June 3, 2024 duration: 5 min

7 reasons for us to choose Chinese hamster ovary (CHO) cells for recombinant protein production

Frequently described as the “workhorse” for therapeutic protein production, Chinese Hamster Ovary (CHO) cells remain the most commonly used mammalian host for large-scale protein production. The majority of recombinant biopharmaceutical proteins, most of which are monoclonal antibodies, are produced using CHO cells.

CHO cells possess several characteristics that make them highly adaptable for industrial-scale protein production, including: (i) their ability to produce recombinant proteins and antibodies with post-translational modifications highly compatible with humans, (ii) CHO cells grow in suspension, in serum-free and chemically defined media devoid of animal-derived components — which maximizes suspension growth in bioreactors and improves batch-to-batch consistency, and (iii) highly amenable to processes due to their tolerance of genetic manipulation.

In this blog, we set out main reasons why CHO cells are our go-to cell type for recombinant protein and antibody production.

Reason 1: CHO cells are a mammalian cell line derived from the ovary of the Chinese hamster, but require no live animals today

Derived from epithelial cells from the ovary of the Chinese hamster, CHO cells are an immortalized cell line routinely employed for therapeutic protein production. The cell line was first established in the 1950s by Theodore Puck at the Roosevelt Institute for Cancer Research, who was keen on the Chinese Hamster model. Their low chromosome number made them a good model for cytogenetic and tissue culture experiments.

Puck could culture ovary cells in an adherent culture grown on a glass surface and maintain long-term cultivation of the cells. Puck and co-authors observed that the cells grew “excellently”, with “no diminution in growth rate or cellular and colonial morphology”¹. These characteristics made CHO cells highly adaptable to biomedical research.

Reason 2: They were employed to produce the first recombinant biotherapeutic protein

The first FDA-approved recombinant protein — tissue plasminogen activator (TPA) (trade name: Activase) from Genentech — was produced using CHO cells and approved in 1987 for treating patients with Acute Ischemic Stroke². Currently, 70% of all approved recombinant protein production relies on CHO cells³, including seven of the top 10 drugs by global sales, which are monoclonal antibodies⁴⁵.

Reason 3: Chemical selection and gene amplification are optimized in this cell type

Different variants have been subcloned since the original CHO cell line was described. One commonly used cell line is CHO-DXB11, which is deficient in dihydrofolate reductase—an enzyme responsible for folate synthesis and linked to the synthesis of glycine, purine, and thymidine.

As DHFR-deficient CHO cells are auxotrophs [lost the ability to synthesize certain substances required for the growth due to mutations] for glycine, hypoxanthine (a purine derivative), and thymidine (GHT), this cell line has proved indispensable for screening and selecting heterologous genes containing a functional copy of the DHFR gene. Clonal selection utilizes growth media devoid of GHT, with the surviving cells containing the gene of interest and DHFR genes in their genomes.

An additional advantage of CHO-DXB11 is they can undergo amplification, which increases the selection pressure to favor high expression of the desired gene. Culturing cells in high levels of the folic acid analogue methotrexate (MTX) blocks DHFR activity. Therefore, low-level DHFR activity is insufficient, and only high-expressing clones are isolated.

Reason 4: CHO cells grow in suspension, which is ideal for recombinant protein production in bioreactors

Although the original CHO cell line was adherent, the CHO-S lineage was adapted for growth in suspension. Suspension cells are essentially single or group cells floating in culture media. Their anchorage-independent properties mean that they are not limited by the area of the substratum available for growth, making them ideal for large-scale culture.

Additionally, suspension cells do not need to be dissociated to be maintained. For commercial biomanufacturing, suspension CHO cells are preferred because of improved handling, productivity, and scalability. The processes involved in maximizing the growth of CHO suspension cells in bioreactors involve retaining cells inside the bioreactor while removing waste media and replenishing cells with fresh media.

Reason 5: They are highly adaptable to serum-free and chemically defined (CD) medium

One of the highest costs associated with biomanufacturing is the cost of the media and supplements, the latter of which is derived from animal sera. Another problem with animal sera is product variation, which arises from factors such as age and geographical origin.

Using serum-free and chemically defined (CD) media reduces batch-batch variability and manufacturing costs and lowers the risk of infectious agents and contamination. One key characteristic of CHO cells is their ability to grow in CD medium without animal-derived products, described as early as 1965 by Ham et al., and others⁶⁷⁸.

Culturing CHO cells in a serum-free medium also allows cells to detach from surfaces and float, which is beneficial in creating high-density suspension cultures in bioreactors.

Reason 6: Antibodies with specific glycan profiles can be produced in CHO cells

Post-translation modifications—such as glycosylation—of recombinant biopharmaceuticals are essential for their efficacy, antigenicity, and serum half-life. A recent example is the glycosylated human-mouse chimeric IgG1κ monoclonal antibody Siltuximab, produced in CHO cells and targeting soluble human IL-6. CHO cells produce human-like glycosylation, which makes the generated products more compatible and bioactive in human hosts⁹.

Additional considerations of employing CHO cells are the cell type may produce glycosylated structures at very low levels that are not present in humans – such as N-glycolylneuraminic acid (Neu5Gc) and galactose-α1,3-galactose (α-gal), which can elicit immune responses in humans¹⁰¹¹.

Clonal stability of CHO culture, which can lead to phenotypic drift, is another area that requires verification when employing CHO cells. Therefore, characterization and monitoring of the glycosylation profile and cell line stability to ensure consistent manufacture of biotherapeutics in CHO cells remains vital.

Reason 7: Validated safety profile with low susceptibility to human viruses

Since the 1980s, the strong safety profile of CHO cells has been extensively validated by the Food and Drug Administration (FDA) and European Medicines Agency (EMA)’s regulatory, safety, and quality requirements. Many human pathogenic viruses, including HIV and influenza, have been shown to be unable to replicate in CHO¹², meaning that contamination of CHO DNA is negligible in downstream purified products.

evitria offers recombinant antibodies expressed in CHO cells

As a leading service provider for recombinant antibody expression in CHO cells, evitria combines Swiss thoroughness with an innovative spirit.

In line with the requirements of today’s market, the objective is to offer both fast project execution and the highest quality standards, in other words, Maximum quality delivered in a minimum of time:

Fast execution from initial idea to purified antibody

… applies to all phases from project inception to shipment of the final product:

Highest quality standards …

… are adhered to and delivered throughout all processes of every project. evitria focuses on one service, namely, the transient antibody expression in CHO cells. This allows customers to expect guaranteed delivery with respect to:

From the Swiss headquarters, evitria delivers worldwide and provides high-quality CHO cells for use in an ever-growing portfolio of biotech drugs.
For more information about our Recombinant Antibody Expression service, please get in touch.

References

1. Puck, E. T., Cieciura, S. J. & Robinson, A. Genetics of Somatic Mammalian Cells. III. Long-term cultivation of euploid cells from human and animal subjects. J Exp Med 108, 945–946 (1958). ↩︎
2. FDA. Drugs@FDA: FDA-Approved Drugs. https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm (2024).
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3. Butler, M. & Spearman, M. The choice of mammalian cell host and possibilities for glycosylation engineering. Current Opinion in Biotechnology vol. 30 Preprint at https://doi.org/10.1016/j.copbio.2014.06.010 (2014).
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4. Bhutani, P. et al. U.S. FDA Approved Drugs from 2015-June 2020: A Perspective. Journal of Medicinal Chemistry vol. 64 Preprint at https://doi.org/10.1021/acs.jmedchem.0c01786 (2021). ↩︎
5. Walsh, G. & Walsh, E. Biopharmaceutical benchmarks 2022. Nat Biotechnol 40, (2022). ↩︎
6. Traustason, B., Cheeks, M. & Dikicioglu, D. Computer-aided strategies for determining the amino acid composition of medium for chinese hamster ovary cell-based biomanufacturing platforms. International Journal of Molecular Sciences vol. 20 Preprint at https://doi.org/10.3390/ijms20215464 (2019). ↩︎
7. Wurm, F. M. CHO quasispecies-Implications for manufacturing processes. Processes vol. 1 Preprint at https://doi.org/10.3390/pr1030296 (2013). ↩︎
8. Ham, R. G. Clonal growth of mammalian cells in a chemically defined, synthetic medium. Proc Natl Acad Sci U S A 53, (1965). ↩︎
9. Kim, J. Y., Kim, Y. G. & Lee, G. M. CHO cells in biotechnology for production of recombinant proteins: Current state and further potential. Applied Microbiology and Biotechnology vol. 93 Preprint at https://doi.org/10.1007/s00253-011-3758-5 (2012). ↩︎
10. Bosques, C. J. et al. Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins. Nature Biotechnology vol. 28 Preprint at https://doi.org/10.1038/nbt1110-1153 (2010). ↩︎
11. Ghaderi, D., Taylor, R. E., Padler-Karavani, V., Diaz, S. & Varki, A. Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol 28, (2010). ↩︎
12. Wiebe, M. et al. A multifaceted approach to assure that recombinant tPA is free of adventitious virus. Dev Biol Stand. 70, 147–151 (1989). ↩︎