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High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies

2010; Springer Science+Business Media; Volume: 37; Issue: 9 Linguagem: Inglês

10.1007/s10295-010-0746-1

1476-5535

Gavin C. Barnard, Angela Kull, Nathan S. Sharkey, Seemab S. Shaikh, Alissa M. Rittenhour, Irina Burnina, Youwei Jiang, Fang Li, Heather Lynaugh, Teresa Mitchell, Juergen H. Nett, Adam Nylen, Thomas I. Potgieter, Bianka Prinz, Sandra Rios, Dongxing Zha, Natarajan Sethuraman, Terrance A. Stadheim, Piotr Bobrowicz,

Monoclonal and Polyclonal Antibodies Research

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.