Host Cell Lines: CHO, HEK293, NS0 – Pros and Cons

The Role of Mammalian Cell Lines in Recombinant Protein Production

Mammalian cell lines are the preferred hosts for producing recombinant proteins that require complex folding, post-translational modifications (PTMs), and secretion. Their ability to perform glycosylation and phosphorylation makes them ideal systems for producing biologically active proteins, especially those used in diagnostics, therapeutics, and advanced biomedical research. 

Among the mammalian systems, Chinese Hamster Ovary (CHO), Human Embryonic Kidney 293 (HEK293), and murine myeloma NS0 cells are commonly used platforms. Their selection depends on several factors, including protein type, PTM requirements, production scale, development timelines, regulatory considerations, and downstream compatibility.

CHO Cells: Industry Standard for Biotherapeutics 

Originally derived from the ovary of the Chinese hamster in the late 1950s, CHO cells have become the dominant platform for recombinant protein production in the pharmaceutical industry. They are used in the manufacture of over 70% of biologics, including the vast majority of therapeutic monoclonal antibodies. 

Advantages of CHO Cells 

• Scalability and Productivity: CHO cells can be cultured in both adherent and suspension formats. They exhibit high volumetric productivity (up to 10 g/L in fed-batch cultures) and robust growth in chemically defined media. 

• Genetic Stability: CHO cell lines such as CHO-K1, CHO-S, CHO-DG44, and CHO-DXB11 are well-characterized and genetically stable, making them suitable for long-term manufacturing. 

• Biosafety Profile: Due to their non-human origin, CHO cells have a lower risk of propagating human viruses. This feature simplifies viral clearance validation and enhances safety. 

• Established Regulatory Acceptance: The extensive use of CHO systems in approved biologics results in a favorable and predictable regulatory path. 

• Adaptability: CHO cells can be genetically manipulated through metabolic selection (e.g., DHFR and GS systems) and are compatible with serum-free, animal-free production. 

• Engineering Potential: Advances in host cell engineering—such as overexpression of anti-apoptotic genes (e.g., BCL2, MCL1) and glycoengineering—have improved productivity and consistency. 

Disadvantages of CHO Cells

• Non-Human Glycosylation Patterns: CHO cells can introduce non-human glycans such as N-glycolylneuraminic acid (NGNA) and galactose-α1,3-galactose (α-Gal), which may elicit immunogenic responses in humans. 

• Lower Transfection Efficiency: CHO cells are less amenable to transient transfection compared to HEK293 cells, extending development timelines. 

• Genomic Instability Risk: Despite their utility, CHO cells are prone to genomic rearrangements under production stress, leading to reduced productivity over time. 

• Production Bottlenecks: Downstream processing can limit the recovery of high titers produced in bioreactors, leading to diminishing returns at very high expression levels. 

Related: Recombinant Protein Expression

HEK293 Cells: High Transfectability and Human-Like PTMs

HEK293 cells, established in 1973 by transforming human embryonic kidney cells with adenovirus 5 DNA, are a versatile tool in molecular biology. Their derivatives, including HEK293T, HEK293F, HEK293E, and HEK293-6E, offer variations tailored for specific applications in gene expression, viral vector production, and transient protein expression. 

Advantages of HEK293 Cells

• Human-Origin PTMs: HEK293 cells produce proteins with PTMs that closely resemble those found in endogenous human proteins, including complex glycosylation and γ-carboxylation. This is especially beneficial for proteins requiring high-fidelity modification (e.g., drotrecogin alfa, recombinant factor IX Fc fusion). 

• High Transfection Efficiency: HEK293 cells are highly amenable to both transient and stable transfection using a wide array of techniques including calcium phosphate, lipofection, and electroporation. 

• Rapid Protein Expression: Their ease of manipulation allows fast protein expression, making them well-suited for early-stage feasibility studies and small-scale production. 

• Versatile Derivatives: Derivatives like HEK293T, which contain the SV40 large T antigen, enhance protein yields and are widely used in retroviral and lentiviral production. 

Disadvantages of HEK293 Cells 

• Virus Susceptibility: The human origin of HEK293 increases the risk of contamination with human-specific pathogens, posing biosafety and regulatory concerns, especially in large-scale production. 

• Aggregation in Suspension: HEK293 cells tend to aggregate at high densities in suspension culture, limiting their scalability and complicating industrial bioreactor processes. 

• Limited Regulatory Track Record: Although approved HEK-derived products exist, their use in commercial-scale biotherapeutic manufacturing is still less established than CHO systems. 

• Selection Constraints: Unlike CHO systems that utilize robust metabolic selection strategies, HEK293 cells often rely on antibiotic selection, which is costlier and subject to regulatory limitations. 

Related: Recombinant Protein Expression

NS0 Cells: Legacy System in Monoclonal Antibody Production

NS0 cells are murine myeloma cells historically used for the commercial production of therapeutic monoclonal antibodies. While their prominence has declined with the rise of CHO systems, they are still used in legacy manufacturing processes and for certain specialized applications. 

Advantages of NS0 Cells

• High IgG Secretion Capacity: NS0 cells are particularly efficient in expressing immunoglobulins, with strong protein secretion mechanisms. 

• Stable Production: They are amenable to stable expression systems and exhibit reproducible results in long-term cultures. 

• Established Methods: Protocols for scaling NS0 cultures are well-developed, and the cell line is compatible with industrial-scale suspension culture. 

Disadvantages of NS0 Cells 

• Immunogenic Glycoforms: NS0 cells can incorporate immunogenic epitopes like α-Gal into recombinant proteins, increasing the risk of adverse immune reactions in patients. 

• Murine Origin: The non-human origin raises additional regulatory hurdles, particularly concerning viral clearance and product safety. 

• Declining Use: Most current development efforts have shifted toward CHO systems, which offer greater flexibility, safety, and genetic tool availability. 

Comparative Summary: CHO vs HEK293 vs NS0

Factors Influencing Host Cell Line Selection

Post-Translational Modifications (PTMs): 
PTMs are essential determinants of protein functionality, stability, immunogenicity, and pharmacokinetics. While CHO cells can perform complex glycosylation, discrepancies from native human structures can compromise therapeutic efficacy and safety. In contrast, HEK293 cells offer near-native PTMs, especially for PTMs like γ-carboxylation that CHO systems cannot perform efficiently. 

Scalability and Production Stability: 
CHO cells are preferred for large-scale, long-term production due to their superior growth in suspension culture and genetic stability. HEK293 cells are mainly used in research-scale applications, where fast expression is prioritized over scalability. NS0 cells, while scalable, are being phased out due to their murine glycan profiles and lower regulatory favorability. 

Regulatory and Biosafety Considerations: 
CHO cells remain the gold standard due to their proven safety record and extensive use in licensed therapeutics. HEK293 cells, although increasingly accepted, carry biosafety risks related to their human origin and are subject to more stringent viral clearance protocols. NS0 cells present similar concerns due to murine virus risk and are less frequently selected for new therapeutic development. 

Trends in CHO Cell Line Optimization 

Ongoing advances in CHO cell engineering aim to improve productivity, stability, and product quality. These include: 

• Targeted Integration Platforms: Use of CRISPR/Cas9 and recombinase-based landing pads for precise and stable transgene expression. 

• miRNA Engineering: Manipulating miRNAs such as miR-2861 or miR-106b to improve stress resistance, productivity, and growth. 

• Glycoengineering: Knockouts of glycosyltransferases (e.g., FUT8, SLC35C1) to achieve consistent glycan profiles and enhanced therapeutic efficacy. 

• High-Throughput Screening: Integration of tools like FACS, VIPS, and the Berkeley Lights Beacon platform for efficient clonal selection. 

• ‘Omics and Systems Biology: Application of transcriptomics and proteomics to guide rational design of improved cell lines for difficult-to-express (DTE) proteins.

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References: 

1. Tihanyi, B., & Nyitray, L. (2020). Recent advances in CHO cell line development for recombinant protein production. Drug Discovery Today: Technologies, 38, 25-34. https://doi.org/10.1016/j.ddtec.2021.02.003

2. Dumont, J., Euwart, D., Mei, B., Estes, S., & Kshirsagar, R. (2015). Human cell lines for biopharmaceutical manufacturing: History, status, and future perspectives. Critical Reviews in Biotechnology, 36(6), 1110. https://doi.org/10.3109/07388551.2015.1084266

3. Sun, 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

4. 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