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Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads

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To date, Chinese hamster ovary (CHO) cell lines are responsible for more than 80% of the approved monoclonal antibodies.1,2 CHO cells are particularly attractive because of their ease of use – vis-à-vis in vitro genetic engineering – and due to high protein expression.

For many years, scientists have leaned on random integration to create transgenic CHO cell lines in order to manufacture biologics or therapeutic proteins.3 However, this randomness brings about a widely heterogeneous cell pool, which necessitates in turn extensive pool and clone screening to identify the clones that carry the desired properties (high productivity, high cell growth, stable expression, etc.). To sidestep random chromosomal insertion and decrease heterogeneity between clones, Evotec Biologics designed 2 targeted integration systems that express high-level expression of recombinant proteins in CHO-K1 cell lines.4

 

Characterization of landing pad clones and integration site analysis

First, 2 clonal cell lines stably expressing enhanced eGFP reporter landing pads in genomic hotspots were generated. The authors then characterized integration events of several donor vectors encoding both monoclonal antibodies and a Fc-fusion molecule using Cre or PhiC31 recombinase mediated integration. With this, correct targeting of landing pads was confirmed by TLA and PCR (Figure 1).

Fig for Evotec blog

Figure 1. Identifying landing pad insertion sites via TLA. TLA read coverage plots (encircled in blue) reveals the genomic locations
of integration sites in Phi31 clone 158 (A), PhiC31 clone 215 (B), and RMCE clone 703 (C)


Interestingly, comparable expression was observed in both systems prior GCV counter selection.
While the authors were able to achieve GCV enrichment with their RMCE system, their PhiC31 system did not see the same success. Of note, GCV enrichment appeared to improve protein expression as well as cell productivity of recombinase mediated cassette exchange (RMCE)-integrated molecules. With that said, GCV counterselection unfortunately did not seem to remove all cells with unsaturated RMCE landing pads.

Circumventing random chromosomal insertion in the development of transgenic CHO cell lines

In this study, Evotec Biologics elaborated 2 recombinase-based targeted integration systems for the manufacturing of exogenous proteins using transposons-mediated landing pads. Neither of these systems have been previously used, and the goal of this study was to compare both options in order to evaluate the one that might be most suitable for in-house usage. In particular, recombination was carried out either by Cre or PhiC31 recombinase. Genetic characterization of the altered cell lines revealed correct targeting of landing pads. Noteworthily, post-integration enrichment for fully saturated landing pads using GCV increased recombinant protein titer and productivity, with potential off-target random integration detected only following GCV enrichment.

Although additional work and optimization is still needed, the perk of their method lies in the predictability with regards to chromosomal integration of transgenes. Indeed, directing predefined locations that promote high expression of an exogenous protein, allows to develop cell lines expressing different recombinant protein therapeutics with a high degree of specificity and reproducibility.

 

The importance of early documentation in cell line engineering projects

Screening clone screening in the cell line development process – in order to select the desired ones and eliminate those that are contaminated (i.e. carries mutations in their transgenes) - will help you to de-risk R&D decisions and accelerate time to clinic.

Recently, we have introduced CHOice: our first fully integrated in-house solution for CHO clone selection. This option will allow you and your colleagues to generate, in-house, our high-quality genetic data in order to select the clone with the desired genetic makeup.

Click below to find out what is included in our CHOice solution and what it can do for you:

CHOice in-house solution

 

Or click here instead, to read a summary of a Novartis study, which led them to describe our TLA-based method as “a powerful and versatile analytical screening platform to support the CHO cell line development process for biopharmaceutical companies.”

Novartis leverages TLA-based genetic QC solutions



References

[1] Jayapal, K.P., Wlaschin, K.F., Hu, W.S., Yap, M.G.S. (2007). Recombinant protein therapeutics from CHO Cells - 20 years and counting. Chem. Eng. Prog. 103, 40–47.

[2] Walsh, G. (2018). Biopharmaceutical benchmarks. Nat. Biotechnol. 36, 1136–1145.

[3] Zhu, J., Hatton, D. (2018). New mammalian expression systems. Adv. Biochem. Eng. Biotechnol. 165, 9–50.

[4] Reese, N. B., Ku, S. (2021). Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads. Current Research in Biotechnology. 3, 269-280.

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