Accelerated cell line development using two-color fluorescence activated cell sorting to select highly expressing antibody-producing clones

Biotechnol Bioeng. 2008 Feb 15;99(3):578-87. doi: 10.1002/bit.21612.


The success of engineered monoclonal antibodies as biopharmaceuticals has generated considerable interest in strategies designed to accelerate development of antibody expressing cell lines. Stable mammalian cell lines that express therapeutic antibodies at high levels typically take 6-12 months to develop. Here we describe a novel method to accelerate selection of cells expressing recombinant proteins (e.g., antibodies) using multiparameter fluorescence activated cell sorting (FACS) in association with dual intracellular autofluorescent reporter proteins. The method is co-factor-independent and does not require complex sample preparation. Chinese hamster ovary (CHO) clones expressing high levels of recombinant antibody were selected on the basis of a two-color FACS sorting strategy using heavy and light chain-specific fluorescent reporter proteins. We were able to establish within 12 weeks of transfection cell lines with greater than a 38-fold increase in antibody production when compared to the pool from which they were isolated, following a single round of FACS. The method provides a robust strategy to accelerate selection and characterization of clones and builds a foundation for a predictive model of specific productivity based upon on two-color fluorescence.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / isolation & purification*
  • Antibodies, Monoclonal / metabolism*
  • CHO Cells
  • Cell Culture Techniques / methods
  • Cell Separation / methods*
  • Cloning, Molecular / methods*
  • Cricetinae
  • Cricetulus
  • Flow Cytometry / methods*
  • Microscopy, Fluorescence, Multiphoton / methods*


  • Antibodies, Monoclonal