Biotherapeutics have become increasingly complex over the last decade, and so has their engineering process.¹ Because of this, the industry demands streamlined cell line development processes to mitigate risks and accelerate early phase testing. Comprehensive genomic analyses have shown to greatly benefit speed and quality of medicine development programs.² Besides, those who seek commercial licensure will need to present solid genetic evidence to the regulatory authorities (including U.S. Food and Drug Administration (FDA) and European Medicines agency (EMA)) to guarantee the quality, safety, and efficacy of their manufactured biological products (once they wish to enter clinical studies or market).^3-6

Given the mounting focus on clonal derivation of mammalian production cell lines in biopharmaceutical development, we dedicated this Opinion & Review to expand on the role of genetic data as part of clonality assessment.

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I. Clonality assessment: a control strategy for product development requested by regulatory authorities

Both industry and regulators want to ensure consistency and quality of biological therapeutics and therefore, their manufacturing process. According to officials, one critical criteria is to assure clonality (i.e. the clonal derivation of master cell bank (MCB)/working cell bank (WCB) used in the manufacturing process). Clonality assurance is thought to minimize the heterogeneity of cell banks and thus, favor a consistent process to manufacture a product.^1,7-9 In fact, the US FDA and EMA reference ICH Q5D and EMA/CHMP in their guidelines, respectively, and both explicitly indicate that biologic products should be derived from a single cell progenitor or monoclonal cell line.

This requirement led industrial players to improve cloning methods and integrate single-cell cloning technologies step as part of their cell line development programs supporting clinical and commercial use.^1,10 Indeed, if the submitted data is unconvincing, additional manufacturing controls will most likely be required. From an upper management perspective, this implies delay and added costs, in a time where speeding up drug development and speed-to-market is more relevant than ever. Such significant and additional efforts, toward the development of adequate control strategies, may then compromise the competitive edge of a product. To evade such unwelcome setback, pharma and biotech companies are racing to identify ways to demonstrate clonal derivation of their existing cell banks more convincingly.

Based on our interactions with experts in this field as well as frequent discussions with our customers, it has become apparent that manufacturers who have developed their cell line via a limiting dilution approach are often asked to provide additional documentation and evidence as part of their regulatory filling process. For this, officials have publicly been encouraging the use of newer cloning methods (e.g. FACS, ClonePix, Solentim's VIPS, etc.), provided that the method is qualified and that its capabilities have been validated.^11,12 While proven to be a suitable approach for gathering evidence to support clonality during IND or BLA filing newly generated cell lines, the above-listed developments prove unsuitable for “legacy cell lines”.¹⁰ There, re-cloning of the MCB - late in development - may introduce significant risk (e.g. affect titer and quality of products) and might also not be advisable due to comparability issues (i.e. difficulty in linking pre-clinical lots with commercial lots).¹³

II. Advantages and limitations of genetic testing methods to support clonal derivation of legacy cell lines

As previously mentioned, while recent developments (e.g. single cell sorting and high-throughput imaging) allow for the acquisition of evidence supporting clonality during (for instance) an IND or BLA filing, such acceptable assurance may not be available for legacy cell lines. To this end, industry leaders like Novartis tapped into our proprietary genetic QC solutions to develop a high-level statistical analysis in order to better assess clonality in recombinant cell lines. The fruitful outcome of this collaboration was put forward in a joint whitepaper.¹⁴

Interestingly and prior to this release, Glenmark Pharmaceuticals also leveraged our proprietary technology to design a method that would allow them to show that a cell population is derived from a single ancestor cell, based on the homogeneity of specific genomic signatures.⁷ Glenmark published their approach in the Journal of Biotechnology. A range of leading pharmaceutical companies discussed with regulatory authorities on the suitability of our approach to prove clonality – after which we successfully performed these projects for them.

Besides our proprietary approach, other molecular technologies can also help you characterize individual subclones, from the MCB, to determine their genetic make-up and determine whether they are genetically similar. Table I (retrieved from Wu et al’s commentary) clearly outlines some alternatives for genetic testing that can be applied toward clonality assessment of legacy cell lines.¹⁰ Besides lower resolution, none of the listed technologies can output all the essential (genetic) characteristics in a single experiment, like TLA-based assays can.

III. Including genetic data as part of your regulatory filings

Although we recognize that genetics only represents one of the components to determine the quality of a product, it lies at the fundament of producing your biologic. In recognition of our unrivalled expertise and TLA-based capabilities, leading biotech and pharma globally outsource the complexities accompanying genetic analyses to our capable hands, allowing them to fully focus instead on what they do best: bringing needed treatments and therapies to the patients.

Indeed, our proprietary TLA-based solutions are ideally positioned to help you and your team navigate the stringent genetic QC hurdles imposed by regulatory authorities. With almost a decade of experience, our assays have been extensively endorsed in public literature (including peer-reviewed publications by Pfizer/Lonza, Janssen R&D, Novartis), have been used in successful IND/BLA applications, and are widely regarded as the gold standard for the genetic characterization of engineered (pharmaceutical) cell lines, be it to:

• QC novel transfection methods
• Select clones with desired genetic characteristics
• Assure monoclonality
• Thoroughly characterize MCB
• Assess genetic stability/drift

These in turn have warranted partnerships with PerkinElmer, FUJIFILM Diosynth Biotechnologies and Genedata Selector. Crucially, if you and your team are on a hunt for a one-stop shop that provides reporting on par with regulatory authorities’ expectations, then look no further: Cergentis is your go-to partner.

To learn more about our Clonality Assurance Package, click below:

References

[1] Frye C, Deshpande R, Estes S, Francissen K, Joly J, Lubiniecki A, Munro T, Russell R, Wang T, Anderson K. Industry view on the relative importance of "clonality" of biopharmaceutical-producing cell lines. Biologicals. 2016 Mar;44(2):117-22. doi: 10.1016/j.biologicals.2016.01.001. Epub 2016 Feb 3. PMID: 26852257.

[2] May, M. (2021, May 25). Accelerating cell line development timelines. Genetic Engineering & Biotechnology News. https://www.genengnews.com/topics/bioprocessing/accelerating-cell-line-development-timelines/

[3] ICH Q5B. (1996). Analysis of the expression construct in cell lines used for production of rDNA-derived protein products. European Medicines Agency. https://www.ema.europa.eu/en/ich-q5b-analysis-expression-construct-cell-lines-used-production-rdna-derived-protein-products

[4] EMA. (2018). Requirements for quality documentation concerning biological investigational medicinal products in clinical trials. European Medicines Agency. https://www.ema.europa.eu/en/requirements-quality-documentation-concerning-biological-investigational-medicinal-products-clinical

[5] ICH Q5D. (1998). ICH Q5D Derivation and characterisation of cell substrates used for production of biotechnological/biological products. European Medicines Agency. https://www.ema.europa.eu/en/ich-q5d-derivation-characterisation-cell-substrates-used-production-biotechnologicalbiological

[6] FDA. (1992). Supplement to the points to consider in the production and testing of new drugs and biologic & produced by recombinant dna technology: nucleic acid characterization and genetic stability. U.S. Food and Drug Administration. https://www.fda.gov/media/70898/download

[7] Aebischer-Gumy C, Moretti P, Little TA, Bertschinger M. Analytical assessment of clonal derivation of eukaryotic/CHO cell populations. J Biotechnol. 2018 Nov 20;286:17-26. doi: 10.1016/j.jbiotec.2018.08.020. Epub 2018 Aug 30. Erratum in: J Biotechnol. 2020 Jan 10;307:208. PMID: 30172783.

[8] Walsh, G., 2014. Biopharmaceutical benchmarks 2014. Nat. Biotechnol. 32 (10), 992–1000.

[9] Zhu, J., 2012. Mammalian cell protein expression for biopharmaceutical production. Biotechnol. Adv. 30 (5), 1158–1170. https://doi.org/10.1016/j.biotechadv.2011.08.022.

[10] Wu P, Hartman T, Almond L, Stevens J, Thrift J, Ojha J, Alves C, Shaw D, Laird MW, Emmins R, Zhu Y, Liu R, Du Z, Koehler R, Jostock T, Anderson K, Campbell C, Clarke H. Advancing Biologics Development Programs with Legacy Cell Lines: Advantages and Limitations of Genetic Testing for Addressing Clonality Concerns Prior to Availability of Late Stage Process and Product Consistency Data. PDA J Pharm Sci Technol. 2020 Mar-Apr;74(2):264-274. doi: 10.5731/pdajpst.2018.009316. Epub 2019 Sep 13. PMID: 31519780.

[11] Welch JT, Arden NS. Considering "clonality": A regulatory perspective on the importance of the clonal derivation of mammalian cell banks in biopharmaceutical development. Biologicals. 2019 Nov;62:16-21. doi: 10.1016/j.biologicals.2019.09.006. Epub 2019 Oct 3. PMID: 31588011.

[12] Jia, A. (2016, September 20). Interview with ex-FDA regulator: Session 1 (of 4). Solentim. https://benchfly.com/video/3093/interview-with-ex-fda-regulator-session-1-of-/

[13] Novak, R. (2017, January 23). Regulatory perspective on the evaluation of clonality of mammalian cell banks. U.S. Food and Drug Administration. https://cdn.ymaws.com/www.casss.org/resource/resmgr/cmc_no_am_jan_spkr_slds/2017_CMCJ_Novak_Rachel.pdf

[14] Bergboer, JGM., Kelder, MJE., van Min, M., Tuta, N., Crček, M., Vogelsang, M. (2020). TLA and NGS combined with qPCR-based breakpoint analysis for the assurance of monoclonality in recombinant cell lines. Cergentis. https://download.cergentis.com/whitepaper/novartis-clonality-assurance

[15] Ho C, Tong, Y & Yang Y (2013). Generation of monoclonal antibody-producing mammalian cell lines. Pharm. Bioprocess. 1(1), 71–87

[16] Pérez A, Sormanni P, Andersen S, Sakhnini S, Rodriguez-Leon I, Bjelke J, Gajhede A, De Maria L, Otzen D, Vendruscolo, M & Lorenzen N (2019). In vitro and in silico assessment of the developability of a designed monoclonal antibody library, mAbs, 11:2, 388 400, DOI: 10.1080/19420862.2018.1556082

[17] Jesús Zurdo Pharm (2013). Developability assessment as an early de-risking tool for biopharmaceutical development Bioprocess. 1(1), 29–50

[18] 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