As antibody formats become increasingly complex, so does their production and quality assurance. In bispecific antibody production, ICH Q5D guidelines require verification that the transgenes encoding all four individual chains of the antibody are inserted correctly into the cell. This can be a challenge when using traditional methods such as FISH or Southern Blot because the sequences of different genes are highly similar.
That’s where Targeted Locus Amplification (TLA) can help by producing high-quality genetic sequence data based on proximity to the inserted genes.
Therapeutic uses of antibodies
Antibodies are widely used for both therapeutic and research purposes, thanks to their ability to specifically target certain biomolecules, known as antigens. Therapeutic applications capitalize on the neutralization, carrier or cytotoxic effects of antibodies, and are used in a range of areas, including cancer, cardiovascular disease, inflammatory disease, infectious diseases and organ transplantation, to name but a few1.
As with all developing technologies, therapeutic antibodies and their production have evolved significantly since the early days of isolating specific B-cell clones from antigen-injected mice. Modern antibody engineering techniques allow scientists to improve the kinetic function, stability and mechanisms of action to specifically enhance antibody functionality.
Altering the basic structure of the antibody can provide novel or enhanced functions. Such engineered antibody fragments reduce native antibodies to their bare functional bones, which can improve tissue penetration and ease manufacturing2.
In addition to the production of simplified antibody fragments, another emerging trend is that of bispecific antibodies. Antibodies, due to their structure of paired heavy and light chains with constant and variable domains, are ideal for alterations that allow them to simultaneously target two different antigens. These provide advanced functions, for example, recruiting cytotoxic immune cells to specific tumor cells by simultaneously targeting receptors on both1.
Bispecific antibodies are usually produced by cloning reverse-transcribed mRNA into mammalian cell lines, such as CHO cells. This can be challenging, since it relies on the correct integration of four transgenes: two light chains and two heavy chains. Ensuring the quality of the final product requires the identification and purification of a single clonal cell line expressing the correctly assembled antibody3.
Traditional methods used to genetically characterize these transgenic cell lines, such as FISH or Southern blots, have difficulty distinguishing between the integration sites of each chain variant because the sequences are so similar. Therefore, a more reliable method of characterization is needed.
Targeted Locus Amplification
Conventional sequencing techniques are hypothesis-driven, since they either require some prior knowledge of the insertion point to allow for primer design, or do not provide in-depth characterization of the sequences at a single nucleotide level.
Targeted Locus Amplification, or TLA, is a method that uses proximity cross-linking, fragmentation and religation, combined with next generation sequencing of a locus of interest, without the need for detailed prior knowledge of the region4.
The resulting sequence information allows nucleotide-level insertion point identification, detection of sequence variants, including point mutations and genomic rearrangements, as well as identifying clones that share the same integration site5.
TLA has been widely adopted by the leading pharmaceutical companies and included in dozens of peer-reviewed articles and regulatory filings.
Therefore, you can rest assured that TLA can be relied upon for the detailed characterization of engineered cell lines and can easily be used to assess the stability of transgenic CHO cell lines.
Let Cergentis help you
Interested in seeing how TLA can improve your bispecific antibody production? For an overview of our technology, visit www.cergentis.com.
To learn about the use of TLA in genetic cell line characterization, visit this page.
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(1) Labrijn AF, et al. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery 18, 585-608; 2019
(2) Nelson AL. Antibody fragments – hope and hype. mAbs 2, 77-83; 2010.
(3) Wang Q, et al. Design and production of bispecific antibodies. Antibodies 8, 43; 2019
(4) De Vree PJP et al. Target sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nature Biotechnology 2014, 32 (10): 1019
(5) Aeschlimann SH, et al. Enhanced CHO clone screening: application of targeted locus amplification and next-generation sequencing technologies for cell line development. Biotechnology Journal14:e1800371, 2019.