, monoclonal antibody to VEGF-A, monoclonal antibody to PSGL-1, monoclonal antibody to CD4, monoclonal antibody to a CD3, monoclonal antibody to EGF, monoclonal antibody to carcinoembryonic antigen (CEA) and monoclonal antibody to IL-2 receptor.   
     
     
         87 . The method of  claim 86 , wherein said first polypeptide is covalently linked to three poly(ethylene glycol) moieties. 
     
     
         88 . The method of  claim 87 , wherein at least two of said three poly(ethylene glycol) moieties are covalently linked to said first polypeptide via an N-linked glycan. 
     
     
         89 . The method of  claim 86 , wherein said first polypeptide is EPO comprising an amino acid sequence according to SEQ ID NO:1, said sequence optionally having at least one mutation selected from the group consisting of Arg 139  to Ala 139 , Arg 143  to Ala 143  and Lys 154  to Ala 154 . 
     
     
         90 . A method of forming a composition comprising a first erythropoietin (EPO) conjugate, said first EPO conjugate comprising a first number of poly(alkylene oxide) moieties covalently linked to said EPO, said method comprising:
 (a) contacting a mixture comprising said first EPO conjugate with an anion exchange medium;   (b) eluting said first EPO conjugate from said anion exchange medium, forming a first eluate comprising said first EPO conjugate;   (c) contacting said first eluate with a hydrophobic interaction chromatography (HIC) medium; and   (d) eluting said first EPO conjugate from said hydrophobic interaction chromatography medium,   
       thereby forming a composition comprising said first EPO conjugate. 
     
     
         91 . The method of  claim 90 , wherein said mixture of step (a) comprises a second EPO conjugate having a second number of poly(alkylene oxide) moieties covalently linked to said EPO, wherein said first number and said second number are different. 
     
     
         92 . The method of  claim 91 , wherein said first number is 3 and said second number is a member selected from 0, 1, 2 and 4. 
     
     
         93 . The method of  claim 90 , wherein said second EPO conjugate is present in said composition at a concentration that is less than about 10%. 
     
     
         94 . The method of  claim 90 , wherein said HIC medium is a member selected from a butyl and a phenyl resin. 
     
     
         95 . The method of  claim 90 , wherein each of said poly(alkylene oxide) moieties is a member independently selected from a poly(ethylene glycol) moiety and a poly(propylene glycol) moiety. 
     
     
         96 . The method of  claim 95 , wherein each of said poly(alkylene oxide) moieties has an independently selected molecular weight between about 1 kDa and about 200 kDa. 
     
     
         97 . The method of  claim 90 , wherein said first EPO conjugate comprises three poly(ethylene glycol) moieties. 
     
     
         98 . The method of  claim 97 , wherein at least two of said three poly(ethylene glycol) moieties are covalently attached to said EPO via an N-linked glycan. 
     
     
         99 . The method of  claim 90 , wherein said EPO comprises an amino acid sequence according to SEQ ID NO:1, said sequence optionally having at least one mutation selected from the group consisting of Arg 139  to Ala 139 , Arg 143  to Ala 143  and Lys 154  to Ala 154 . 
     
     
         100 . The method of  claim 90 , wherein said EPO conjugate comprises at least one poly(alkylene oxide) moiety that is covalently linked to said EPO via a glycosyl linking group, wherein said glycosyl linking group is covalently linked to an amino acid residue of said EPO or is covalently linked to a glycosyl moiety of said EPO. 
     
     
         101 . The method of  claim 100 , wherein said glycosyl linking group is an intact glycosyl linking group. 
     
     
         102 . The method of  claim 101 , wherein said intact glycosyl linking group is a member selected from a GlcNH moiety, a GlcNAc moiety, and a sialic acid moiety. 
     
     
         103 . The method of  claim 90 , further comprising:
 (e) eluting said first EPO conjugate from a cation exchange chromatography medium.   
     
     
         104 . The method of  claim 103 , wherein step (e) is performed after step (d). 
     
     
         105 . The method of  claim 90 , further comprising: contacting said EPO and a modified sugar nucleotide having a glycosyl moiety covalently attached to a poly(alkylene oxide) moiety, in the presence of a glycosyltransferase under conditions sufficient for said glycosyltransferase to form a covalent bond between said glycosyl moiety and said EPO, thereby forming said first EPO conjugate. 
     
     
         106 . The method of  claim 105 , wherein said glycosyl moiety is a sialic acid moiety and said glycosyltransferase is a sialyltransferase. 
     
     
         107 . The method of  claim 105 , further comprising: contacting in a single reaction vessel said EPO and a nucleotide-N-acetylglucosamine (GlcNAc) molecule and a nucleotide galactose (Gal) molecule in the presence of a N-acetylglucosamine transferase selected from GnT1 and GnT2, and a galactosyl transferase, under conditions sufficient for said N-acetylglucosamine transferase and said galactosyl transferase to form a terminal -GlcNAc-Gal moiety covalently linked to said EPO.