CHO Cell Genetic Instability: From Transfection to Stable Cell Line

Chinese hamster ovary (CHO) cells are the predominant host cell type used in the production of recombinant therapeutic proteins. They are chosen as hosts, because of their ability to create, fold and modify proteins in a manner that makes them compatible with the human immune system. Moreover, CHO cells are tried and tested model organisms for bioprocess platforms, meaning regulatory body approval for new therapeutics is relatively easy to achieve. CHO cells are inherently genetically unstable, which can lead to a decline in productivity and poses a threat to product quality heterogeneity of stable cell lines. The primary aim of this thesis was to characterise genomic instability of a CHOK1SV cell line and measure directly the impact this genetic instability has on the fidelity of recombinant plasmid copies. The impact of this would be two-fold: Firstly, an accurate quantification of genetic instability type and frequency would be established. Secondly, the techniques used to characterise genetic instability would be evaluated as tools for the detection of instability in cell line development processes. Microsatellite analysis and karyotype analysis were used to assess CHO cell genomic instability at the base pair / gene copy number (GCN) level and the chromosome level respectively. Microsatellites were found to be effective markers for genetic drift and cell line relatedness. However, there was no substantial evidence of microsatellite mutational change, and so it could not be concluded that microsatellites are an effective marker for deficient DNA replication / DNA damage or mismatch repair. Microsatellite change did not correlate with changes in GCN or cell specific productivity (qP). There was substantial evidence of chromosomal aberration from Karyotype analysis, which showed considerable levels of aneuploidy and chromosome breakage/fusion events. It was concluded that CHO cells have an inherent chromosomal instability and that karyotyping is a promising tool for genetic instability cell line development assessments. However, there was no substantial association found between changes in CHO karyotype and changes in qP or GCN. In order to generate a stable GFP cell line for the investigation of recombinant plasmid genetic instability it was necessary to optimise an electroporation protocol. Preliminary experiments indicated that standard industry conditions were suboptimal and so a Design of Experiments (DoE) – based strategy was used to optimise electroporation. Final optimal conditions (termed 320-26) improved transfection efficiency by 17%. The final results chapter outlines a novel single-molecule real time (SMRT) sequencing analysis platform, which maximises the sensitivity of the technology, enabling mutation calling from individual molecules at a 0.01% frequency. One mutation was present at high levels throughout the study, a C T transition in the bacterial origin of replication, which is assumed to have originated from the original plasmid stock. There was no evidence of mutations arising in plasmid cloning or as a result of the pre-integration CHO cell environment. Substantial levels of point mutation were found in recombinant plasmid copies. Mutations were randomly distributed along the length of the plasmid and were apparently not influenced by natural selection. G and C residues were mutated to a greater extent than A and T residues, with G.C A.T transitions predominating. This final assessment of CHO cell genetic instability shows the requirement for product quality checks during cell line development.