The process of stable cell line generation is a laborious process reserved for the large-scale production of monoclonal antibody pharmaceuticals. In this article, we explain our recommendations and approaches to monoclonal antibody production. We detail the average lead times for this process. Check out other frequently asked questions (FAQs) about stable cell lines for monoclonal antibody production on our dedicated page.
Before starting: recommendation to maximize production yields
The generation of stable cell lines is a mandatory step in the successful clinical development of monoclonal antibodies. At ProteoGenix, we recommend the extensive early testing of antibody leads before committing to large-scale production.
Early developability assessment is a well-established and successful approach used to ensure an antibody is as stable, reactive, and effective as it can before developing a custom and optimized production protocol. In this way, we can bypass many stable expression hurdles and ensure optimal production at reasonable costs. Antibody characteristics such as aggregation, affinity and avidity, specificity and selectivity, and glycosylation profile are known to have the greatest impact on developability and can be measured at our facilities.
Full-length monoclonal antibodies are often produced in mammalian expression systems such as Chinese hamster ovary (CHO) or mouse myeloma (NS0) cells. At ProteoGenix, we specialize in CHO cell lines offering a large diversity if hosts including CHO-DG44 (a cell line lacking both alleles of DHFR, suitable for metabolic selection), CHO-S, and a proprietary GS- CHO cell line, among others.
Although we have over 28 years of experience in protein production in different expression systems (mammalian, bacterial, yeast, insect), CHO-based stable production continues to be our system of choice. CHO cells are more tolerant to changes of pH, temperature, pressure, oxygen concentrations. Plus, they are more amenable to gene amplification leading to production levels comparably higher than those achieved by other production systems. Plus, CHO cells produce proteins with glycosylation profiles similar to humans resulting in lower immunogenicity and optimal therapeutic efficacy.
In contrast, antibody fragments devoid of glycans may be produced faster at high titers in simpler systems such as bacterial cells (e.g. Escherichia coli or Bacillus subtillis) known for their high turnover rates. The simpler genetic background of bacteria makes them more amenable to manipulation and scale-up. However, antibodies in E. coli are produced in the cytoplasm or periplasm requiring harsher extraction methods.
Timeframes differ between the two approaches.
How long does it take to generate stable mammalian cell lines for monoclonal antibody production?
When starting from an optimized expression vector, stable cell line generation starts at 4-6 months. First, antibody genes need to be optimized (i.e. codon optimization) and cloned into a high-expression vector. The vector is then transfected or co-transfected in parallel with another vector containing the selective markers (i.e. DHFR or GS are the two most widely used in CHO-based production) into competent mammalian cells. We carry out vector linearization to increase stable integration efficiency.
Successful transfection can be achieved by chemical (e.g. PEG or other reagent to increase membrane permeability) or physical methods (e.g. electroporation) in non-selective medium and optimal conditions. The process of transfection takes about 24-48 hours.
After transfection, the process of selection starts; cells are amplified in selective medium (for several weeks) and positive clones subsequently isolated either by:
- Limiting dilution (most conventional method)
- Verified In-Situ Plate Seeding (VIPS™)
Independently of the method used for single clone selection, the process is laborious and complex and starts at 4 months. Subsequent steps comprise assessing the production yield, cell line stability, and antibody properties in several promising single clones to select the best clone for further development.
Further development includes process optimization (4-5 weeks), cell bank preparation and stability studies (variable), and production scale-up.
How long does it take to generate recombinant bacterial cell lines for monoclonal antibody production?
- coli is the most widely used host used for the production of licensed biologicals, including antibody fragments for therapeutic applications. In contrast to mammalian cells, bacteria can replicate plasmids provided they carry an adequate origin of replication. For this reason, they maintain plasmids at high copy numbers when cultured in selective media.
Given that bacteria require no stable integration, this simplifies the process of monoclonal antibody production and significantly reduces lead times. Starting from an expression vector, competent E. coli cells can be transformed in non-selective medium in only 2 to 14 hours. Given that single cells can be isolated by growing in solid medium, the process of selection and single clone isolation takes about 48 hours.
The successful transformation can be assessed by using reporter genes (positive and negative colonies display distinct colors or fluorescence) and PCR amplification of the gene of interest. Small-scale production tests and process optimization are subsequently conducted with variable timeframes (starting at 2-3 weeks). Finally, production scale-up and cell line transference can be accomplished in 2-3 weeks.
Stable production of monoclonal antibodies in CHO cells is still the method of choice for the production of most biopharmaceuticals. However, generating stable cell lines is a laborious and time-consuming process starting at 4-6 months. For this reason, we highly recommend extensive early testing and antibody lead optimization before committing to large-scale development.
In contrast, E. coli is the alternative system for the production of licensed antibody fragments. This process takes a fraction of the time to achieve compared to CHO-based production. However, bacteria are limited by their inability to perform post-translational modifications required by most monoclonal antibody therapies. For this reason, researchers continue investigating alternative systems in order to reduce production costs and lead times.