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

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.