Chromosomal Instability Drives Convergent and Divergent Evolution Traits in Mammalian CHO Lineages

Product quality control to avoid lot-to-lot variation is critical in biotherapeutic protein manufacturing and remains a challenge. Intrinsic genetic instability of Chinese hamster ovary (CHO) cells, as the primary expression host, has been implicated in production inconsistency yet the mechanism is poorly defined. Using a multi-omics approach, we systemically analyzed the variations of CHO lineages derived from a common CHO-K1 line derived from the original parental Puck lab ancestry. We identify a unique equilibrium between continuous random genetic variation of the CHO genome and select heritable traits driven by culture adaptation conditions, selection criteria, and genetic linkage. The inherited changes are associated with the selection pressures related to serum removal, adherent/suspension culture transition, protein expression and secretion capacity. Clonal cells continuously evolve demonstrating both unique and surprisingly common chromosome (Chr) structural rearrangement across lineages yielding signatures varying in frequency, magnitude and selectivity and highlighting that genetic and phenotypic inheritance is affected by gene location and linkage. We observed that a universally conserved haploid reduction of a region of Chromosome (Chr) 2 (36-60 mB), present after serum-free adaptation in suspension culture, was consistently inherited, suggesting a common chromosomal rearrangement for general adaptation of serum-free suspension culture. Genetic variations also included approximately ~200s insertions/deletions (INDELs), ~1000s single nucleotide polymorphisms (SNPs), and ~300-2000s copy number variations (CNVs), which are exacerbated after gene editing. Global staining patterns of H3K9 tri-methylation showed that Chr 9, 10 and X are significantly more heterochromatic as compared to other chromosomes, which is reverse correlated with integration frequency of recombinant DNA. Varying epigenetic mechanisms provide transcriptional regulation, resulting in stable inheritance of 11% - 64% transcriptomic changes through lineages. Together, these observations demonstrate a highly plastic, multi-dimensional ‘omics’ signature for CHO cells and points the way towards future chromosomal engineering and other synthetic biology interventions for rational design of optimal bioproduction hosts.