Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch

J Biotechnol. 2017 Aug 10;255:16-27. doi: 10.1016/j.jbiotec.2017.06.009. Epub 2017 Jun 15.

Abstract

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHOBRI/rcTA) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHOBRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHOBRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins.

Keywords: CHO; Cell pools; Inducible expression system; Methionine sulfoximine; Polyethylenimine; Recombinant proteins.

MeSH terms

  • Animals
  • Batch Cell Culture Techniques
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Gene Expression
  • Genetic Vectors
  • Methionine Sulfoximine / pharmacology
  • Plasmids / genetics*
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • Protein Engineering / methods*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*

Substances

  • Recombinant Proteins
  • Methionine Sulfoximine