Journal of Industrial Microbiology & Biotechnology

, Volume 37, Issue 9, pp 961–971

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

  • Gavin C. Barnard
  • Angela R. 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 E. Rios
  • Dongxing Zha
  • Natarajan Sethuraman
  • Terrance A. Stadheim
  • Piotr Bobrowicz
Original Paper

DOI: 10.1007/s10295-010-0746-1

Cite this article as:
Barnard, G.C., Kull, A.R., Sharkey, N.S. et al. J Ind Microbiol Biotechnol (2010) 37: 961. doi:10.1007/s10295-010-0746-1

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

AntibodyYeastScreeningPichia pastorisFermentation

Copyright information

© Society for Industrial Microbiology 2010

Authors and Affiliations

  • Gavin C. Barnard
    • 1
  • Angela R. Kull
    • 2
  • Nathan S. Sharkey
    • 2
  • Seemab S. Shaikh
    • 2
  • Alissa M. Rittenhour
    • 2
  • Irina Burnina
    • 2
  • Youwei Jiang
    • 2
  • Fang Li
    • 2
  • Heather Lynaugh
    • 2
  • Teresa Mitchell
    • 2
  • Juergen H. Nett
    • 2
  • Adam Nylen
    • 2
  • Thomas I. Potgieter
    • 2
  • Bianka Prinz
    • 2
  • Sandra E. Rios
    • 2
  • Dongxing Zha
    • 2
  • Natarajan Sethuraman
    • 2
  • Terrance A. Stadheim
    • 2
  • Piotr Bobrowicz
    • 1
  1. 1.Adimab Inc.LebanonUSA
  2. 2.GlycoFi Inc. (wholly owned subsidiary of Merck & Co, Inc.)LebanonUSA