Engineering Antibodies on the Surface of CHO Cells

Methods Mol Biol. 2020;2070:397-422. doi: 10.1007/978-1-4939-9853-1_22.


While antibody libraries are traditionally screened in phage, bacterial, or yeast display formats, they are produced in large scale for pharmaceutical and commercial use in mammalian cell lines. The simpler organisms used for screening have significantly different folding and glycosylation machinery than mammalian cells; consequently, clones resulting from these libraries may require further optimization for mammalian cell expression. To streamline the antibody discovery process, we developed a Chinese hamster ovary (CHO) cell-based selection system that allows for long-term display of antibody Fab fragments. This system is facilitated by a semi-stable Epi-CHO episomal platform to maintain antibody expression for up to 2 months and is compatible with standard PCR-based mutagenesis strategies. This protocol describes the simple and accessible use of CHO display coupled with flow cytometry to enrich for antibody variants with increased ligand-binding affinity from large libraries of ~106 variants, using HER2-binding antibodies as an example.

Keywords: Antibody engineering; CHO; Glycosylation; Mammalian; Surface display.

MeSH terms

  • Animals
  • Antibodies, Monoclonal* / biosynthesis
  • Antibodies, Monoclonal* / chemistry
  • Antibodies, Monoclonal* / genetics
  • CHO Cells
  • Cell Surface Display Techniques*
  • Cricetulus
  • Genetic Vectors / chemistry
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Humans
  • Immunoglobulin Fab Fragments* / biosynthesis
  • Immunoglobulin Fab Fragments* / chemistry
  • Immunoglobulin Fab Fragments* / genetics
  • Mutagenesis
  • Plasmids / chemistry
  • Plasmids / genetics
  • Plasmids / metabolism
  • Protein Engineering*


  • Antibodies, Monoclonal
  • Immunoglobulin Fab Fragments