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Structural Analysis of Random Transgene Integration in CHO Manufacturing Cell Lines by Targeted Sequencing

Biotechnology and Bioengineering
December 21, 2021

Chinese Hamster Ovary (CHO) cells are extensively used to produce biotherapeutics. This is because of their robustness, high cell-specific productivity, and ability to express proteins with human-like structure and post-translational modifications. However, CHO cells are prone to genetic instability which can result in changes in productivity or undesired phenotypic characteristics. It is thus important to screen regularly throughout host cell engineering to optimize biologics manufacturing.

Stadermann et al. set out to explore the impact of random transgene integration on the productivities of CHO cell lines. To do this, they leveraged Targeted Locus Amplification (TLA), a valuable Next-Generation Sequencing (NGS) based technology as it enables accurate detection and characterization of the genomic positions of transgene insertion sites and potentially occurring structural rearrangements resulting from the transgene integration.

The results of this study demonstrated that the integration site impacts the performance of CHO cells. The effects of supertransfection (repeated transfection) were also explored and led to boosted production if the integration site was different from the original clone. The analysis of protein-coding genes directly affected by the transgene integration revealed that the transgenes often integrated with genes associated with and relevant to cellular functions like cell growth and proliferation. This emphasizes the importance of analyzing CHO cell lines for events that could alter their phenotype. Furthermore, the analysis of TLA data could clearly identify clones originating from the same mother clone. However, the exact preserved genetic make-up of the integration sites does not necessarily predict the phenotypic behavior of the child clones.

Early genetic analysis of several clones streamlines the cell line development process. TLA proved highly valuable in supporting this. In this work, the workflows of Genedata Selector®  were used for the TLA data processing including quality control. The data analysis was also conducted in the platform to enable the generation of circular plots and the detection of specific integration site.