Original Paper

Journal of Industrial Microbiology & Biotechnology

, Volume 37, Issue 9, pp 961-971

First online:

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

  • Gavin C. BarnardAffiliated withAdimab Inc.
  • , Angela R. KullAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
  • , Nathan S. SharkeyAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
  • , Seemab S. ShaikhAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
  • , Alissa M. RittenhourAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
  • , Irina BurninaAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
  • , Youwei JiangAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
  • , Fang LiAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
  • , Heather LynaughAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Teresa MitchellAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Juergen H. NettAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Adam NylenAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Thomas I. PotgieterAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Bianka PrinzAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Sandra E. RiosAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Dongxing ZhaAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Natarajan SethuramanAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)
    • , Terrance A. StadheimAffiliated withGlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.) Email author 
    • , Piotr BobrowiczAffiliated withAdimab Inc.

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Abstract

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.

Keywords

Antibody Yeast Screening Pichia pastoris Fermentation