Biologics 4.0: Emergence of the CHO Biofoundry

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Accelerated homology-directed targeted integration of transgenes in CHO cells via CRISPR/Cas9 and fluorescent enrichment

Development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. With draft genome of several CHO cell lines and targeted genome editing technologies, rCHO cell line development based on site-specific integration has the potential to overcome the limitations of clonal heterogeneity. In a previous study, we demonstrated efficient and precise targeted gene integration in CHO cells using CRISPR/Cas9 technology and homology-directed repair (HDR) pathway1). However, it requires a selection process, which limits targeted integration of multiple transgenes at multiple sites due to a limited number of selection markers and a lengthy selection process. Here, we improved the targeted integration platform by applying fluorescent enrichment of transfected cells. The improved system is based on a fluorescent protein A linked Cas9 together with sgRNA towards the integration site and donor DNA harboring a fluorescent gene B outside homology regions. Involvement of fluorescent markers in constructs confers FACS enrichment of cells transfected with both Cas9 and donor DNA. Simultaneous introduction of GFP 2A peptide-linked Cas9 and sgRNA expression vectors together with donor plasmid has enabled precise targeted integration of large transgenes encoding model proteins including antibody, following transient expression and FACS enrichment. Subsequent selection of non-fluorescent clonal cells allowed for excluding cells with randomly integrated donor DNA. In this way, we not only shortened the clone screening time, but also generated clonal CHO cell lines with site-specific, marker-free (no antibiotic selection needed), and clean (no unwanted DNA present) targeted integration of GOI. Further improvement in targeted integration efficiency was additionally assessed by chemical treatment toward cell cycle arrest or nonhomologous end joining inhibition combined with fluorescent enrichment. Taken together, the present platform has the huge potential to accelerate targeted generation of stable production CHO cell lines in a rational way

Polyclonal production of antibodies using CHO cell line mixtures

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Transcriptional response to recombinant protein production in isogenic multi-copy CHO cells

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Expression of anti-apoptotic genes to enhance rAAV production

Recombinant Adeno-associated viruses (rAAV) are becoming popular as viral vector delivery systems for gene therapy. Currently, two rAAV-based products are available on the market and several clinical trials are on-going with promising results for diseases such as hemophilia or muscular dystrophy1,2. The most common method for manufacturing rAAV products is by triple transfection with a plasmid containing the gene of interested flanked by ITR sequences, a plasmid with adenovirus helper genes and a plasmid with AAV helper genes. The scalability of transfection processes has been disputed for a long time. However, in rAAV production it might be the only feasible option until a proficient stable cell line is developed solving all current challenges including, but not limited to: low cell density at transfection and interruption of cell replication. Another bottleneck in all cell culture-based process is the duration of the culture. Typically, the viable cell density increases until a stationary phase is reached in which, after a specific amount of time depending on the cell line, apoptosis begins and viability rapidly falls. In rAAV production processes, apoptosis is triggered after transfection by AAV helper genes (mostly Rep78 and Rep 52) which are toxic for mammalian cells, reducing the duration of the cell culture decreasing the productivity of the process3,4 . To overcome this challenge, several anti-apoptotic genes from different origins, such as human (bcl-2, bcl-xL and mcl-1), hamster (bcl-2 and bcl-xL) and viral genes (bhrf-1, p25 and vBcl-2), have been expressed in this study as well as a recombinant protein-based product to mimic the production of rAAV. Even though some genes were already reported in the literature, a detailed comparison could not be performed as random integration was used in the development of most of the cell lines. In this novel study, targeted integration was used to develop isogenic cell lines with one copy of both the anti-apoptotic gene and the protein product. As expected, the expression of anti-apoptotic genes increased the duration of the cell culture proving its potential as a tool to enhance the performance of rAAV production processes. Consistent with the literature, some genes were also able to improve the specific productivity of the protein product by a significant amount. In fact, one gene that was not studied previously outperformed the other candidates. Regarding the metabolism, consumption rates of glucose and production rates of lactate were analyzed to have a better understanding of the effect of these genes on the metabolism of the mammalian cell. Considering all the available results, we propose the use of anti-apoptotic genes to enhance the production of rAAV with mammalian cells. References 1. Verdera HC, Kuranda K, Mingozzi F. AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Mol Ther. 2020;28(3):723-746. doi:10.1016/j.ymthe.2019.12.010 2. Van Vliet KM, Blouin V, Brument N, Agbandje-McKenna M, Snyder RO. The Role of the Adeno-Associated Virus Capsid in Gene Transfer. Vol 437.; 2008. doi:10.1007/978-1-59745-210-6_2 3. Matsushita T, Okada T, Inaba T, Mizukami H, Ozawa K, Colosi P. The adenovirus E1A and E1B19K genes provide a helper function for transfection-based adeno-associated virus vector production. J Gen Virol. 2004;85(Pt 8):2209-2214. doi:10.1099/vir.0.79940-0 4. Schmidt M, Afione S, Kotin RM. Adeno-Associated Virus Type 2 Rep78 Induces Apoptosis through Caspase Activation Independently of p53. J Virol. 2000;74(20):9441-9450. doi:10.1128/jvi.74.20.9441-9450.200

Expression of anti-apoptotic genes to enhance rAAV production

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Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells

Characterisation of two snake toxin-targeting human monoclonal immunoglobulin G antibodies expressed in tobacco plants

Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies

Enhancing CHO cell productivity through a novel dual selection system using Aspg and Gs in glutamine free medium

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