Chinese hamster ovary (CHO) cell lines remain the most popular expression system for the manufacturing of recombinant proteins (e.g. monoclonal antibodies). With the rise of biopharmaceutical candidates in the pipeline, there is an ever-increasing need to streamline cell line development processes to mitigate risks and accelerate early phase testing.
Optimization strategies for an improved CHO expression system based on glutamine
Since the first launch of the GS (glutamine synthetase) Gene Expression System® in 19921, Lonza has continued to push boundaries and has tirelessly been searching for ways to improve its platform by increasing the productivity of their expression system. Efforts towards this goal have led to discovering that site-specific integration (SSI) helps reduce the variability in the expression of manufactured biotherapeutic products2.
Reducing expression variability with site-specific integration to create better recombinant mammalian cell lines
The classical approach of random integration (RI) - to create commercial-ready cell lines - is believed to promote position variegation effect, genome plasticity, and clonal variation2,3. As such, scientists at Lonza decided to exploit SSI to temper this variability and were committed to create a CHOK1SV GS-KO® derived SSI host (HD7876), via Flp-Frt based targeting. More specifically, the generation of HD7876 contained: the stable Fer1L4 locus, a CHOK1SV® glutamine synthetase knockout (GS-KO) host as well as a targetable and genetically well-defined landing pad. Of note, the GS-KO host cell line is also derived from the firm’s suspension-adapted CHOK1SV™ line, in which both alleles for GS have been ablated, thereby requiring glutamine.
A rapid, reliable, and cost-effective approach to accelerate timelines in the cell line development cycle
To evaluate the fidelity of the Flp recombinase mediated cassette exchange (RMCE) event in HD7876 host, scientists at Lonza and Pfizer opted TLA technology as the method of choice for the in-depth genetic characterization of their manipulation40. More specifically, homology directed integration strategy was chosen to favor targeted insertion into a landing pad that is flanked with incompatible Frt sequences in the Fer1L4 gene. The vector used harbored a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line.
Via PCR amplification of the vector-landing pad junctions, results confirmed the expected vector integration in the landing pad, in the RMCE-generated pools. Furthermore, TLA analysis showed no evidence of a reverse insertion.
In the present study, Lonza and Pfizer have generated and subsequently performed thorough evaluation of their novel CHOK1SV GS-KO® derived SSI host, HD7876. Results suggested that, in terms of expression and generational stability, their innovative cell line displayed an excellent and improved performance. In fact, data showed that the concentration of monoclonal antibody secreted are akin or superior to the best cell lines derived from a RI process and the 144E12 host.
Unique TLA-based solution is shaking up the cell line development scene
An important objective in product supply is to minimize cell bank heterogeneity for the consistent production of biologics. This motivation is further fueled by regulatory authorities' expectations, who place heavy emphasis on the assurance of clonal derivation of producer cell lines, to safeguard the quality, safety, and efficacy of all manufactured products. As a result, cell line identity and clonality verifications require thorough analytical genetic characterization.
By combining TLA with breakpoint-based qPCR analysis, we can help assess monoclonality. Furthermore, the complete genetic characterization of your cell lines at various generation times will allow you to gain better genetic insights into the consistent production of your biologics. Crucially, we offer detailed reporting suitable for use in the regulatory application for Pharmaceuticals for Human Use, according to FDA and EMA expectations (ICH guidelines Q5B5 and Q5D6,7,8, CTD Quality module, section 3.2.S.2.39).
Contact us to find out what our full TLA reporting includes and/or click here to learn more about the structure of our various packages.
 DePalma, Angelo. “Weighing Protein Expression Levels with Cell Growth.” Genetic Engineering & Biotechnology News, 1 Nov. 2017, https://www.genengnews.com/magazine/303/weighing-protein-expression-levels-with-cell-growth/. Accessed 26 Apr 2021.
 Feary, M, Moffat, MA, Casperson, GF, Allen, MJ, Young, RJ. CHOK1SV GS‐KO SSI expression system: A combination of the Fer1L4 locus and glutamine synthetase selection. Biotechnol Progress. 2021;e3137. https://doi.org/10.1002/btpr.3137
 Feng YQ, Lorincz MC, Fiering S, Greally JM, Bouhassira EE. Position effects are influenced by the orientation of a transgene with respect to flanking chromatin. Mol Cell Biol. Jan 2001;21(1):298-309. doi:10.1128/MCB.21.1.298-309.2001
 de Vree PJ, de Wit E, Yilmaz M, et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Research. Nature biotechnology. Oct 2014;32(10):1019-25. doi:10.1038/nbt.2959
 ICH Q5B - Analysis of the Expression Construct in Cells Used for Production of r-DNA Derived Protein Products ICH, November 1995
 ICH Q5D - Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/ Biological Products ICH, July 1997
 Christopher Frye et al. Industry view on the relative importance of “clonality” of biopharmaceutical-producing cell lines Biologicals 44(2): 117-122 (2016)
 Christel Aebischer-Gumy et al. Analytical assessment of clonal derivation of eukaryotic/CHO cell populations J. Biotechnol, 286:17-26 (2018)
 ICH Topic M 4 Q - Common Technical Document for the Registration of Pharmaceuticals for Human Use - Quality, July 2003