A framework to quantify karyotype variation associated with CHO cell line instability at a single‐cell level

Abstract

Chinese hamster ovary (CHO) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. However, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. Based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with production instability and that these events can be characterized. We developed a production instability model using secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) as well as a framework to quantify chromosomal rearrangements by karyotyping. In the absence of methotrexate (MTX), CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes. In contrast, when MTX was re-introduced, the growth rate and SEAP productivity reversed to the initial values, demonstrating the reversibility of production instability in CHO-SEAP cells. Fluorescence in situ hybridization analysis identified that the SEAP genes were incorporated in the chromosomal rearrangement (insertion) part of the der(Z9) chromosome. Karyotype analysis indicated that the insertion ratio of the der(Z9) chromosome decreased in the CHO-SEAP cells grown without MTX, demonstrating a correlation between chromosomal rearrangement and production instability. Our results support a mechanism for production instability, wherein a randomly generated chromosomal rearrangement (or genotype) results in cells with a growth advantage that is also associated with non (or low)-producing traits. As a result, the non-producing cells grow faster and thereby outgrow the producing population. Biotechnol. Bioeng. 2017;114: 1045-1053. © 2016 Wiley Periodicals, Inc.

Growth Rate Changes in CHO Host Cells Are Associated with Karyotypic Heterogeneity

“…The P25 cells exhibited a higher specific growth rate, maximum viable cell density, and integrated viable cell density by 18%, 30%, and 24% when compared to the P5 cells, respectively (Figure ), demonstrating that CHO‐DUK cells display unexpected phenotypic changes at late passages. Moreover, the higher growth rate of CHO‐DUK cells in the late passage is consistent with a previous observation in CHO‐SEAP cells; when the only selection pressure is the growth rate (no selective drug), faster growing cells are more likely to be selected and enriched in late passages.…”

“…Chinese hamster ovary (CHO) cells have been widely used for biopharmaceutical manufacturing. While many efforts have been made to achieve improved productivity and product quality of recombinant proteins produced in CHO cells, a number of studies have described cell line instability, such as a decrease in productivity, reduced culture longevity, or varied host cell protein expression during extended cell cultures . Cell line instability results in variability in the biomanufacturing process that can affect cost or regulatory compliance, yet the causes and mechanisms of cell line instability remain elusive.…”

“…Cell line instability results in variability in the biomanufacturing process that can affect cost or regulatory compliance, yet the causes and mechanisms of cell line instability remain elusive. Given the hypothesis that cell line instability is associated with chromosomal rearrangements, we previously developed and applied a karyotype‐based framework to quantify chromosomal rearrangements in secreted alkaline phosphatase (SEAP)‐producing CHO cells that exhibited production instability . Karyotyping and fluorescence in situ hybridization analysis demonstrated that the SEAP production instability was associated with a particular chromosomal rearrangement, suggesting a mutation‐and‐selection mechanism .…”

CHO‐Omics Review: The Impact of Current and Emerging Technologies on Chinese Hamster Ovary Based Bioproduction

“…The future of omics for bioproduction likely entails employing multiple omics technologies on individual CHO cell lines rather than an all‐encompassing consensus model. While a consensus CHO model integrating multiple omics datasets is useful and predictive for CHO cell engineering and media development, the variability of parental CHO cell lines accompanied by genomic instability resulting from the introduction of transgenes calls for an approach that uses omics to scrutinize individual CHO production lines . Similar to the idea of personalized medicine, focusing omics strategies on individual CHO lines, beginning from the parental strain through to the final production clone, will maximize the gains in productivity and quality resulting from cell line engineering, and medium and process optimizations for each individual cell line.…”

“…Incorporating CHO single cell heterogeneity data into systems models is currently not possible since extensive data are currently lacking. In this regard, emerging methods of acquiring and analyzing data from single cells will be necessary …”