Assessing the probability of clonality achieved by single‐cell cloning of CHO cells through cell deposition combined with imaging using distinguishable cells

Abstract Mammalian cell lines used for clinical studies and post‐approval production of recombinant DNA‐derived biotherapeutics are expected to be derived from a single cell, and regulatory submissions are expected to provide robust evidence of monoclonality. Imaged single‐cell deposition followed by whole‐well imaging using specialized instruments has, in many cell line development labs, replaced the “gold standard” of two rounds of limiting dilution due to its increased speed and the assurance of clonality provided by orthogonal images. However, there is still a lack of information on how the procedures used to define these clonal cell lines perform. Here we use a mixture of two distinguishable Chinese hamster ovary (CHO) cells to document that a greater than 99% probability of clonality can be obtained from our single‐cell cloning method that uses our preparation procedures, the VIPS® single‐cell deposition instrument, the Cell Metric® whole‐well imager, and a comprehensive visual review. Together with the assurance of cell/well images, the determination of the probability of clonality of our VIPS+Cell Metric method provides a strong package of evidence of single‐cell derivation of a recombinant CHO cell line.