Methods to produce single glycoform antibodies

Abstract

An enzymatic method is provided for restructuring an affinity ligand bound heterogenous glycoform antibody sample to a substantially homogenous single desired glycoform antibody sample for therapeutic uses and kits for performing the methods. A method for enzymatically altering the Fc region of an affinity ligand bound antibody from a heterogenous glycoform to a substantially homogenous single glycoform comprises: contacting the affinity ligand bound heterogeneous glycoform antibody with a reaction buffer designed for a particular glycoform modification for a time sufficient and under conditions to modify the glycoform of the Fc region to a substantially homogeneous single form; optionally adding one or more nucleotide sugars and/or cofactors; and releasing the substantially homogeneous single glycoform antibody sample from said affinity ligand. The invention also encompasses biopharmaceuticals comprising single glycoform mAbs and polyclonal antibodies enzymatically produced for the treatment of cancers and immune disorders as well as compositions comprising the single glycoform antibodies as a biopharmaceutical.

1 . A method to enzymatically alter the Fc region of an affinity ligand bound antibody from a heterogenous glycoform to a substantially homogenous single glycoform, the method comprising:
 (a) contacting said affinity ligand bound heterogeneous glycoform antibody having exposed glycan of the Fc region with a reaction buffer designed for a particular glycoform modification for a time sufficient and under conditions to modify the glycoform of the Fc region to a substantially homogeneous single form;   (b) optionally adding one or more nucleotide sugars and/or cofactors; and   (c) releasing a substantially homogeneous single glycoform antibody from said affinity ligand.   
     
     
         2 - 68 . (canceled) 
     
     
         69 . The method of  claim 1 , wherein said antibody comprises polyclonal antibody, a monoclonal antibody, human monoclonal antibodies, humanized monoclonal antibodies, recombinantly produced monoclonal antibodies, single chain antibodies or any fragments thereof. 
     
     
         70 . The method of  claim 69 , wherein said monoclonal antibody is selected from the group consisting of cetuximab, rituximab, muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, 1-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101 (Aphton), volociximab (Biogen Idee and PDL BioPharm), Anti-CD80 mAb (Biogen Idee), Anti-CD23 mAb (Biogen Idel), CAT-3888 (Cambridge Antibody Technology), CDP-791 (Imclone), eraptuzumab (Immunomedics), MDX-010 (Medarex and BMS), MDX-060 (Medarex), MDX-070 (Medarex), matuzumab (Merck), CP-675,206 (Pfizer), CAL (Roche), SGN-30 (Seattle Genetics), zanolimumab (Serono and Genmab), adecatumumab (Sereno), oregovomab (United Therapeutics), nimotuzumab (YM Bioscience), ABT-874 (Abbott Laboratories), denosumab (Amgen), AM 108 (Amgen), AMG 714 (Amgen), fontolizumab (Biogen Idee and PDL BioPharm), daclizumab (Biogent Idee and PDL BioPharm), golimumab (Centocor and Schering-Plough), CNTO 1275 (Centocor), ocrelizumab (Genetech and Roche), HuMax-CD20 (Genmab), belimumab (HGS and GSK), epratuzumab (Immunomedics), MLN1202 (Millennium Pharmaceuticals), visilizumab (PDL BioPharm), tocilizumab (Roche), ocrerlizumab (Roche), certolizumab pegol (UCB, formerly Celltech), eculizumab (Alexion Pharmaceuticals), pexelizumab (Alexion Pharmaceuticals and Procter & Gamble), abciximab (Centocor), ranibizimumab (Genetech), mepolizumab (GSK), TNX-355 (Tanox), and MYO-029 (Wyeth). 
     
     
         71 . The method of  claim 70 , wherein said antibody is selected from monoclonal antibodies: EG2-hFc, Cetuximab and aIL8-hFc or polyclonal human serum IgGs. 
     
     
         72 . The method of  claim 1 , wherein the glycoform modification is configured for the treatment of a cancer and comprises the presence of galactose in the glycans of the Fc region, or wherein the glycoform modification is configured for the treatment of an autoimmune disorder or inflammatory disorders and comprises the presence of terminal sialic acid in the glycans of the Fc region. 
     
     
         73 . The method of  claim 1 , wherein the glycoform modification is selected from G2 glycoform, GO glycoform, M3 glycoform, S2 glycoform, A2B glycoform, A2BG2 glycoform or SI glycoform. 
     
     
         74 . The method of  claim 1 , wherein said reaction buffer comprises one or more enzymes, which effect galactosylation, sialylation, degalactosylation, desialylation or conversion to core non-galactosylated or core mannose structures. 
     
     
         75 . The method of  claim 74 , wherein said enzymes are Mannosyl-glucosamine transferases selected from MGAT1, MGAT2 or MGAT3; Galactosyltransferases selected from p GalT1, p4GalT2, p4GalT3, p4GalT4, p4GalT5, 4GalT6, or p4GalT7; Sialyltransferases selected from ST6Gal1 or ST6Gal2; Mannosidases selected from a Mannosidase-I, a Mannosidase-II, a(1-2) Mannosidase, a(1-6) Mannosidase, a(1-2,3) Mannosidase, or a(1-2,3,6) Mannosidase); Hexosaminidases selected from β-N-acetylhexosaminidase, β-N-Acetylglucosaminidase, or a-N-Acetyl glucosaminidase; Galactosidases selected from β-galactosidase, β(1-4) galactosidase or a(1-3,6) galactosidase; Sialidases selected from (2-3,6,8) sialidase or ot(2-3) sialidase; fucosidases selected from a-L-Fucosidase, a(1-6) fucosidase, a(1-2) fucosidase, a(1-3,4) fucosidase or a(1-2,3,4) fucosidase) or any combinations of the foregoing. 
     
     
         76 . The method of  claim 74 , wherein said reaction buffer is provided at a pH of 6.0-8.0 or at a pH of 7.2. 
     
     
         77 . The method of  claim 76 , wherein said reaction buffer is provided at a temperature of 25° C. to 40° C. or at a temperature of 37° C. 
     
     
         78 . The method of  claim 1 , wherein the nucleotide sugars are UDP-Glc, UDP-Gal, UDP-GalNAc, UDP-GlcNAc, UDP-GlcUA, UDP-Xyl, GDP-Man, GDP-Fuc, CMP-Neu5Ac, CMP-Neu5Gc or any combination thereof. 
     
     
         79 . The method of  claim 78 , wherein the nucleotide sugars are provided in a concentration of 0.5 to 5 mM or 1 to 1.5 mM. 
     
     
         80 . The method of  claim 1 , wherein the cofactor is selected from Mn, Ca, Mg, Na, K, α-Lactalbumin or any combination thereof and said cofactor is provided in a range of from 2 to 10 mM. 
     
     
         81 . The method of  claim 1 , wherein said affinity ligand is selected from protein A, protein G, protein A/G, protein L or any combination thereof and said affinity ligand is provided linked to a solid support selected from agarose, sepharose, polyacrylic, polystyrene or other synthetic polymers. 
     
     
         82 . The method of  claim 1 , wherein contacting said affinity ligand bound heterogeneous glycoform antibody with a reaction buffer is done for up to 72 hours and wherein said releasing is done using an elution buffer. 
     
     
         83 . The method of  claim 1 , wherein the method further comprises washing with a wash buffer at pH of 6.0-8.0 after (a) and/or (b) to wash the affinity ligand bound heterogeneous glycoform antibody or the substantially homogeneous single glycoform antibody, wherein said wash buffer comprises PBS, TRIS buffer, BIS-TRIS buffer, MES buffer, BES buffer, MOPS buffer or HEPES buffer. 
     
     
         84 . The method of  claim 1 , wherein said heterogeneous glycoform antibody is provided from a cell culture supernatant, from serum or a biological sample. 
     
     
         85 . The method of  claim 1 , further comprising formulating said substantially homogeneous single glycoform antibody into a composition that is formulated for the treatment of a cancer, automimmune disorder or an inflammatory disorder, said composition optionally comprising one or more of a pharmaceutically acceptable carrier, an optional further therapeutic agent, an isotonizing agent or an adjuvant. 
     
     
         86 . A method of generating mAbs having a desired single glycosylation profile in the Fc region formulated for cancer immunotherapy; said method comprising:
 (a) adding a heterogeneous mAb population from a sample to a solid phase support comprising an affinity ligand which binds to said heterogenous mAb to immobilize the mAb on said solid phase support in a manner that exposes glycans of the Fc region;   (b) optionally washing said solid phase with a buffer to wash away any unbound mAb and non-target proteins from said biological sample; and,   (c) contacting said bound heterogenous mAb population on said solid phase support with a reaction buffer comprising one or more enzymes to perform galactosylation and/or desialylation of said mAb;   (d) optionally repeating step (c) to further alter the glycan profile obtained to a new single glycan profile; and   (e) recovering a galactosylated and/or desialylated single glycoform mAb formulated for cancer immunotherapy.   
     
     
         87 . A method of generating antibodies having a desired single glycoform profile formulated for immune disorder or inflammatory immunotherapy from a heterogeneous glycoform antibody population; said method comprising:
 (a) adding said heterogeneous glycoform Ab population from a sample to a solid phase support comprising an affinity ligand which binds to said antibody to immobilize the antibody on said solid phase support in a manner that exposes glycans of the Fc region;   (b) optionally washing said solid phase with a buffer to wash away any unbound antibody and non-target proteins from said biological sample; and,   (c) contacting said bound antibody on said solid phase support with a reaction buffer comprising one or more enzymes to perform galactosylation and sialylation of said antibody at the Fc region;   (d) optionally repeating step (c) to further alter the glycan profile obtained to a new single glycan profile; and   (e) recovering the galactosylated and sialylated single glycoform antibody formulated for immune disorder or inflammatory disorder immunotherapy.