Schematic representation of the production of CHO cell-based anti-target monoclonal antibody. HC and LC indicates heavy chain and light chain, respectively. DOI: 10.1186/s12896-022-00738-6
Monoclonal antibodies (mAbs) are the largest class of recombinant proteins used as therapeutics, playing a critical role in treating cancers, autoimmune diseases, and infectious conditions. Producing recombinant mAbs requires a host system that can efficiently express, fold, and modify these complex proteins while maintaining their efficacy and safety. Several factors influence the selection of a host system, including yield, post-translational modifications (PTMs), scalability, regulatory compliance, and cost efficiency.1
Key Considerations for Host Selection
Yield and productivity are primary considerations, as commercial-scale mAb production must achieve high expression levels to meet market demand. Post-translational modifications, particularly glycosylation, are necessary for stability, half-life, and biological function. Scalability is another key factor, as the chosen system must support large-scale production in bioreactors while maintaining consistency and reproducibility. Regulatory acceptance is also critical since certain host-derived contaminants and immunogenicity risks can impact therapeutic applications. Finally, cost efficiency plays a role in host selection, with factors such as media costs, infrastructure requirements, and downstream processing expenses influencing the overall economic feasibility of production.
Among the various expression systems available, mammalian cells remain the dominant platform for recombinant mAb production due to their ability to perform human-like glycosylation and generate fully functional antibodies.
Mammalian Cell Systems
Mammalian cells are the industry standard for producing therapeutic mAbs because they support proper protein folding, assembly, and post-translational modifications. The most widely used cell lines for recombinant mAb production are Chinese Hamster Ovary (CHO) cells and HEK293 cells, each with distinct advantages and applications.
CHO Cells: The Industry Standard
CHO cells have been the preferred host for mAb production for over three decades due to their ability to generate human-compatible glycosylation patterns, which are essential for stability, immunogenicity reduction, and receptor binding. They can achieve high production yields, often reaching gram-per-liter levels in optimized fed-batch and perfusion bioreactors. Additionally, CHO cells have a long history of regulatory approval, making them a trusted platform for biopharmaceutical manufacturing. Many FDA-approved therapeutic mAbs, including blockbuster drugs, originate from CHO-based systems.
On the other hand, CHO cells have a higher cost of production due to expensive media requirements and complex culture conditions. CHO cells have a relatively long doubling time of 18-24 hours, which extends development timelines, particularly when generating stable cell lines, a process that can take six months to a year. However, advancements in cell line engineering, such as CRISPR/Cas9-mediated genetic modifications and optimized vector systems, have improved CHO cell productivity and reduced bottlenecks in development.
HEK293 cells are another commonly used mammalian host, particularly for transient mAb expression. These cells have a faster doubling time (12-16 hours) and higher transfection efficiency, making them ideal for research applications, early-stage development, and rapid screening of mAb candidates. HEK293 cells are particularly useful for producing mAbs that are difficult to express in CHO cells.
However, HEK293 cells are less commonly used for large-scale commercial production due to their lower stability and lower yield in comparison to CHO-based systems. Additionally, while HEK293 cells perform human-like glycosylation, subtle differences in glycan profiles may affect therapeutic efficacy and stability. Their regulatory acceptance is also more limited than CHO cells, with fewer FDA-approved biologics originating from HEK293-based systems.
In addition to CHO and HEK293 cells, other mammalian cell lines have been explored for mAb production. Murine myeloma cell lines, such as NS0 and SP2/0, were historically used for mAb production but have largely been replaced by CHO cells due to concerns about murine-derived contaminants and limited regulatory acceptance.
DOI: 10.1007/s40259-018-0319-9
Advancements in Mammalian Expression Systems
Recent advancements in mammalian cell engineering have focused on increasing yield, improving cost efficiency, and enhancing product quality. Glycoengineering strategies have been developed to eliminate non-human glycosylation patterns, improving therapeutic efficacy and reducing immunogenicity. Metabolic engineering approaches aim to optimize nutrient utilization and reduce toxic byproducts, enhancing cell viability and productivity. Innovations in expression vector design, including stronger promoters and codon optimization, have further improved transcription and translation efficiency.
In parallel, advances in bioprocessing have contributed to the increased efficiency of mammalian cell cultures. Perfusion bioreactor systems allow continuous production with higher cell densities, improving yield while reducing overall production time. These innovations ensure that mammalian expression systems remain the gold standard for recombinant mAb production.
While mammalian cells dominate therapeutic mAb production, microbial systems such as Escherichia coli and yeast offer cost-effective alternatives, particularly for antibody fragments like Fab and single-chain variable fragments (scFv).
E. coli is widely used for recombinant protein expression due to its rapid growth and ease of genetic manipulation. However, it lacks the machinery for glycosylation and has difficulty correctly folding full-length antibodies, often leading to the formation of insoluble inclusion bodies. To address these challenges, researchers have developed engineered strains with enhanced folding and secretion capabilities, though full-length mAb production remains difficult in bacterial systems.
Yeast expression systems, particularly Pichia pastoris and Saccharomyces cerevisiae, offer a promising alternative to mammalian cells. Yeasts are eukaryotic, allowing them to perform some post-translational modifications, and they can achieve high expression levels in cost-effective culture conditions. However, a major challenge in yeast-based mAb production is glycosylation, as yeast-derived glycans differ from human glycans, potentially affecting antibody efficacy and stability. Advances in glycoengineering have helped mitigate this issue, making yeast a viable option for specific mAb applications.
DOI: 10.1016/j.ijbiomac.2024.131379
Plant and Insect-Based Expression Systems
Plant-based expression systems, sometimes referred to as "plantibodies," use hosts such as Nicotiana benthamiana, rice, and soybean to produce recombinant mAbs. Plants offer a scalable and pathogen-free production system with lower costs compared to mammalian cell cultures. However, glycosylation differences and relatively low expression levels remain challenges for widespread adoption.
Insect cell systems, particularly the baculovirus expression vector system (BEVS) using Spodoptera frugiperda (Sf9, Sf21) cells, provide another alternative. While insect cells can perform some human-like glycosylation, yields are typically lower than CHO cells, and batch variability can pose manufacturing challenges.
Alternative and Emerging Hosts
In addition to traditional expression systems, researchers are exploring novel platforms for mAb production. Cell-free protein synthesis (CFPS) enables rapid, on-demand production of recombinant proteins without the need for living cells, but scalability remains a challenge. Other emerging hosts, such as filamentous fungi and algae, are under investigation for their potential to produce mAbs cost-effectively while performing post-translational modifications.
Choosing the Right System for Specific Applications
Selecting the optimal host for mAb production depends on the intended application. For therapeutic mAbs, CHO cells remain the preferred choice due to their human-like glycosylation and regulatory acceptance. For research and diagnostic mAbs, HEK293 or microbial systems can provide rapid and cost-effective expression. In biosimilar development, CHO cells are often required to match the glycosylation patterns of the original biologic.
At Biointron, we are dedicated to accelerating antibody discovery, optimization, and production. Our team of experts can provide customized solutions that meet your specific research needs, including CHO-K1 Stable Cell Line Generation and Recombinant Protein Expression in Mammalian Cells & E. Coli. Contact us to learn more about our services and how we can help accelerate your research and drug development projects.
References:
Das, P. K., Sahoo, A., & Veeranki, V. D. (2024). Recombinant monoclonal antibody production in yeasts: Challenges and considerations. International Journal of Biological Macromolecules, 266, 131379. https://doi.org/10.1016/j.ijbiomac.2024.131379
