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 proteins1-6, 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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