When working in pharmaceutical cell line development, you typically seek rapid development of a high producer clone, while ensuring stable product quality.1-3 Even though cell line development is a critical building block in biomanufacturing, it is often considered as the bottleneck within the pharmaceutical supply chain.4 Therefore, thinking about (risk-) mitigation strategies early on will be key to safeguard the successful commercialization of your products.5,6 In fact, recent COVID-19 events have further fueled the desire to simplify production and shorten time to clinic.
To date, clonal Chinese hamster ovary (CHO) cell banks remain the workhorse for therapeutic protein production (e.g. monoclonal antibodies). However, the plasticity of their genomes make them susceptible to genomic rearrangements, which can affect transgenes (especially when concatemerized) and thus, translate into production instability.7,8 Hence, the need for extensive genetic screening of candidate clones during cell line development is not only highly recommended but often, inescapable due to regulatory standards.1,2,9 With an intrinsic motivation for speed to market, biotherapeutic manufacturers are racing to streamline cell line development in order to meet the increasing demand for high amounts of biologics.8,10 Unfortunately, despite being an essential component in risk mitigation, genetics is often overlooked.
In this Opinion & Review, we will elaborate on the benefits of early analytical clone screening in cell line development, weigh in on tools commonly employed to evaluate CHO clones’ genetic fingerprint and explore the advantages of adopting a fully integrated in-house platform for integration site analysis (ISA) to expedite manufacturing and commercialize biotherapeutics.
Genetic characterization: a crucial metric in CHO clone selection
Recombinant expression of therapeutic proteins typically requires stable integration of transgenes into the host genome, whereby higher number of integration sites lead to higher titer.2 Therefore, identifying the exact genomic coordinates of insertion site(s) within the host genome and assessing the genetic integrity of the integrated vector sequence will help certify that the selected CHO clone does not harbor any undesired integration events (e.g. concatemers, which are unstable and prone to rearrangements) or mutations in their GOI (LC/HC).11-15
For these reasons, we have been witnessing a growing interest from manufacturers to identify and adopt high-throughput genetic screening platforms that will enable thorough and rapid selection of stable producer CHO clones, by shedding light on the integrity of integrated GOI sequences.1-3
Traditional methods for comprehensive genetic characterization in CHO clones
Traditionally, scientists resort to a combination of Southern blotting (SB), fluorescence in situ hybridization (FISH) and Sanger sequencing to look at integration sites and genomic integrity of their CHO clones. These technologies, however, only answer part of the questions that need to be answered, and blots may often be difficult to interpret. Therefore, standard techniques are known to yield incomplete results with regards to transgene integration and are limited in their ability to supply information regarding structural variations at the insertion site.16,17 A combination of these technologies with an NGS-based approach is therefore the gold standard expected by regulators.
As for whole-genome sequencing (WGS), the approach generates coverage across the entirety of the genome and can answer questions about transgene integration and genomic integrity. However, the sheer amount of information produced here will only complexify data analysis and interpretation. In fact, it may take more than a week to plough through a single data set. Thus, if you and your team wish to zero in on specific genomic regions (e.g. transgene integration sites), this approach would be superfluous as it would not prove to be time-efficient nor cost-effective. Furthermore, equal coverage of the entire genome moreover means you either get low coverage (and thus confidence) across your region of interest (ROI), or you require astronomical amounts of sequence coverage.3
As previously mentioned, there is today a growing need to streamline cell line development processes to mitigate risk and accelerate early phase testing.5,6 As such, new tools that can yield faster, higher throughput and/or can deliver additional readouts compared to existing approaches will likely attract manufacturers’ attention. With that said, developers want to be able to present – above all – solid genetic evidence to regulators (including FDA and EMA) and more convincingly demonstrate the genetic quality of their products as part of their filings. In 2019, Novartis reported for the first time the applicability and benefits of our TLA/NGS-based workflow as part of their CHO clone selection and described how the method can process up to 24 CHO clones in less than 7 workdays. As a result, this publication illustrates how comprehensive genomic analyses can greatly benefit speed and quality of medicine development programs.
Even though cell culture conditions can be optimized to reduce mistranslations later in development, we sought to underscore here the importance of screening DNA mutations early on, to minimize negative impacts on development in the long run and reduce cost during the CHO cell line development for biopharmaceutical companies.3
A need to adopt robust bioanalytical assays during CHO clone selection
We recognize that genetics represents only a fraction of what is important to CHO manufacturers. In fact, cell line optimization mostly aims at achieving higher productivity and product yields. On this note, several publications have shown the benefits of transient or stable overexpression of keys genes implicated in cellular metabolism, protein biosynthesis and glycosylation towards growth rates, productivity and better product quality, respectively.8,18 While the advantages of high titers or intact proteins are undisputable, it will be equally important to not disregard the added value of genetics in identifying and eliminating clones producing aberrant protein products at an early stage. As evidenced throughout, selecting the CHO clone with the preferred genetic make-up is a crucial step in the cell line development process toward producing stable and high-quality biotherapeutic proteins. The necessity for early clone screening also continues to be heavily propounded by big pharmaceutical companies (e.g. Novartis, Boehringer Ingelheim) and global CDMOs (e.g. FUJIFILM Diosynth Biotechnologies) through various (peer-reviewed) publications, open forums and/or at global conferences.1-3 Moreover, with increasing number of complex novel protein therapeutics entering the pipelines, their associated production and quality assurance processes also become more complex. As a case in point, bispecific antibody production is particularly challenging because it requires the integration of multiple genes and typically involves highly homologous vector sequences.8 Therefore, a method that can easily discriminate the integration sites of each chain variant would prove highly valuable.
A case for routine and early analytical clone screening in CHO cell line development
Looking forward, we would strongly advise not to postpone genetic characterization of your CHO cell lines as genetic QC not only (1) reflects good practice to assess risk prior to clinical testing but can also (2) ensure more expedited time-to-clinic while preventing setbacks. From a process characterization perspective, it will be essential to continue identifying and adopting robust analytical assays to accurately probe Critical Quality Attributes (CQA) not only in final top clones but also, as an overall QC strategy of the selection process and certainty prior advancing into the GMP phase. On this note, genetic characterization can help serve as a business risk indication before reaching clinical trial and is therefore, highly relevant for biosimilar manufacturing.
Recently, Cergentis has expanded its product portfolio to include a new in-house solution: CHOice®. Click below to find out what our new fully integrated in-house solution for CHO clone selection can do for you and your team.
Alternatively, click below to learn watch the recording of our recent webinar to learn how TLA-based solutions are applied by our customers for CHO clone selection.
References
[1] 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.
[2] Pybus LP, Bergboer J, van Min M, Saunders FL. 2020. Application of targeted locus amplification for enhanced Apollo X CHO clone screening. (FUJIFILM Diosynth biotechnologies application note) https://download.cergentis.com/application-note/application-of-targeted-locus-amplification-for-chinese-hamster-ovary-clone-screening
[3] Aeschlimann SH, Graf C, Mayilo D, Lindecker H, Urda L, Kappes N, Burr AL, Simonis M, Splinter E, van Min M, Laux H. Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next-Generation Sequencing Technologies for Cell Line Development. Biotechnol J. 2019 Jul;14(7):e1800371. doi: 10.1002/biot.201800371. Epub 2019 May 15. PMID: 30793505.
[4] The Dish. (2020, December 3). Platform Approach to Accelerating Cell Line and Process Development. Cell Culture Dish. https://cellculturedish.com/platform-approach-to-accelerating-cell-line-and-process-development/
[5] Jesús Zurdo Pharm (2013). Developability assessment as an early de-risking tool for biopharmaceutical development Bioprocess. 1(1), 29–50
[6] Bailly M, Mieczkowski C, Juan V, et al. Predicting Antibody Developability Profiles Through Early Stage Discovery Screening. MAbs. 2020;12(1):1743053. doi:10.1080/19420862.2020.1743053
[7] Torres M, Dickson AJ. Reprogramming of Chinese hamster ovary cells towards enhanced protein secretion. Metab Eng. 2022 Jan;69:249-261. doi: 10.1016/j.ymben.2021.12.004. Epub 2021 Dec 17. PMID: 34929420.
[8] Tihanyi B, Nyitray L. (2020). Recent advances in CHO cell line development for recombinant protein production. Drug Discov Today Technol. Dec;38:25-34. doi: 10.1016/j.ddtec.2021.02.003. Epub 2021 Apr 12. PMID: 34895638.
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[15] Kaas CS, et al. Deep Sequencing Reveals Different Compositions of mRNA Transcribed from the F8 Gene in a Panel of FVIII-Producing CHO Cell Lines. Biotechnol. J. 10(7) 2015: 1081–1089.
[16] 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.
[17] 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.
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