Monoclonal antibody production can be achieved in different systems. At ProteoGenix, we advise our customers to choose recombinant mammalian systems because these systems achieve higher production yields, higher purity levels, and secrete antibodies to the culture supernatant making them easier to purify. Learn more about our monoclonal antibody production timelines and discover why we recommend recombinant mammalian systems for most applications. Check out other frequently asked questions (FAQs) page about monoclonal antibodies on our dedicated page.

Monoclonal antibody production in hybridoma cell cultures

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:

  • Securing antibody sequence information (even if hybridomas are lost during storage, they can still be recombinantly expressed in other systems)
  • Achieving higher purity levels
  • Obtaining higher and more consistent production yields

 

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.

Recombinant antibody production in mammalian systems

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:

  1. Antibody sequencing when sequences are not yet available (2-3 weeks for variable regions and leader sequences; 3-4 weeks for the full-length sequence)
    1. From purified proteins (mass spectra based sequencing)
    2. From hybridoma cell lines (transcriptomic-based sequencing)
  2. Choice of the adequate expression vector, gene synthesis, and cloning of antibody-encoding genes – starting at 2-3 weeks
  3. Transfection
    1. Transient expression for short-term projects or small/medium-scale production (starting at 3-4 weeks)
    2. Stable cell line generation for long-term projects, commercial and large-scale production (starting at 5 months)
  4. Production scale-up and purification (lead times are quantity-dependent)

 

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:

  • Monoclone selection using the limiting dilution method (starting at 4 months) – this method is the conventional approach to monoclone selection and it requires several rounds of dilution and amplification to limit cell density in each well to a minimum (ideally one cell per well). Although less expensive than other methods, limiting dilution has important drawbacks including the fact that it can be extremely labor-intensive and time-consuming. Moreover, limiting the number of cells per well by dilution will only enable the isolation of the most abundant clones – which might not be the most productive ones.
  • Monoclone selection using the Verified In-Situ Plate Seeding (VIPS™) method (starting at 4 months) – the VIPS™ method is an advanced approach to monoclone selection. It is less labor-intensive than the conventional approach; however, it requires the use of specialized equipment. The VIPS™ equipment was designed to automatically seed single cells into 96 or 384 well microplates. In contrast to the traditional method, most single clones are isolated and evaluated before amplification enabling researchers to choose the best candidates – which might not be the most abundant ones.

 

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.

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