Monoclonal antibodies (mAbs) currently dominate the biopharmaceutical market. In fact, 84% of mAbs stem from Chinese hamster ovary (CHO) expression system1,2, rendering it the most popular cell factory for the production of recombinant biotherapeutics.
However, the CHO genome is notorious for undergoing a constant and high-rate rearrangement. Its unstable transgene expression therefore presents challenges during manufacturing and scale up. As a result, scientists at Janssen R&D, the University of Natural Resources and Life Sciences in Vienna and the Austrian Centre of Industrial Biotechnology sought to dissect the molecular drivers of expression instability to pinpoint the inefficient sites for integration.3
Studying factors that govern phenotypic heterogeneity in CHO cells to steer away from instability in the future
Traditional strategies based on random integration (RI) - for the development of a high-expressing recombinant CHO cell line - are regarded as one of the causes for instability and heterogeneity.4,5 Hence, the link between transgene integration and molecular repercussions - vis-à-vis the disturbance of protein production - has been the subject of many studies for many years. Past research have shown that genomic rearrangements occurring at the transgene integration site can lead to loss of [transgene] copies over time.6 In turn, epigenetic modifications are thought to drive “transcription silencing” of the promoters. Moreover, if transgenes are found near telomeres or heterochromatin regions, “position effect” is then anticipated.7 All in all, CHO cell lines with unstable transgene expression are more susceptible to apoptosis and are more likely to experience reduced efficiency with regards to post-transcriptional processes.8
To fathom out the factors driving phenotypic heterogeneity, stable and unstable phenotypes of Horizon Discovery CHO-K1 (HD-BIOP3) derived production cell lines were compared in this study. More specifically, the cell lines were sorted into 6 phenotypes corresponding to low, medium or high copy number with stable and unstable transgene expression. For each category, a subclone was selected and sequenced by WGS, RNA-Seq and TLA (to sequence the entire genome, transcriptome, and QC the transgene construct as well as the neighboring regions, respectively).3
Assessing the precise location of integration events and the integrity of transgenic loci with TLA
13 cell lines sampled from early passage culture were analyzed by TLA. To assess whether variants may have arisen during cell passages, the main 6 cells were also evaluated at P10.
Upon screening a low copy cell line with unstable transgene expression (5G10), TLA data picked up 2 integration sites, whereby one brought about an inversion and the other occasioned a deletion event in the host genome. Additionally, TLA spotted partial integration at a third site at P10.
Based on all the results, it was inferred that products from multiple integration sites that spawn structural rearrangements and precipitate incomplete transcripts at late passage is likely to interfere with the host transcriptome and can, hence, trigger instability.
Expression stability of CHO cell lines can be regulated at 3 levels
In conclusion, it appears that consistent productivity and stability can be controlled at 3 levels9,10:
1) The location of the integration site. Regions with low genomic variability and high transcriptional activity are favorable sites to target.
2) Transgene integrity and concatemerization. The precise arrangement of the integrated transgene/vector sequences should have low transgene fusions and should not be accompanied by large structural rearrangements.
3) Stress related cellular processes. Differential expression of genes associated with such processes should be absent.
Relevance of TLA-based solutions in cell line development
This consequential work underscores the importance of early documentation in cell line engineering projects. Ultimately, the authors advise that proper clone screening and thorough genetic characterization should be performed and considered (for the choice of a subclone) already at early stages of cell line development. With that said, TLA-based solutions can be relevant at several stages of cell line development:
1) To assist in the optimization and validation of new technologies
2) To select clones with desired (clean) integrations
3) To assess clonality and genetic stability of your cell lines
For more information, we invite you to consult our Cell Line Development page.
References
[1] Walsh G. Biopharmaceutical benchmarks 2018. Nat Biotechnol 2018;36:1136–45.
[2] Jayapal K, Wlaschin K, Hu W, Yap M. Recombinant protein therapeutics from CHO Cells - 20 years and counting. Chem Eng Prog 2007;103:40–7.
[3] Dhiman H, Campbell M, Melcher M, Smith KD, Borth N. Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations. Comput Struct Biotechnol J. 2020 Nov 12;18:3632-3648. doi: 10.1016/j.csbj.2020.11.008. PMID: 33304461; PMCID: PMC7710658.
[4] Hamaker NK, Lee KH. Site-specific integration ushers in a new era of precise CHO cell line engineering. Curr Opin Chem Eng 2018;22:152–60.
[5] Lee JS, Kildegaard HF, Lewis NE, Lee GM. Mitigating Clonal Variation in Recombinant Mammalian Cell Lines. Trends Biotechnol 2019. https://doi.org/10.1016/j.tibtech.2019.02.007.
[6] Bandyopadhyay AA, O’Brien SA, Zhao L, Fu H-Y, Vishwanathan N, Hu W-S. Recurring genomic structural variation leads to clonal instability and loss of productivity. Biotechnol. Bioeng.
[7] Wilson C, Bellen HJ, Gehring WJ. Position effects on eukaryotic gene expression. Annu Rev Cell Biol 1990;6:679–714.
[8] Dorai H, Corisdeo S, Ellis D, Kinney C, Chomo M, Hawley-Nelson P, et al. Early prediction of instability of Chinese hamster ovary cell lines expressing recombinant antibodies and antibody-fusion proteins. Biotechnol Bioeng 2012;109:1016–30.
[9] Kohli A, Melendi PG, Abranches R, Capell T, Stoger E, Christou P. The quest to understand the basis and mechanisms that control expression of introduced transgenes in crop plants. Plant Signal Behav 2006;1:185–95.
[10] Papapetrou EP, Schambach A. Gene insertion into genomic safe harbors for human gene therapy. Mol Ther 2016;24:678–84.
