Development and characterization of an automated imaging workflow to generate clonally‐derived cell lines for therapeutic proteins

In the development of biopharmaceutical products, the expectation of regulatory agencies is that the recombinant proteins are produced from a cell line derived from a single progenitor cell. A single limiting dilution step followed by direct imaging, as supplemental information, provides direct evidence that a cell line originated from a single progenitor cell. To obtain this evidence, a high‐throughput automated imaging system was developed and characterized to consistently ensure that cell lines used for therapeutic protein production are clonally‐derived. Fluorescent cell mixing studies determined that the automated imaging workflow and analysis provide ∼95% confidence in accurately and precisely identifying one cell in a well. Manual inspection of the images increases the confidence that the cell line was derived from a single‐cell to >99.9%. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:584–592, 2018

[1]  R Staszewski,et al.  Murphy's law of limiting dilution cloning. , 1990, Statistics in medicine.

[2]  Edmund N C Newman,et al.  Rapid automated selection of mammalian cell colonies by cell surface protein expression , 2007 .

[3]  Jorge Quiroz,et al.  Statistical analysis of data from limiting dilution cloning to assess monoclonality in generating manufacturing cell lines , 2016, Biotechnology progress.

[4]  J. O’Neill,et al.  A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): development and definition of the system. , 1977, Mutation research.

[5]  Quality of biotechnological products: derivation and characterisation of cell substrates used for production of biotechnological/biological products. ICH Harmonised Tripartite Guideline. , 1998, Developments in biological standardization.

[6]  I. Johnson,et al.  The molecular probes handbook : a guide to fluorescent probes and labeling technologies , 2010 .

[7]  Gary Walsh,et al.  Biopharmaceutical benchmarks 2014 , 2014, Nature Biotechnology.

[8]  C. Demaria,et al.  Accelerated Clone Selection for Recombinant CHO Cells Using a FACS‐Based High‐Throughput Screen , 2007, Biotechnology progress.

[9]  B. Snedecor,et al.  Slashing the timelines: Opting to generate high‐titer clonal lines faster via viability‐based single cell sorting , 2016, Biotechnology progress.

[10]  I Lefkovits,et al.  Limiting dilution analysis of the cells of immune system I. The clonal basis of the immune response. , 1984, Immunology today.

[11]  Jianwei Zhu,et al.  Mammalian cell protein expression for biopharmaceutical production. , 2012, Biotechnology advances.

[12]  Gary Walsh,et al.  Biopharmaceutical benchmarks , 2000, Nature Biotechnology.

[13]  James Savery,et al.  Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging , 2015, Biotechnology progress.

[14]  A. Li,et al.  Simplification of the CHO/HGPRT mutation assay through the growth of Chinese hamster ovary cells as unattached cultures. , 1981, Mutation research.

[15]  R Staszewski.  Murphy's law of limiting dilution cloning revisited. , 1990, Statistics in medicine.

[16]  H. Coller,et al.  Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybridoma monoclonality. , 1986, Methods in enzymology.