Method for producing proteins in pichia pastoris that lack detectable cross binding activity to antibodies against host cell antigens

Abstract

Methods for producing proteins and glycoproteins in Pichia pastoris that lack detectable cross binding activity to antibodies made against host cell antigens are described. In particular, methods are described wherein recombinant Pichia pastoris strains that do not display a β-mannosyltransferase 2 activity with respect to an N-glycan or O-glycan and do not display at least one activity selected from a β-mannosyltransferase 1, 3, and 4 activity to produce recombinant proteins and glycoproteins. These recombinant Pichia pastoris strains can produce proteins and glycoproteins that lack detectable α-mannosidase resistant β-mannose residues thereon and thus, lack cross binding activity to antibodies against host cell antigens. Further described are methods for producing bi-sialylated human erythropoietin in Pichia pastoris that lack detectable cross binding activity to antibodies against host cell antigens.

1 . A method for producing a recombinant glycoprotein in  Pichia pastoris  that lacks detectable cross binding activity with antibodies made against host cell antigens, comprising:
 (a) providing a recombinant  Pichia pastoris  host cell which does not display β-mannosyltransferase 2 activity with respect to an N-glycan or O-glycan and does not display at least one activity selected from β-mannosyltransferase 1 activity and β-mannosyltransferase 3 activity with respect to an N-glycan or O-glycan and which includes a nucleic acid molecule encoding the recombinant glycoprotein;   (b) growing the host cell in a medium under conditions effective for expressing the recombinant glycoprotein; and   (c) recovering the recombinant glycoprotein from the medium to produce the recombinant glycoprotein that lacks detectable cross binding activity with antibodies made against host cell antigens.   
     
     
         2 . The method of  claim 1 , wherein the host cell does not display β-mannosyltransferase 2 activity, β-mannosyltransferase 1 activity, and β-mannosyltransferase 3 activity with respect to an N-glycan or O-glycan. 
     
     
         3 . The method of  claim 1 , wherein the host cell further does not display β-mannosyltransferase 4 activity with respect to an N-glycan or O-glycan. 
     
     
         4 . The method of  claim 1 , wherein the detectable cross binding activity with antibodies made against host cell antigens is determined in a sandwich ELISA. 
     
     
         5 . The method of  claim 1 , wherein the detectable cross binding activity with antibodies made against host cell antigens is determined in a Western blot. 
     
     
         6 . The method of  claim 1 , wherein:
 (i) the recombinant glycoprotein is a therapeutic glycoprotein   (ii) the therapeutic glycoprotein is selected from the group consisting erythropoietin (EPO); cytokines such as interferon α, interferon β, interferon γ, and interferon ω; and granulocyte-colony stimulating factor (GCSF); GM-CSF; coagulation factors such as factor VIII, factor IX, and human protein C; antithrombin III; thrombin; soluble IgE receptor α-chain; immunoglobulins such as IgG, IgG fragments, IgG fusions, and IgM; immunoadhesions and other Fc fusion proteins such as soluble TNF receptor-Fc fusion proteins; RAGE-Fc fusion proteins; interleukins; urokinase; chymase; and urea trypsin inhibitor; IGF-binding protein; epidermal growth factor; growth hormone-releasing factor; annexin V fusion protein; angiostatin; vascular endothelial growth factor-2; myeloid progenitor inhibitory factor-1; osteoprotegerin; α-1-antitrypsin; α-feto proteins; DNase II; kringle 3 of human plasminogen; glucocerebrosidase; TNF binding protein 1; follicle stimulating hormone; cytotoxic T lymphocyte associated antigen 4—Ig; transmembrane activator and calcium modulator and cyclophilin ligand; glucagon like protein 1; and IL-2 receptor agonist;   (iii) the host cell is genetically engineered to produce glycoproteins that have human-like N-glycans; and/or   (iv) the host cell is genetically engineered to produce glycoproteins that have predominantly an N-glycan selected from Man 5 GlcNAc 2 , Man 3 GlcNAc 2 , GlcNAcMan 5 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 , NANAGalGlcNAcMan 5 GlcNAc 2 , GlcNAcMan 3 GlcNAc 2 , GlcNAc (1-4) Man 3 GlcNAc 2 , Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2 , and NANA (1-4) Gal (1-4) GlcNAc (1-4) Man3GlcNAc2.   
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . A composition comprising one or more recombinant glycoproteins obtained by the method of  claim 1 . 
     
     
         11 . A method for producing a mature human erythropoietin in  Pichia pastoris  comprising predominantly sialic acid-terminated biantennary N-glycans and having no detectable cross binding activity with antibodies made against host cell antigens, comprising:
 (a) providing a recombinant  Pichia pastoris  host cell genetically engineered to produce sialic acid-terminated biantennary N-glycans and does not display a β-mannosyltransferase 2 activity with respect to an N-glycan or O-glycan, and does not display at least one activity selected from a β-mannosyltransferase 1 activity and a β-mannosyltransferase 3 activity with respect to an N-glycan or O-glycan and which includes two or more nucleic acid molecules, each encoding a fusion protein comprising a mature human erythropoietin fused to a signal peptide that targets the ER and which is removed when the fusion protein is in the ER;   (b) growing the host cell in a medium under conditions effective for expressing and processing the first and second fusion proteins; and   (c) recovering the mature human erythropoietin from the medium to produce the mature human erythropoietin comprising predominantly sialic acid-terminated biantennary N-glycans and having no detectable cross binding activity with antibodies made against host cell antigens.   
     
     
         12 . The method of  claim 11 , wherein the host cell does not display β-mannosyltransferase 2 activity, β-mannosyltransferase 1 activity, and β-mannosyltransferase 3 activity with respect to an N-glycan or O-glycan. 
     
     
         13 . The method of  claim 12 , wherein the host cell further does not display β-mannosyltransferase 4 activity with respect to an N-glycan or O-glycan. 
     
     
         14 . The method of  claim 11 , wherein the signal peptide is a  S. cerevisiae  αMATpre signal peptide or a chicken lysozyme signal peptide. 
     
     
         15 . The method of  claim 11 , wherein at least one nucleic acid molecule encodes a fusion protein wherein the erythropoietin is fused to the  S. cerevisiae  αMATpre signal peptide and at least one nucleic acid molecule encodes a fusion protein wherein the erythropoietin is fused to the  S. cerevisiae  αMATpre signal peptide a chicken lysozyme signal peptide. 
     
     
         16 . The method of  claim 11 , wherein the codons of the nucleic acid sequence of the nucleic acid molecule encoding the erythropoietin is optimized for expression in  Pichia pastoris.    
     
     
         17 . The method of  claim 11 , wherein the detectable cross binding activity with antibodies made against host cell antigens is determined in a sandwich ELISA. 
     
     
         18 . The method of  claim 11 , wherein the detectable cross binding activity with antibodies made against host cell antigens is determined in a Western blot. 
     
     
         19 . The method of  claim 11 , wherein recovering the mature human erythropoietin comprising predominantly sialic acid-terminated biantennary N-glycans and having no detectable cross binding activity with antibodies made against host cell antigens from the medium includes a cation exchange chromatography step. 
     
     
         20 . The method of  claim 11 , wherein recovering the mature human erythropoietin comprising predominantly sialic acid-terminated biantennary N-glycans and having no detectable cross binding activity with antibodies made against host cell antigens from the medium includes a hydroxyapatite chromatography step. 
     
     
         21 . The method of  claim 11 , wherein recovering the mature human erythropoietin comprising predominantly sialic acid-terminated biantennary N-glycans and having no detectable cross binding activity with antibodies made against host cell antigens from the medium includes an anion exchange chromatography step. 
     
     
         22 . A composition comprising a mature human erythropoietin comprising predominantly sialic acid-terminated biantennary N-glycans and having no detectable cross binding activity with antibodies made against host cell antigens obtained from the method of  claim 18  and a pharmaceutically acceptable salt. 
     
     
         23 . The composition of  claim 22 , wherein the mature human erythropoietin comprising predominantly sialic acid-terminated biantennary N-glycans and having no detectable cross binding activity with antibodies made against host cell antigens is conjugated to a hydrophilic polymer.