Mammalian cell line generation is foundational to protein-based therapeutic product development. Ensuring production cell line “clonality” (i.e., clonal derivation of the production cell line) is a requirement for product registration. Many single-cell deposition approaches have been developed since the inception of traditional limiting-dilution. However, regulatory expectations, in line with the capability of this original cloning technique, still lead many institutions to either execute multiple rounds of single-cell plating or conduct extensive screening immediately post-cloning to demonstrate the single-cell origin of a production cell line. Using fluorescence-activated cell sorting (FACS), we have developed a strategy to verify single-cell deposition that, when combined with statistical analysis, creates a highly-effective and efficient process for generating clonally-derived production cell lines in just one round of plating…