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Cell Line Development

Detailed TLA reporting for clonality assurance suitable for use in regulatory filings

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4 min read

During the production of recombinant biologics, it is important to ensure consistent quality throughout the entire life cycle of a product. In fact, regulatory agencies ask to demonstrate the clonal derivation of existing cell banks used in the manufacturing process.1,2,3

The more traditional technique of 2 rounds of limiting dilution4,5- with low seeding densities – is not only time-consuming but also costly. Other acceptable procedures include the use of the ClonePix system6, flow cytometry-mediated single cell sorting7,8 and automated cell imaging systems.9 With that said, some ongoing clinical programs employ legacy cell lines that were created before the industry had access to such practices and methods. Therefore, their cell line may not satisfy current regulatory expectations for clonality when filing for market access. In these cases, rather than changing altogether their cell line to ensure compliance, TLA-based solutions offer the opportunity to still perform monoclonality assessment.


Assessing clonal derivation of CHO cells with TLA published by Glenmark

In 2018, Glenmark published an article in which TLA - in combination with NGS - confirmed that 2 independently generated Chinese hamster ovary (CHO) cell lines were clonally derived, with an upper 95% confidence interval limit of a potentially present contaminating population of 1.3%.

Designing a monoclonality assessment

Together with Novartis, we have published a whitepaper in which we propose an efficient method to assess the probability of clonality in recombinant cell lines This collaborative work culminated in the extension of our product portfolio, with the introduction of the: Clonality Assurance Package.

To describe it simply, the package is made up of 2 distinct parts. In the first instance, we will dissect the unique genetic features of your master cell bank (MCB), including breakpoint sequences between genome and vector (in any species), which characterizes integration sites or vector-vector junctions of an integrated concatemer. Given that a CHO cell bank was analyzed in this case study and that the intrinsic plasticity of their genome can result in the loss of specific genetic sequences of the MCB in subclones12,13,14,15, we therefore suggest (as part of our package) to analyze at least 2 MCB-specific breakpoints.

In the second part, we leverage MCB-specific breakpoint qPCR assays and high-level statistical analyses, to verify for the presence of the identified breakpoint reads.

As such, our clonality package offers a rapid, reliable, and cost-effective approach to analytically assess the probability of monoclonal derivation from MCB. Noteworthily, our studies are also performed and reported to be acceptable for regulatory filing in accordance with FDA and EMA requirements (ICH guidelines Q5B and Q5D, CTD module 3.2.S.2.3).4,16,17,18,19

TLA-based solutions make strides in cell line development

With almost a decade of experience, TLA-based solutions have been widely adopted by the pharmaceutical industry at several stages of cell line development.10,20,21,22 Moreover, our newly introduced clonality package has also witnessed growing popularity, thanks to the fact that our detailed reporting of TLA data for clonality assurance is suitable for use in regulatory filings.

Click here to learn more about the structure of our clonality package. If you found this article insightful, you may also be interested in our Genetic Stability Package.


For more information on how TLA can support your cell line development process, watch our latest webinar:

A single NGS-based solution for (CHO) clone selection and comprehensive characterization of transgene integrity, clonality and stability for cell line development

Recorded Thursday 20 May 2021




[1] Frye, C., Deshpande, R., Estes, S., Francissen, K., Joly, J., Lubiniecki, A., et al., 2016. Industry view on the relative importance of "clonality" of biopharmaceutical-producing cell lines. Biologicals 44 (2), 117–122. https://doi.org/10.1016/j.biologicals.2016.01.001.

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

[3] 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.

[4] Kennett S. (2014). Establishing Clonal Cell Lines – A Regulatory Perspective Black Cell, Blue Cell, Old Cell, New Cell? WCBP

[5] Wu P et al. (2018) Tools and methods for providing assurance of clonality for legacy cell lines. Cell Culture Engineering XVI

[6] Newman ENC and Whitney D. (2007) Rapid automated selection of mammalian cell colonies by cell surface protein expression. Nat Methods. 4, 462

[7] Misaghi S et al. (2016) Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting. Biotechnol Prog. 32:198–207

[8] DeMaria CT et al (2007). Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen. Biotechnol Prog. 23:465–472

[9] Evans K et al. (2015). Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging. Biotechnol Prog. 31:1172–1178

[10] Aebischer-Gumy, C, et al. Analytical Assessment of Clonal Derivation of eukaryotic/CHO Cell Populations. Journal of Biotechnology, vol. 286, 2018, pp. 17-26.

[11] Bergboer, J GM., Kelder, M JE., van Min, M., Tuta, N., Crček, M., Vogelsang, M. (2020). Targeted Locus Amplification and NGS combined with qPCR-based breakpoint analysis for the assurance of monoclonality in recombinant cell lines. Retrieved from: https://www.cergentis.com/technology/appnotes

[12] Barnes, L.M., Bentley, C.M., Dickson, A.J., 2003. Stability of protein production from recombinant

[13] Vcelar, S., Melcher, M., Auer, N., Hrdina, A., Puklowski, A., Leisch, F., et al., 2018. Changes in chromosome counts and patterns in CHO cell lines upon generation of recombinant cell lines and subcloning. Biotechnol. J. 13 (3), e1700495. https://doi.org/10.1002/biot.201700495.

[14] Wurm, F., 2013. CHO quasispecies—implications for manufacturing processes. Processes 1 (3), 296–311. https://doi.org/10.3390/pr1030296.

[15] Barnes LM et al. (2003). Stability of protein production from recombinant mammalian cells. Biotechnol. Bioeng. 81:631–639

[16] ICH FDA (1998). Topic Q5D Quality of Biotechnological Products: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products. CPMP/ICH/294/95

[17] EMA (2016). Development, production, characterisation and specifications for monoclonal antibodies and related products. EMA/CHMP/BWP/532517/2008

[18] Novak R. (2017). Regulatory perspective on the evaluation of clonality of mammalian cell banks. CDER/OPQ/OBP/DBRRI

[19] Welch J. (2017). Tilting at clones: A regulatory perspective on the importance of “Clonality” of mammalian cell banks. CDER/OPQ/OBP/DBRRIV

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