CHO versus HEK293: Which cell line is right for my protein expression?

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• Introduction – Relevance of CHO and HEK293
• HEK293 Cell Line
• CHO Cell Line
• Choosing the right cell line
• Conclusions
• References
  

Recombinant proteins are increasingly used in biomedical research, diagnostics, and as biotherapeutics, playing an important role in advancing our understanding and treatment of many diseases. The production of complex recombinant proteins relies on mammalian cell lines, which can secrete complex proteins in large quantities without the need for cell lysis. [1].

Since mammalian cell lines used in recombinant protein expression closely resemble human cells, or can even consist of human cells, they are an ideal choice for producing proteins that require complex post-translational modifications (PTMs)such as glycosylation and phosphorylation. Mammalian cells can fold, modify, and process proteins in a way that closely mimics the human cellular post-translational machinery, resulting in biologically active and functional proteins.

Two of the most commonly used mammalian cell lines for recombinant protein expression are Chinese Hamster Ovary (CHO) and Human Embryonic Kidney 293 (HEK293) cells. Both cell lines exhibit desirable characteristics for recombinant protein production, possessing high efficiency in protein synthesis, consistent reproducibility across various batches, rapid replication, and the capacity to generate PTMs. Furthermore, both cell types have been adapted for suspension growth, facilitating large-scale production in both serum-based and serum-free media. Consequently, these cell lines have become indispensable tools in biotechnology, biopharmaceuticals, and various research projects.

However, the choice between HEK293 and CHO cells is not a one-size-fits-all scenario. Each cell line has its own set of advantages and disadvantages to consider when designing recombinant protein expression studies. This article explores the differences between these popular cell lines and helps scientists make an informed decision when selecting the ideal cell line for their specific needs.

The HEK293 cell line, established in 1973, resulted from the transformation of human embryonic kidney cells with sheared adenovirus type 5 DNA. HEK293 is so named because it took precisely 293 attempts before the experiment was successful [2]. Since then, this robust and fast-growing cell line and its derivatives have been used extensively in receptor signaling, cancer research, the development of viral vaccines, and large-scale protein production.

• Rapid growth and adaptability: HEK293 cells can adapt to various culture conditions and are easy to maintain, making them a flexible choice.
• Reproducibility of results: HEK293 cells offer consistent, reliable, and reproducible results.
• Transfection amenability: HEK293 cells are highly amenable to transfection and can be efficiently transfected using various physical and chemical methods.
• PTMs: HEK293 cells are able to generate complex PTMs including glycosylation. This capability makes HEK293 cells particularly well-suited for producing difficult-to-express proteins when compared to CHO cells [3].

• Susceptible to viral contamination: Like all human cell lines, HEK293 cells carry a potential risk of human-specific viruses, which can compromise the safety and efficacy of the final product.
• Tendency to aggregate: HEK293 cells tend to aggregate in suspension culture, especially at higher cell densities, which may restrict large-scale production [4].

This cell line was first isolated from the ovary of a Chinese hamster in the late 1950s. Scientists quickly realized these cells were fast-growing, resilient, and amenable to genetic manipulation by mutagenesis. This led to CHO cells becoming the workhorse of the biopharma industry, with over 70% of biopharmaceuticals, and almost all antibodies, produced within this cell line [5].

CHO cell lines have a longstanding history in the biopharmaceutical industry, and continue to be a popular choice for recombinant protein expression, with unique advantages and disadvantages:

• Proven track record: CHO cells have a well-established history of safe and efficient protein production, making them a trusted choice for biopharmaceutical manufacturing.
• Scalability: CHO cells grow well as adherent culture and in suspension, which makes them ideal for large-scale culture and industrial production.
• Stability: CHO cells are known for their genetic stability, which is essential for maintaining consistent protein quality.
• Adaptability and tolerance: They are adaptable for animal-free and serum-free culture conditions. Additionally, they have a high tolerance to variations in parameters such as pH, oxygen levels, temperature, or cell density.
• Low risk of virus contamination: Due to the hamster origin, the risk of propagation of human viruses is decreased, reducing production loss and increasing biosafety.

• Limited post-translational modifications: While CHO cells can perform some PTMs, disparities exist between CHO cells and human cell lines. Notably, CHO cells have been observed to introduce non-human glycan structures like N-glycolylneuraminic acid (NGNA) and the galactose-alpha-1,3-galactose group (α-Gal) onto glycoprotein products, increasing the risk of immunogenicity [6,7]. Additionally, CHO cells are not able to completely reproduce human glycostructures and other essential PTMs as they lack certain enzymes found in human cells [8,9].
• Long development time: CHO cell lines typically have lower transfection efficiencies than HEK293 cells, resulting in longer and more resource-intensive development [10, 11].

From small-scale feasibility studies to industrial production of recombinant proteins, the choice of mammalian host system can significantly impact the success of the bioprocess and the quality of the final product. While CHO and HEK293 cells share similar properties, they also have distinct characteristics that can render them more or less suitable for specific applications. As such, the choice between employing CHO or HEK293 cells depends on the unique demands of the project, such as the nature of the protein, the required PTMs, and scalability.

PTMs are a crucial aspect to consider when choosing your host system for protein production and research projects as they can significantly impact the biological activity, stability, pharmacokinetics, and immunogenicity of recombinant proteins [8, 12]. Therefore, controlling PTMs is a fundamental aspect of protein expression systems at any scale, to ensure the consistent quality and batch-to-batch uniformity of biologics, in addition to minimizing undesirable effects like decreased stability and increased immunogenicity.

While CHO cells can produce proteins with PTMs similar to those found in humans, they can also introduce variations that can compromise product stability, immunogenicity, and potency [6-9], which could be particularly concerning in the case of biotherapeutics. In contrast, human cell lines like HEK293 can yield therapeutics with native PTMs, a critical consideration for proteins requiring specific modifications. For example, the therapeutic agents drotrecogin alfa and recombinant factor IX Fc fusion protein require gamma (γ)-carboxylation, a PTM that HEK293 cells, but not CHO cells, can efficiently produce [13].

Another crucial factor to consider is scalability. CHO cells are typically the preferred choice for large-scale production due to their robustness and adaptability to industrial-scale bioreactors. CHO cells can be cultured in shake flasks and bioreactor suspension which is advantageous for high-density cell culture and large-scale production. Like CHO cells, HEK293 cells grow rapidly in suspension and in serum-free culture, plus they can express proteins both transiently and stably, and are highly efficient at protein production. However, the growth and production properties of the HEK293 cell line are still considered to be inferior to that of CHO cells due to their tendency to aggregate in suspension, limiting their ability to be cultured at larger scales [14]. As a result, HEK293 cells are typically preferred for smaller-scale research endeavors, or for limited protein production purposes.

When it comes to regulatory approval, both CHO and HEK293 cells are a good choice for recombinant protein expression. Both cell lines have a well-established presence in the biopharmaceutical industry, being responsible for producing the majority of therapeutic proteins. Over the last three decades, CHO and HEK293 cells have been shown to be safe hosts and as such, they are approved by most regulatory authorities such as the FDA and EMA.

Selecting the most appropriate cell line is a critical step toward the successful expression of your protein of interest, and making an informed decision will ultimately lead to more effective and efficient research or production processes. The choice between CHO and HEK293 cell lines for recombinant protein expression should be based on careful consideration of your specific needs, including the protein type, PTMs, scale of production, and time.

In summary, HEK293 cells excel in delivering fast and efficient production of recombinant proteins with complex post-translational modifications, while CHO cells offer stability, scalability, and a proven track record. However, it's crucial to emphasize that ongoing efforts are dedicated to enhancing the adaptability of both cell lines for improved production capabilities. As the demand for mammalian cell-based protein expression continues to grow, the right choice of host cell line can ensure the quality, efficacy, and safety of these life-saving diagnostics, therapeutics, and more.

1. un, H., Wang, S., Lu, M., Tinberg, C. E., & Alba, B. M. (2023). Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research. PLOS ONE, 18(6), e0285971. https://doi.org/10.1371/journal.pone.0285971

2. Stepanenko, A. A., & Dmitrenko, V. V. (2015). HEK293 in cell biology and cancer research: Phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene, 569(2), 182–190. https://doi.org/10.1016/j.gene.2015.05.065

3. Malm, M., Kuo, C.-C., Barzadd, M. M., Mebrahtu, A., Wistbacka, N., et al. (2022). Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins. Metabolic Engineering, 72, 171–187. https://doi.org/10.1016/j.ymben.2022.03.009

4. Seidel, S., Maschke, R. W., Mozaffari, F., Eibl-Schindler, R., & Eibl, D. (2023). Improvement of HEK293 Cell Growth by Adapting Hydrodynamic Stress and Predicting Cell Aggregate Size Distribution. Bioengineering, 10(4), 478. https://doi.org/10.3390/bioengineering10040478

5. Lalonde, M.-E., & Durocher, Y. (2017). Therapeutic glycoprotein production in mammalian cells. Journal of Biotechnology, 251, 128–140. https://doi.org/10.1016/j.jbiotec.2017.04.028

6. Ghaderi, D., Taylor, R. E., Padler-Karavani, V., Diaz, S., & Varki, A. (2010). Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nature Biotechnology, 28(8), 863–867. https://doi.org/10.1038/nbt.1651

7. Fliedl, L., Grillari, J., & Grillari-Voglauer, R. (2015). Human cell lines for the production of recombinant proteins: On the horizon. New Biotechnology, 32(6), 673–679. https://doi.org/10.1016/j.nbt.2014.11.005

8. Goh, J. B., & Ng, S. K. (2018). Impact of host cell line choice on glycan profile. Critical Reviews in Biotechnology, 38(6), 851–867. https://doi.org/10.1080/07388551.2017.1416577

9. Berkner, K. L. (1993). Expression of recombinant vitamin K-dependent proteins in mammalian cells: Factors IX and VII. Methods in Enzymology, 222, 450–477. https://doi.org/10.1016/0076-6879(93)22029-f

10. Vatandoost, J., & Dolatabadi, B. (2017). Stable and Transient Expression of Human Coagulation Factor IX in Mammalian Expression Systems; CHO Versus HEK Cells. Gene, Cell and Tissue, 4(2), Article 2. https://doi.org/10.5812/gct.13096

11. Suen, K. F., Turner, M. S., Gao, F., Liu, B., Althage, A., et al. (2010). Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure. Protein Expression and Purification, 71(1), 96–102. https://doi.org/10.1016/j.pep.2009.12.015

12. Zhou, Q., & Qiu, H. (2019). The Mechanistic Impact of N-Glycosylation on Stability, Pharmacokinetics, and Immunogenicity of Therapeutic Proteins. Journal of Pharmaceutical Sciences, 108(4), 1366–1377. https://doi.org/10.1016/j.xphs.2018.11.029

13. Dumont, J., Euwart, D., Mei, B., Estes, S., & Kshirsagar, R. (2016). Human cell lines for biopharmaceutical manufacturing: History, status, and future perspectives. Critical Reviews in Biotechnology, 36(6), 1110–1122. https://doi.org/10.3109/07388551.2015.1084266

14. Abaandou, L., Quan, D., & Shiloach, J. (2021). Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes. Cells, 10(7), 1667. https://doi.org/10.3390/cells10071667

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