At ProteoGenix, we can produce monoclonal antibodies using different expression systems depending on the intended applications. For research and diagnostics, it is often sufficient to produce these molecules natively in hybridomas or using recombinant systems with high turnover rates, such as Escherichia coli which are unable to perform glycosylation.
Growing hybridoma cell lines in suspension and harvesting antibodies from the culture supernatant remains an important approach for small-scale applications. Typically, hybridomas can be grown in medium with or without serum. The former allows a very quick production (about 3-10 days) of antibodies containing residual contamination (animal protein and unspecific IgG); while the latter requires acclimatization of 8-12 days before hybridomas can produce monoclonal antibodies efficiently in suspension. This results in a total lead time of 10-22 days.
However, some hybridoma cell lines are unable to thrive under laboratory conditions. In these cases, the ascites method can be used. Despite achieving significant production yields, the use of the ascites method is increasingly discouraged because it does not comply with EU directives and recommendations regarding animal safety. Moreover, hybridomas often suffer from genetic drift making them prone to losing antibody-encoding genes. In recent years, it has also become evident that hybridomas often produce additional antibody chains, reducing the overall purity and yield.
For these reasons, when monoclonal antibodies are intended for routine research and diagnostic analysis, we advise our customers to choose recombinant expression for their projects. Recombinant expression has the advantages:
Despite the large diversity of antibody expression systems, bacterial systems are still sparsely used due to the risk of forming inclusion bodies which severely hinder antibody purification. In contrast, mammalian systems provide a more robust approach to monoclonal antibody production.
Monoclonal antibodies can be produced in many different cell lines at our facilities. But we favor Chinese hamster ovary (CHO) or Human embryonic kidney (HEK) cell lines due to their robust growth and ability to perform human-like glycosylation on the Fc domain of monoclonal antibodies.
Our optimized workflow and timelines for monoclonal antibody production can be detailed as follows:
Transient expression of monoclonal antibodies is sufficient for most applications. In these cases, depending on the required yield and purity levels, the transfection, production, purification stages (steps 4 and 5 in the process depicted above) start at 3 to 4 weeks. Monoclonal antibodies produced by transient expression are used in preliminary characterization studies, research projects, and preclinical tests.
In contrast, when antibodies are intended for therapeutic applications or to be used as components of in vitro diagnostics and medical devices, stable cell lines must be generated. In these cases, the transfection, screening, and subcloning start at 5-6 months. The major challenges of any stable cell line generation process derive from the labor-intensive and time-consuming process of monoclone selection and stability confirmation.
In detail, upon vector’s transfection, stable pools (a mixture of several clones) are obtained and amplified. Preliminary characterization of these pools enables researchers to identify those with the highest and most consistent production yield. Subsequently, highly productive pools are selected for monoclone isolation. Monoclones are defined as a specific subpopulation originated from a single cell, thus, monoclones share the same genetic background with stably integrated vectors and similar production yields.
Ensuring “clonality” can be particularly challenging when working with mammalian cell lines due to the difficulty in isolating single cells. Currently, monoclone isolation at ProteoGenix can be performed using one of two major strategies:
Given the lower number of clone amplification cycles, the VIPS™ method allows a faster and more robust approach to monoclone screening and selection. Moreover, stable cell line generation, which starts at 5-6 months, can be successfully reduced by several weeks.