Optimized dual assay for the transgenes selection and screening in CHO cell line development for recombinant protein production

Biotechnol Lett. 2019 Sep;41(8-9):929-939. doi: 10.1007/s10529-019-02711-4. Epub 2019 Jul 18.

Abstract

Objective: To develop a simple robust methodology of screening multiple CHO cell clones secreting recombinant proteins to assess their specific productivity.

Results: We developed a dual assay based on immunoassay measurements of a recombinant protein expression combined with staining of viable cells with resazurin. Following this approach, colonies can be simultaneously assessed for cell growth rate and for production of a recombinant protein. Combination of these two assays enables to estimate productivity of a recombinant protein per cell from the very early stages of a cell line development process (CLD) and exclude poor producers from further steps. Comparison of the dual assay with a standard CLD protocol followed by only analysis of protein expression level showed at least 10-20% increase in the amount of clones that can be included into pool of high-producers at early stages. This shortens duration of a typical CLD scheme from 23 to 19 weeks.

Conclusions: Our method: (i) allows to include into workflow clones that demonstrate slow growth during single cell cloning but producing high amounts of a target protein, which otherwise would be lost in standard protocols of cells screening; (ii) can be applied for testing of DNA vectors for transfection and protein production; (iii) can be used for monitoring the heterogeneity of cell population and analysis of stable pools productivity.

Keywords: CHO DG44; DHFR; Optimization; Recombinant mAbs; Recombinant proteins; Resazurin; Selection.

MeSH terms

  • Animals
  • Biotechnology / methods*
  • CHO Cells*
  • Cell Proliferation*
  • Cricetulus
  • Cytological Techniques / methods*
  • Mass Screening / methods*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Staining and Labeling / methods

Substances

  • Recombinant Proteins