Expression of Recombinant Antibodies

Frenzel, André; Hust, Michael; Schirrmann, Thomas

Inefficient secretion of larger heterologous proteins (>30 kDa), proteolysis of secreted proteins during high-cell density fermentation, and inappropriate glycosylation of human glycoproteins are serious issues which required engineering of yeast strains. Overexpression of the chaperone immunoglobulin binding protein (BiP) or protein disulfide isomerase (PDI) in S. cerevisiae increased scFv secretion titers twofold to eightfold, with an average yield of 20 mg/L in shake flask culture (107). Yeasts tend to hyperglycosylate heterologous proteins even at positions not glycosylated in the native mammalian host, which can influence activity of antibodies and is a potential source of immunogenicity or adverse reactions in human patients. P. pastoris exhibits much lower hyperglycosylation than S. cerevisiae, and its N -linked carbohydrate structures are already similar to the mammalian high-mannose core unit Man 5-6 GlcNAc 2 (108). Moreover, genetically modified glyco-engineered P. pastoris strains have been generated which www.frontiersin.org produce humanized glycosylation patterns (109-113). The therapeutic IgG antibodies produced in glyco-engineered yeast achieved results that were comparable to its counterpart Trastuzumab that has been produced in mammalian cells (114). Unlike IgGs produced in wildtype yeast, those produced in glyco-engineered yeasts were able to mediate antibody-mediated effector functions. Production processes employing glyco-engineered yeasts are currently optimized for commercial antibody production (115) as well as for high throughput screening (116).