Monoclonal antibodies can be produced in a variety of recombinant systems including mammalian, microbial (bacterial and yeast), or insect cells. However, the choice of an adequate production system depends on the antibody’s intended application. Read the complete article to find out how to choose the best production system for each application and check out other frequently asked questions (FAQs) page about monoclonal antibodies on our dedicated page.

Monoclonal antibodies are complex glycoproteins consisting of an antigen-binding region (Fab) and a conserved region also known as the crystallizable fragment (Fc). The latter is considered particularly complex due to the glycans that coat its surface giving antibodies immunomodulating properties.

It is often the nature of these glycans and the intended application of the monoclonal antibody that defines the choice of a production system. For instance, the production of antibodies for therapeutic applications is typically reserved for mammalian systems capable of human-like glycosylation. In contrast, the production of diagnostic antibodies is more flexible given that the presence and distribution of glycans are not essential properties for accurate antigen detection.

Therapeutic monoclonal antibodies production

Given the requirement for adequate glycan structure and distribution on the surface of the Fc region, therapeutic antibodies are often produced in well-studied mammalian systems such as:

  • Chinese hamster ovary (CHO) cells
  • Human embryonic kidney (HEK293) cells
  • Baby hamster kidney (BHK-21) cells
  • Mouse myeloma (NS0) cells

 

From this list of widely used cell lines for monoclonal antibody production, CHO and HEK293 lineages continue to be the most popular choices. The reason for this preference stems from the wealth of knowledge available on these cell lines and the robustness of transfection and selection protocols.

Transfection, somewhat comparable to bacterial transformation, consists of introducing plasmid DNA into mammalian cells. However, considering these cells are unable to recognize the bacterial origin of replication, plasmid copy numbers per cell decrease with each new division cycle. This decrease leads to the reduction and finally arrest of antibody expression in these systems – also known as transient expression.

Transient expression is useful for a wide variety of applications including antibody production for preclinical studies. However, when large-scale production is envisaged, stable cell lines must be generated.

In contrast to transient transfection, stable transfection requires foreign DNA to be successfully incorporated in the genomes of mammalian expression systems. A variety of strategies have been developed to force the incorporation of exogenous DNA molecules into the genome, these can be divided in:

 

  • Random integration – the DNA is integrated into a random location. This approach carries a high risk of generating low-production clones since the DNA may be integrated into a low-expression region.
  • Site-specific integration – the DNA is purposely integrated into a high-expression region. This approach is optimal in comparison to random integration; however, it requires a vast knowledge of the host’s genomics.

 

Selection systems for positively transfected mammalian cells are typically metabolic in contrast with the antibiotic-based selection employed in microbial systems. The two main metabolic systems used for antibody production include:

  • Glutamine synthetase (GS) system – GS is a vital enzyme for glutamine synthesis. To exploit that pathway, cell lines are either engineered to lack this enzyme (GS-), or its expression is heavily impaired prior to transfection. In these cases, positive clones can be selected by growing in a medium lacking glutamate (GS substrate) and using vectors with an extra copy of GS.
  • Dihydrofolate reductase (DHFR) system – DHFR catalyzes the conversion of folate to tetrahydrofolate, a precursor of de novo synthesis of nucleotides. To exploit this pathway, the same principle as the one used in the GS system is applied.

Diagnostic monoclonal antibodies production

A vast number of antibodies used in classic diagnostic applications are produced in hybridoma cell lines grown in suspension or, sparsely, using the ascites production method. However, hybridoma cell lines are often prone to genetic drift or loss of antibody-encoding genes. For this reason, diagnostic antibody production is increasingly abandoning native expression systems and evolving to recombinant production.

Given that many diagnostic applications are ex-situ (e.g. ELISA, flow cytometry, WB, immunocyto- or immunohistochemistry, etc.), monoclonal antibodies used in these applications often don’t require the presence of glycans.

For this reason, diagnostic antibodies are more easily produced in microbial systems including bacteria and yeast. Since many vectors can replicate in these microbial systems, exogenous DNA does not need to be permanently integrated into the genome to ensure robust expression levels, thus, abolishing the time-consuming and labor-intensive process of stable cell line generation.

The selection and isolation of positively transformed clones is also more straightforward in comparison to antibody production in mammalian systems, and antibiotic selection markers are typically preferred.

Concluding remarks

Multiple monoclonal antibody production systems are currently available. The choice of production system depends on the antibody’s intended use.

For therapeutic applications, antibodies are primarily produced in mammalian systems (i.e. CHO or HEK) able to perform human-like glycosylation. In contrast, for diagnostic applications, antibodies rarely depend on the presence of glycans to detect specific antigens. In these cases, microbial systems that are generally unable to perform human-like glycosylation can be used for robust antibody production.

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