In cell line development, early clone screening is important. This helps to certify that the selected clone does not harbor any undesired genetic variants. Because DNA mutations or erroneous DNA replication and mistranslation during protein synthesis can give rise to defective proteins^1-6, such events can affect the safety or efficacy of the final therapeutic product.^1,7 Therefore, clone selection carries a lot of weight in safeguarding stability and product quality.

Mass spectrometric approaches (e.g., LC–MS/MS and LC–MS) are commonly the first ones called into action to retrieve information on location, identity as well as relative quantification of sequence variants at the protein level (for peptide mapping).⁸ These techniques, however, are laborious and yield low throughput, especially when detection of low‐level sequence variants is required.^2,3,9,10 As a result, NGS presents itself as an attractive and alternative candidate, in terms of speed, cost, throughput and sensitivity. In a paper published by Novartis, researchers describe the use of TLA kits in-house for CHO clone selection, towards the manufacturing of monoclonal antibodies.¹¹

Genetic QC of antibody-expressing CHO clones with TLA

In this study, TLA:

• Located the exact positions of integration sites
• Confirmed the integrity of integrated sequences in the CHO clones
• Reliably detected high‐level SNVs within the antibody gene (e.g. missense point mutation in the heavy‐chain gene)

In some cases, TLA also found genetic rearrangements. Of note, TLA data was shown to be in line with results from other standard analytical methods. Furthermore, the detection limits for SNVs calling for clone screening and selection was also performed via CHO‐cell‐mixing experiments in this study.¹¹ All in all, TLA proved to be a robust screening method that presents unique advantages for routine clone analytics during cell line development. With regards to the turnaround time, a TLA kit can process up to 24 CHO clones and deliver results in less than a week.¹¹

A single platform that outputs a range of analytical (genomic) screening parameters

Implementing a robust analytical assay - as part of the cell line development workflow - can help accelerate development timelines and reduce manufacturing costs. In this publication, TLA demonstrated its ability to address an important need and proved its capability to deliver additional genomic readouts compared to other NGS-based techniques.^2,9,10

In fact, TLA enables the selective amplification and thus, the targeted sequencing of any locus of interest (without requiring prior detailed knowledge of the region). Thanks to its unique capabilities, TLA can:

• Identify integration sites
• Validate transgene integrity and vector integrated sequences
• Reliably detect all genetic variation, including structural variants, in and around GOI¹²

As such, these unique insights will allow to identify clones that share the same integration site(s), validate the integrity of the genome-integrated vector sequences and ultimately, identify contaminated clones (e.g., those that contain undesired genomic rearrangement, which can in turn express aberrant proteins) to eliminate them early on. In conclusion, TLA can play an essential role in monitoring important quality parameters for clone selection during cell line development.¹¹

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References

[1] Ren D, Zhang J, Pritchett R, Liu H, Kyauk J, Luo J, Amanullah A. Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody. J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Oct 1;879(27):2877-84. doi: 10.1016/j.jchromb.2011.08.015. Epub 2011 Aug 22. PMID: 21900054.

[2] Zhang S, Bartkowiak L, Nabiswa B, Mishra P, Fann J, Ouellette D, Correia I, Regier D, Liu J. Identifying low-level sequence variants via next generation sequencing to aid stable CHO cell line screening. Biotechnol Prog. 2015 Jul-Aug;31(4):1077-85. doi: 10.1002/btpr.2119. Epub 2015 Jun 12. PMID: 26033952.

[3] Yu XC, Borisov OV, Alvarez M, et al. Identification of codon-specific serine to asparagine mistranslation in recombinant monoclonal antibodies by high-resolution mass spectrometry. Analytical Chemistry. 2009 Nov;81(22):9282-9290. DOI: 10.1021/ac901541h.

[4] Wen D, Vecchi MM, Gu S, Su L, Dolnikova J, Huang YM, Foley SF, Garber E, Pederson N, Meier W. Discovery and investigation of misincorporation of serine at asparagine positions in recombinant proteins expressed in Chinese hamster ovary cells. J Biol Chem. 2009 Nov 20;284(47):32686-94. doi: 10.1074/jbc.M109.059360. Epub 2009 Sep 25. PMID: 19783658; PMCID: PMC2781684.

[5] R. J. Harris, A. A. Murnane, S. L. Utter, K. L. Wagner, E. T. Cox, G. D. Polastri, J. C. Helder, M. B. Sliwkowski, Nat. Biotechnol. 1993, 11, 1293.

[6] Dorai H, Santiago A, Campbell M, Tang QM, Lewis MJ, Wang Y, Lu QZ, Wu SL, Hancock W. Characterization of the proteases involved in the N-terminal clipping of glucagon-like-peptide-1-antibody fusion proteins. Biotechnol Prog. 2011 Jan-Feb;27(1):220-31. doi: 10.1002/btpr.537. Epub 2011 Jan 7. PMID: 21312369.

[7] Zhu J. Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv. 2012 Sep-Oct;30(5):1158-70. doi: 10.1016/j.biotechadv.2011.08.022. Epub 2011 Sep 24. PMID: 21968146.

[8] Jiang T, Song H, Slaney TR, et al. Codon-Directed Determination of the Biological Causes of Sequence Variants in Therapeutic Proteins. Analytical Chemistry. 2017 Dec;89(23):12749-12755. DOI: 10.1021/acs.analchem.7b02914.

[9] Wright C, Groot J, Swahn S, McLaughlin H, Liu M, Xu C, Sun C, Zheng E, Estes S. Genetic mutation analysis at early stages of cell line development using next generation sequencing. Biotechnol Prog. 2016;32(3):813–17. doi:10.1002/btpr.2263.

[10] Cartwright JF, Anderson K, Longworth J, Lobb P, James DC. Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing. Biotechnol Bioeng. 2018 Jun;115(6):1485-1498. doi: 10.1002/bit.26561. Epub 2018 Feb 26. PMID: 29427433.

[11] 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.

[12] de Vree, P., de Wit, E., Yilmaz, M. et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32, 1019–1025 (2014). https://doi.org/10.1038/nbt.2959