High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies
The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.
KeywordsAntibody Yeast Screening Pichia pastoris Fermentation
- 15.Bobrowicz P, Davidson RC, Li H, Potgieter TI, Nett JH, Hamilton SR, Stadheim TA, Miele RG, Bobrowicz B, Mitchell T, Rausch S, Renfer E, Wildt S (2004) Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose. Glycobiology 14:757–766CrossRefPubMedGoogle Scholar
- 18.Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Li H, Choi BK, Hopkins D, Wischnewski W, Roser J, Mitchell T, Strawbridge RR, Hoopes J, Wildt S, Gerngross TU (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313:1441–1443CrossRefPubMedGoogle Scholar
- 19.Li H, Sethuraman N, Stadheim TA, Zha D, Prinz P, Ballew N, Bobrowicz P, Choi BK, Cook WJ, Cukan M, Houston-Cummings NR, Davidson R, Gong B, Hamilton SR, Hoopes JP, Jiang Y, Kim N, Mansfield R, Nett JH, Rios S, Strawbridge R, Wildt S, Gerngross TU (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215CrossRefPubMedGoogle Scholar
- 25.Frachon E, Bondet V, Munier-Lehmann H, Bellalou J (2006) Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols. Appl Environ Microbiol 72:5225–5231CrossRefPubMedGoogle Scholar
- 32.Potgieter TI, Cukan M, Houston-Cummings NR, Drummond JE, Jiang Y, Li F, Lynaugh H, Mallem M, McKelvey T, Mitchell T, Nylen A, Rittenhour A, Stadheim TA, Zha D, d’Anjou M (2009) Production of monoclonal antibodies by glycoengineered Pichia pastoris. J Biotechnol 139(4):318–325CrossRefPubMedGoogle Scholar