The practice of genetically engineering Chinese hamster ovary (CHO) cell lines, to transform them into biotherapeutic protein factories, is widespread.1 In fact, CHO cells remain the preferred mammalian expression because they (1) exhibit a high degree of robustness, (2) can be subjected to various cultivation conditions and (3) are capable of high cell-specific productivities.1 However, CHO cell systems are inherent to genetic instability. As such, their (genomic) plasticity can drive production instability, thereby warranting the need for comprehensive genetic evaluation during cell line development.4-6 In this study, Boehringer Ingelheim applied TLA-based assays to identify the exact position of transgene integration and verify whether structural variations may have potentially occurred post-integration. Therefore, several CHO production cell lines expressing different monoclonal antibodies were genetically compared based on TLA fingerprints.7
Generation of high (CHO) producers via standard transfection displayed a single integration region
3 stable CHO cell clones - each previously transfected with a single expression vector harboring coding sequences for different monoclonal antibody (mAb) and for the CHO glutamine synthetase that act as metabolic selection marker - were selected per mAb and analyzed by TLA and ddPCR. Results indicated that these high producers contained a single integration. Moreover, copy number assessment suggested concatenation within those integration regions. In some cases, genomic rearrangements (e.g. deletions or translocations) even occurred.7
Successive transfection can significantly increase (CHO) product titers
Remarkably, it was found that the generation of CHO cell lines derived from an established high-producing CHO that had undergone consecutive transfection have great potential to significantly boost antibody titers. Surprisingly, for the clones found to be genetically identical following supertransfection, the copy numbers and product titer did not appear to correlate. 7
Complex structural variants induced by transgene integration might provide superior traits to high performing (CHO) clones
Next, the authors investigated whether protein-coding genes affected by transgene integration (e.g. truncated, deleted or translocated) might have conferred superior phenotypes to the high-performing clones that were analyzed. With only one exception, each integration region was linked to at least one gene implicated in cellular processes key to proliferation and growth. While gene function was not further assessed, authors maintain that “knowledge generated in this way by TLA will certainly impact host cell engineering approaches or allow for the early prediction of clone characteristics in the future.”7
Comparative genomic fingerprints of several CHO production cell lines with TLA-based assays
This study sought to genetically characterize transgene insertions and compare multiple CHO cell lines expressing different monoclonal antibodies. For this, the authors resorted to the unique capabilities of TLA, which provide the added benefit of assessing whether structural changes may have accompanied engineering.
In sum, TLA-based assays provided meaningful insights and enabled deeper understanding of transgene integration into high-producing CHO cell lines as well as their ramifications on phenotype. In fact, results from this study underscore the need to draw on multiple sophisticated (clone) screening assays - as well as examining many CHO clones – in order to deepen our comprehension of the processes/genetic events governing strong (clone) performance of CHO production cell lines.
"A valuable tool for comprehensive characterization of CHO production clones early in cell line development"
The use of conventional technologies such as Southern blot or FISH often yield incomplete (genetic) insights.8-9 Conversely, our TLA-based approach can output all critical (genetic) quality attribute (CQA) in a single experiment, as shown in this technological comparison table.
Notably, the recently updated Chinese Hamster reference genome assembly (CriGri-PICRH-1.0 released June 2020, more info on NCBI) has allowed higher-quality genomic analyses. For this reason, our experts are now applying this new reference genome on all incoming projects. In fact, the new reference genome contains chromosomes instead of scaffolds allowing to better visualize results in case of structural rearrangements, and makes it possible to compare TLA results to e.g. FISH data. Also, the quality of this genome meets the same standards as used for other species (e.g. mouse). Finally, as the gene annotation is available, this allows getting information on endogenous genes and/or gene disruption by the introduction of transgenic sequences.
For those eager to accelerate time to clinic and de-risk clone selection early in the CHO cell line development process while keeping their materials and data in-house, you are just in luck. We have recently expanded our product portfolio to include a new in-house solution: CHOice®. Click below to learn more about our latest offering and to find out what CHOice® can do for you!
References
[1] Walsh, G. (2018). Biopharmaceutical benchmarks 2018. Nature Biotechnology, 36, 1136–1145.
[2] Puck, T. T., Cieciura, S. J., & Robinson, A. (1958). GENETICS OF SOMATIC MAMMALIAN CELLS. Journal of Experimental Medicine, 108, 945–956.
[3] Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology, 22, 1393–1398.
[4] Frye, C., Deshpande, R., Estes, S., Francissen, K., Joly, J., Lubiniecki, A., Munro, T., Russell, R., Wang, T., & Anderson, K. (2016). Industry view on the relative importance of “clonality” of biopharmaceuticalproducing cell lines. Biologicals, 44, 117–122. [5] F. M. Wurm & Wurm, 2017
[6] Wurm, M. J., & Wurm, F. M. (2021). Naming CHO cells for biomanufacturing: Genome plasticity and variant phenotypes of cell populations in bioreactors question the relevance of old names. Biotechnology Journal, 2100165. 16
[7] Stadermann A, Gamer M, Fieder J, Lindner B, Fehrmann S, Schmidt M, Schulz P, Gorr IH. Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing. Biotechnol Bioeng. 2022 Mar;119(3):868-880. doi: 10.1002/bit.28012. Epub 2022 Jan 19. PMID: 34935125.
[8] Lattenmayer, C., Loeschel, M., Steinfellner, W., Trummer, E., Mueller, D., Schriebl, K., Vorauer‐Uhl, K., Katinger, H., & Kunert, R. (2006). Identification of transgene integration loci of different highly expressing recombinant CHO cell lines by FISH. Cytotechnology, 51, 171–182.
[9] Li, S., Gao, X., Peng, R., Zhang, S., Fu, W., & Zou, F. (2016). FISH‐based analysis of clonally derived CHO cell populations reveals high probability for transgene integration in a terminal region of chromosome 1 (1q13). PLoS ONE, 11:e0163893.
