Significant strides have been made in cell line development (CLD) and have consequently ushered Chinese hamster ovary (CHO) cell line engineering into a new era.1-3 Although random integration (RI) strategy is classically employed in CHO CLD, several reasons might render the approach less desirable than targeted integration (TI). To name a few, RI can prove to be labor-intensive and unpredictable, since gene integration post-transfection is uncontrolled. In contrast, site specific recombinase systems - a type of TI process - allow inserting a gene of interest to a predetermined genomic location and therefore, presents itself as an attractive alternative for therapeutic antibody manufacturing.
To develop a TI host, a landing pad capable of efficient recombinase-mediated cassette exchange (RMCE) needs to be inserted into a transcriptionally active site. Given that CHO cell lines are workhorses for biotherapeutic production, Genentech embarked on a genome wide search to localize hotspots in the CHO-K1-M genome by either RI or PiggyBac (PB) transposase-based integration. This study was published in Biotechnology Progress.4
Generation of stable and productive antibody-expressing cell lines
In this study, 2 antibody expressing cell lines (i.e., CHO-K1-M derive TI host cells) were generated via Cre/Lox RMCE system to replace the screening antibody expression cassette from the CHO genome with a GFP landing pad. Both TI hosts carry a GFP-expressing landing pad flanked by 2 incompatible LoxP recombination sites (L3 and 2L). Additionally, a third incompatible LoxP site (LoxFAS) was inserted in the GFP landing pad to facilitate a 2-plasmid based RMCE strategy (i.e. targeting 2 separate vectors to a single locus simultaneously). Based on the RMCE efficiency as well as antibody productivity, 2 final TI host candidates (i.e., one per screening method) were then selected and subsequently, fully characterized.4
TLA-based method was leveraged for integration site identification as well as for the analysis of the flanking genomic sequences at the insertion sites. TLA data showed no antibody sequences in both hosts, and the result was further validated by WGS. The researchers also availed themselves of FISH technology to determine the chromosomal location of the GFP landing pad and the result was corroborated by PCR fragment sequencing quantitative real time PCR. Here, a single GFP landing pad gene copy in the genome with correct recombination sites was uncovered.4
All in all, cell lines generated via TI system displayed (1) akin or superior productivity, (2) better stability, and (3) harbored fewer sequence variant (SV) than RI cell lines.4
Targeted integration-based cell lines reveal improved stability profiles for antibody expression
In this study, Genentech’s TI system uniquely took advantage of 3 incompatible LoxP sites to facilitate either single-plasmid or two-plasmid based RMCEs. In fact, this project is thought to be the first known successful generation of a TI system, which directly targets 2 vectors simultaneously to a single locus using the Cre/Lox RMCE system. As such, Genentech’s distinctive approach confers flexibility for chain ratio optimization towards increasing productivity and product quality. While higher productivity can be attained through increased copy numbers (of HC and LC at the locus), it must be noted that productivity can also be influenced by other factors such as: arrangement of the chains at the locus (i.e., position effect). Therefore, evaluating and selecting the best configuration can help maximize productivity and assembly efficiency.4
Ultimately, Genentech’s TI cell lines displayed markedly better stability profiles - for antibody expression - than their RI counterparts. Comparable stability was observed between cell lines carrying either a single or multiple antibody copy number, thereby suggesting that high copy number does not automatically result in (expression) instability. Moreover, given that the selectable markers solely serve to enrich cells with correct TI rather than sorting out for high expression, selective agents frequently employed yet mutagenic (e.g. MTX and MSX) can be sidestepped, to favor instead selectable markers that are not DNA synthesis inhibitors in order to reduce the risk of SV occurence37,38 Based on these promising results, Genentech concludes: “With improved predictability and consistency of TI cell line performance, an optimized production process can be applied to improve TI cell line titers. With the need to screen fewer clones to obtain clinical cell lines, our TI system significantly reduces resources spent on cell line generation.”4
Relevance of TLA-based solutions in (CHO) cell line development
As more complex biotherapeutic formats are being explored and developed, there is a surge in interest from manufacturers to push for more streamlined and reliable CLD processes to divert risk and accelerate early phase testing.5
Our proprietary TLA-based solutions are specifically designed to help you alleviate genetic characterization pains and to help you and your team satisfy the stringent genetic QC requirements mandated by regulatory authorities (e.g. FDA and EMA). With almost 10 years of experience under our belt, our unmatched capabilities continue to be heavily endorsed and extensively described by the likes of Pfizer & Lonza, Janssen and Novartis in scientific publications. Noteworthily, our proprietary assays have also successfully contributed in IND/BLA applications.
Here is a brief overview of the different stages where TLA-based solutions can be applied to in CLD:
- QC novel transfection methods
- Selecting clones with desired genetic characteristics
- Clonality assessment
- Comprehensive characterization of master cell banks (MCB)
- Genetic stability/drift evaluation
Head over here to check out our extensive publication list, which boasts over 50 peer-reviewed scientific papers:
References
[1] Zhou M, Crawford Y, Ng D, et al. Decreasing lactate level and increasing antibody production in Chinese hamster ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases. J Biotechnol. 2011;153(1–2):27-34.
[2] Haryadi R, Ho S, Kok YJ, et al. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells. PLoS One. 2015;10(2):e0116878.
[3] Roman R, Miret J, Scalia F, Casablancas A, Lecina M, Cairó JJ. Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNalpha2 signal peptide. J Biotechnol. 2016;239:57-60.
[4] Ng D, Zhou M, Zhan D, et al. Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase-mediated cassette exchange system. Biotechnol Progress. 2021;e3140. https://doi.org/10.1002/btpr.3140
[5] Dillon M, Yin Y, Zhou J, et al. Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells. MAbs. 2017;9(2):213-230.
