Amino acid sequence variation in protein therapeutics requires close monitoring during cell line and cell culture process development. A cross-functional team of Pfizer colleagues from the Analytical and Bioprocess Development departments worked closely together for over 6 years to formulate and communicate a practical, reliable sequence variant (SV) testing strategy with state-of-the-art techniques that did not necessitate more resources or lengthen project timelines. The final Pfizer SV screening strategy relies on next-generation sequencing (NGS) and amino acid analysis (AAA) as frontline techniques to identify mammalian cell clones with genetic mutations and recognize cell culture process media/feed conditions that induce misincorporations, respectively. Mass spectrometry (MS)-based techniques had previously been used to monitor secreted therapeutic products for SVs, but we found NGS and AAA to be equally informative, faster, less cumbersome screening approaches. MS resources could then be used for other purposes, such as the in-depth characterization of product quality in the final stages of commercial-ready cell line and culture process development. Once an industry-wide challenge, sequence variation is now routinely monitored and controlled at Pfizer (and other biopharmaceutical companies) through increased awareness, dedicated cross-line efforts, smart comprehensive strategies, and advances in instrumentation/software, resulting in even higher product quality standards for biopharmaceutical products.

The inherent nature of cloned CHO cell lines includes the presence of genetic and phenotypic drift that leads to heterogeneous populations. The genetic heterogeneity exhibited by these cells can be exploited to understand the population dynamics of cloned cell lines. Understanding the interplay between heterogeneity, cell culture conditions, and population dynamics will allow for critical assessment of overarching cell line development methods and strategies in terms of population and monoclonality. Sequence variants (SVs) are protein isoforms of the gene-of-interest that contain unintended amino acid substitutions, extensions, or truncations that may contribute to heterogeneity. In this case, SVs are unique sequences in the genome of the integrated transgene that can be used as biomarkers to understand the heterogeneity of a monoclonal cell line and how production process conditions can impact population dynamics. In this study, orthogonal genetic and analytical methods were used to examine the variability of SV levels in four different SV-containing cell lines under varied culture conditions and generational ages. Culture conditions tested had little to no impact on SV levels. However, generational age studies showed two distinct trends: stability of SV levels out to approximately 100 generations in cell lines with higher level SVs (>10%) and a progressive decrease of SV levels as the cells age to approximately 100 generations in cell lines with lower level SVs (<10%). The results suggest that the four SV-containing cell lines fall into two distinct population models; SVs present in the whole population of cells and SVs present in only a sub-population of cells. The data presented here are one of the first studies to not only analyze and compare SV levels in both genetic and protein material but also to utilize SVs as biomarkers to probe distinct populations of cloned cell lines. Biotechnol. Bioeng. 2017;114: 1744-1752. © 2017 Wiley Periodicals, Inc.