A high-fidelity, dual site-specific integration system in CHO cells by a Bxb1 recombinase.

Site-specific integration (SSI) via recombinase mediated cassette exchange (RMCE) has shown advantages over random integration methods for expression of biotherapeutics. As an extension of our previous work developing SSI host cells, we developed a dual-site SSI system having two independent integration sites at different genomic loci, each containing a unique landing pad (LP). This system was leveraged to generate and compare two RMCE hosts, one (dFRT) compatible with the Flp recombinase, the other (dBxb1) compatible with the Bxb1 recombinase. Our comparison demonstrated that the dBxb1 host was able to generate stable transfectant pools in a shorter time frame, and cells within the dBxb1 transfectant pools were more phenotypically and genotypically stable. We further improved process performance of the dBxb1 host, resulting in desired fed batch performance attributes. Clones derived from this improved host (referred as 41L-11) maintained stable expression profiles over extended generations. While the data represents a significant improvement in the efficiency of our cell line development process, the dual LP architecture also affords a high degree of flexibility for development of complex protein modalities.

HIF-1 Signaling Pathway Implicated in Phenotypic Instability in a Chinese Hamster Ovary Production Cell Line.

Monitoring genotypic and phenotypic stability is crucial during the development of recombinant Chinese hamster ovary (CHO) cell lines. Although genotypic instability is well-studied, there are few reports on phenotypic instability. Here, a case study of two clonal cell lines derived from Pfizer's site-specific integration expression platform that expresses the same monoclonal antibody is described. It is shown that both cell lines (herein referred to as "Cell Line A" and "Cell Line B") are genotypically stable up to 130 generations. However, when both cell lines are run side-by-side in a fed-batch production assay, productivity from Cell Line A later generation cells is much lower when compared to earlier generation cells. Phenotypically, later generation Cell Line A cells display increased lactate production, decreased productivity, and decreased cell viability. Metabolic analysis reveals that Cell Line A exhibits increased glycolysis activity and capacity at higher generational age. Whole transcriptomic sequencing shows significant upregulation of the hypoxia-inducible factor 1-alpha (HIF-1α) signaling pathway and associated downstream targets. Furthermore, Western blot analysis confirms elevated HIF-1α protein in Cell Line A cells at later generation. These results suggest a novel role for HIF-1α in the age-associated metabolic changes that result in the phenotypic instability of a recombinant CHO cell line.