The stable production of antibodies, particularly therapeutic monoclonal antibodies, has long been dominated by mammalian recombinant systems. Despite their complex genetic background, cell lines like Chinese hamster ovary (CHO) and murine myeloma cells, are very amenable to growth in suspension in serum-free medium and highly tolerant to changes in pH, temperature, oxidative stress, etc. – making them uniquely adapted to industrial production processes. Check out other frequently asked questions (FAQs) about stable cell lines for monoclonal antibody production on our dedicated page.
Main mammalian cell lines used for stable monoclonal antibody production
Stable production of monoclonal antibodies for commercialization is mainly achieved in mammalian systems. These cells are known to perform proper protein folding and complex post-translational modifications (including human-like glycosylation), vital to ensure optimal antibody reactivity and therapeutic effectiveness.
For the past decades, Chinese hamster ovary (CHO) and mouse myeloma (NS0 and Sp2/0) cells have been considered the industry’s gold standard for therapeutic antibody production. These cells are well-adapted to growth in suspension in serum-free medium and many are compatible with well-established systems of selection – a crucial step in stable cell line generation for monoclonal antibody production.
The majority of currently licensed antibody therapeutics are produced by either CHO or NS0 cell lines, and only a minority of therapeutic antibodies are expressed by bacteria (Escherichia coli) or yeast (Pichia pastoris) (antibodies approved until 2020).
Of these, all antibodies produced in E. coli approved for clinical use consist of antibody fragments: brolucizumab (humanized scFv), caplacizumab (humanized dsFv), moxetumomab pasudotox (murine dsFv), and certolizumab pegol (humanized Fab). Moreover, the sole antibody produced in P. pastoris approved for clinical use is eptinezumab (Vyepti™), approved only in 2020 for migraine prevention.
Why are CHO cells favored for therapeutic antibody production?
The most common CHO cell lines used for the production of licensed antibody pharmaceuticals include CHO-S, CHO-K1, and CHO-DG44. Besides the advantages listed above, many methods for recombinant production (particularly large-scale stable expression) have been optimized for the unique genetic background and nutritional requirements of CHO cells. This expression system is also extremely tolerant to changes in oxygen concentrations, pH, temperature, and pressure – crucial properties for industrial production processes.
Additionally, CHO cells can secrete proteins with complex post-translational modifications very similar to those produced by human cells. Plus, there are several well-established systems for gene amplification in CHO cells leading to higher titers of recombinant proteins.
CHO cells are constantly being engineered and optimized for protein production and they are typically selected using metabolic markers such as:
- Dihydrofolate Reductase (DHFR) system
- Glutamine Synthetase (GS) system
The choice of selection system heavily depends on the genetic background of the cell line being used. For instance, CHO-DG44 cells were engineered from CHO-K1 cells by gamma radiation in the 1960s to eliminate both alleles of DHFR. For this reason, when antibody genes are co-transfected with exogenous DHFR, positive clones are easily selected by eliminating glycine, hypoxanthine, and thymidine (GHT) from the growth medium. Alternatively, methotrexate (MTX) can be used to inhibit endogenous DHFR in DHFR+ CHO cell lines.
CHO cell lines deficient in GS have only been recently developed. For this reason, GS-based selection is still recurrently made using methionine sulphoximine (MSX) able to inhibit endogenous GS.
Despite all the advantages of CHO cell lines for monoclonal antibody production, these cells still produce slightly different glycans to those produced by human cells (α-gal and NGNA glycans which may cause adverse reactions in humans), plus, they lack α[2-6] sialyltransferase α[1-3/4] fucosyltransferases (human enzyme). These limitations continue driving the search for alternative production systems.
Alternative stable production systems for monoclonal antibodies
The regulatory process for licensing new therapeutic antibodies can take up to 10-12 years. For this reason, the cell lines that currently dominate the market aren’t necessarily the ones being used by developers with monoclonal antibodies on the clinical development pipeline.
In recent years, alternative expression systems have been tested for the production of monoclonal antibodies including:
- Baby hamster kidney (BKH21) cells
- HEK including HEK293T
- C6 cells
Each of these cell lines has a complex genetic background and thus requires very specific protocols for successful stable integration and stable antibody production. Notably, human cell lines like HEK are particularly attractive alternatives given their ability to synthesize all human glycans, thus reducing the risks of eliciting adverse reactions (immunogenicity).
However, the greatest disadvantage of these cells is the lack of species barrier. Human cell lines may harbor and potentially contribute to the transmission of human viruses. Although viral charges can be inactivated or eliminated by processes such as nanofiltration, these additional steps increase production costs. Plus, HEK cell lines pose a moderate health hazard to production plant workers and thus require tighter containment and security measures.
For this reason, plant and yeast cells represent a desirable alternative and we may soon witness the rise of licensed antibodies produced by these systems.
Stable cell lines are conventionally developed for the production of licensed biotherapeutics such as monoclonal antibodies. Since the approval of the first antibody in 1986 (muromonab), most antibodies approved for clinical use are either produced in CHO (hamster) or NS0 (mouse) cells.
CHO cells in particular are still the industry’s gold standard for antibody production given their superior tolerance to pH, temperature, and oxidative stress variations. Moreover, well-established cell lines lacking important enzymes (DHFR or GS) make the selection of positively transfected clones more straightforward.
Nevertheless, bacterial systems are still recurrently used to produce antibody fragments even for therapeutic applications. Moreover, given the production of α-gal glycans by CHO cells (potentially immunogenic to humans), alternative systems such as yeast and plant systems are continuously being exploited for the stable expression of monoclonal antibodies.
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