APR May/June 2023 - 68

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preferably in the form of mRNA, both of which can be co-transfected
into CHO cell lines. Upon translation of the mRNA, the resulting transposase
catalyses the transgene excision and integration into the cell
line genome. One advantage of the transposon system compared to
random integration expression vectors, is that it allows intact integration
of the expression cassette at each integration site (Figure 1) and
is therefore devoid of any fragmentation (Figure 2B), or rearrangement
(Figure 2C) or concatemerization (Figure 2D). As a result, directed integration
of the GOI at transcriptionally active sites within the genome
of the cell line leads to homogeneity and clonal stability of greater
than 95%.
Summary - Future Insights
8.
In the past couple of decades, large molecule drug development has
advanced along both upstream and downstream stages in the context
of streamlining processes, establishment of Critical Quality Attributes
(CQAs), compliance requirements, as well as technologies that support
the workflows. From a biological and technical perspective, upstream
cell line development for the production of large drug molecules is
a critical and founding step that not only contribute to the product
titer yields and scale up, but also defines the technologies that will be
required to eliminate cellular artifacts as well as the final purification
of the drug.
As discussed above, the CHO cell line has been widely used for a long
time for protein drug expression and production. Recent gene editing
and GOI delivery vectors, and related mechanisms have enabled
targeted and consistent cell line engineering.
Transposon technologies have offered specific launch pads to
simplify development and reduce manufacturing effort. Additional
advancement and standardization of cell counting and clone
selection methods via automated cell evaluation technologies will
further streamline the end-to-end workflow during CLD. Integration
of processes and protocols with automation, detection and cell
imaging technologies, as well as data analysis and management will
redefine the process to scale upstream cell line development. Machine
learning will further facilitate advanced predictive and data models to
establish best practices and eliminate process gaps. A combinatorial
and collaborative approach across various functional and process
areas will lead to next-generational biologics models for precision,
reliability, and efficacy.
References
1. Mistry, R.K., Kelsall, E., Sou, S.N., Barker, H., Jenns, M., Willis, K., Zurlo, F., Hatton, D., and
Gibson, S., 2021. A novel hydrogen peroxide evolved CHO host can improve the expression
of difficult to express bispecific antibodies. Biotechnol. Bioeng., 118:2326.
2.
Budge, J.D., Knight, T.J., Povey, J., Roobol, J., Brown, I.R., Singh, G., Dean, A., Turner, S.,
Jaques, C.M., Young, R.J., Racher, A.J., and Smales, C.M., 2020. Engineering of Chinese
hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant
biotherapeutic protein production. Metab. Eng. 57:203.
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| May/June 2023
9.
3.
Scarcelli JJ, Shang TQ, Iskra T, Allen MJ, Zhang L. Strategic deployment of CHO expression
platforms to deliver Pfizer's Monoclonal Antibody Portfolio. Biotechnol Prog. 2017
Nov;33(6):1463-1467. doi: 10.1002/btpr.2493. Epub 2017 Jun 2. PMID: 28480558.
4.
Stuible, M., Lier, F.V., Croughan, M.S., & Durocher, Y. (2018). Beyond preclinical research:
production of CHO-derived biotherapeutics for toxicology and early-phase trials by
transient gene expression or stable pools. Current Opinion in Chemical Engineering.
5. Wright C, Alves C, Kshirsagar R, Pieracci J, Estes S. Leveraging a CHO cell line toolkit to
accelerate biotherapeutics into the clinic. Biotechnol Prog. 2017 Nov;33(6):1468-1475. doi:
10.1002/btpr.2548. Epub 2017 Sep 7. PMID: 28842948.
6. Wei, M., Mi, C-L., Jing, C-Q., and Wang, T-Y, 2022. Progress of transposon vector system for
production of recombinant therapeutic proteins in mammalian cells.
7.
Sandoval-Villegas, N., Nurieva, W., Amberger, M., Ivics, Z., 2021. Contemporary transposon
tools: A review and guide through mechanisms and applications of sleeping beauty,
piggyBac and tol2 for genome engineering. Int. J. Mol. Sci. 22:5084.
Matasci, M., Baldi, L., Hacker, D.L., and Wurm F.M., 2011. The piggyBac transposon
enhances the frequency of CHO stable cell line generation and yields recombinant lines
with superior productivity and stability. Biotechnol. Bioeng. 108:2141.
Narayanavari, S.A., Izsvák, Z., 2017. Sleeping Beauty transposon vectors for therapeutic
applications: advances and challenges. Cell Gene Therapy Insights 2017; 3(2), 131-158,
10.18609/cgti.2017.014.
10.
Patel, T., Ingham, C., Fleming, J., Surve, T., Cougot, D., Ashtley, H., and Zurdo, J., 2022.
CHOSOURCE™ ADCC+ cell line for enhanced therapeutic potency. https://horizondiscovery.
com/-/media/Files/Horizon/resources/Posters/chosource-chok1-adcc-cell-line-poster.
pdf. Accessed on 23 May 2023
11.
Surve, T., Rebocho, A., Fleming, J., Pinto, N., Patel, T., Sandeman, K., Claudia, C., Cougot,
D., Astley, H., and Zurdo, J., 2022. Integration of next-generation transposon vectors with
novel host cell lines. https://horizondiscovery.com/ /media/Files/Horizon/resources/
Posters/chosource-tnt-poster.pdf. Accessed on 23 May 2023.
Author Biographies
Anis H. Khimani, PhD, is Senior Strategy Leader with
the Life Sciences strategy group at Revvity. Anis has prior
academic and research background in molecular biology,
viral pathogenesis, vector development, and vaccine
studies from Harvard Medical School and Dana-Farber Cancer Institute.
After his academics and research, he pursued research and development
in genomics and assay development, as well as strategy and product
management in informatics at PerkinElmer. Subsequently, his focus has
been in strategy and market segment development within Life Sciences.
Tanaya Surve is Senior Scientist with the Bioproduction
group at Revvity. Tanaya has over 11 years of experience
in cell line development and has contributed to the drug
product development and launch of several molecules of
therapeutic importance. Her current role focuses on vector development
and cell line engineering to create improved host cell platforms. She
holds a Master's in Biotechnology and a diploma in International
Business Management.

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APR May/June 2023

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