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Recombinant antibodies are monoclonal antibodies produced in recombinant expression systems. Their production process starts by taking the antibody-encoding genes from different sources and transfecting them into highly productive systems, while the term monoclonal antibody is more commonly used to describe antibodies natively produced by hybridomas. Check out other frequently asked questions (FAQs) about recombinant antibodies on our dedicated page.
Recombinant antibodies are produced by transforming/transfecting highly productive expression systems with vectors carrying antibody-encoding genes. The process of recombinant expression allows researchers to protect the antibody sequences (e.g. in case the hybridoma is lost) and achieve more consistent and higher levels of production.
Recombinant technology has allowed the standardization of antibody reagents for multiple applications because it eliminated batch-to-batch variability and improved production yields. Moreover, recombinant production is currently the only way to produce antibodies in sufficient quantity and purity for commercial purposes (i.e. therapeutics, diagnostics, etc.).
The process of recombinant antibody production involves different steps:
Antibodies are Y-shaped glycoproteins consisting of two heavy and light chains, each divided into a constant and variable (antigen-binding) region. The variable regions (VL and VH) are responsible for interacting directly with the antigen, thus, obtaining the sequences of these regions is often sufficient for most applications.
However, for some specific applications, such as therapeutics, the full-length antibody sequence may need to be obtained. In these cases, the constant region contains crucial information regarding the distribution of glycans on the surface of this domain – which dictate the effector functions (immune system-engaging) of therapeutic antibodies.
Both the sequences for the variable regions and the full-length antibodies can be recovered from hybridomas (starting from mRNA), single-cells (using cell sorting technology), peripheral blood mononuclear cells (PBMCs), or even the purified antibody (using mass spectrometry technology).
From bacterial to plants or complex mammalian systems, antibodies can be produced in a wide variety of recombinant expression systems. Despite the vast choice, most antibodies are produced in recombinant mammalian cells optimized throughout decades of cell engineering and culture optimization.
The preference for these systems stems from their ability to perform adequate post-translational modifications (human-like glycosylation) and to the fact that they secrete these molecules into the culture supernatant. The latter greatly simplifies the process of antibody purification.
Chinese hamster ovary (CHO) and Human embryonic kidney (HEK) cells are the most frequently used cell lines for recombinant antibody production. Due to the wealth of knowledge regarding the expression machinery and secretory pathways in these cells, antibodies can be produced in a cost-efficient way for a wide variety of applications.
Bacterial and yeast systems have for long been considered as suitable alternatives for recombinant antibody production. However, bacteria are unable to perform post-translational modifications and often produce antibodies in inclusion bodies, significantly lowering the purifications yields. Moreover, bacterial-produced antibodies often need to be submitted to an additional step – endotoxin removal.
In contrast, yeast cells can be engineered for correct glycosylation and proper protein secretion. Given that their genetic machinery is simpler than that of mammalian cells, the use of yeast cells continues to gain ground over other production methods for the manufacture of recombinant antibodies.
Antibodies can be expressed in multiple formats including scFv (single-chain antibody fragments), Fab (antigen-binding fragment), VHH (single-domain antibodies from camelids), or full-length antibodies (IgG, IgM, among others).
One of the challenges of expressing these antibody formats in mammalian cells is their inability to replicate exogenous DNA. Bacteria and yeast are capable of recognizing specific origins of replication and can replicate plasmids alongside their genomes provided the proper selective pressure is maintained, but mammalian cells cannot.
Two strategies are currently used to overcome this limitation: (i) transient expression and (ii) stable cell line development. In transient expression, a vector is transfected into the cells and its copy number is expected to decline over time. Besides antibody-encoding genes, these vectors also contain a reporter gene which allows the detection of the antibody genes within the cells and estimation of transfection efficiencies. Transient expression vectors are usually devoid of selection markers.
In stable expression, the vector carries genetic signals that allow the integration of antibody-encoding genes into the genome of the mammalian cell. This type of recombinant expression is significantly more time-consuming and labor-intensive than transient expression. For this reason, stable expression is reserved only for large-scale commercial applications.
Selection markers are mostly used for stable expression or recombinant expression in non-mammalian systems.
The protocol for successful transfection/transformation depends on the expression system. The general principle of the process implies the permeabilization of cell membranes without compromising cell viability.
The process of permeabilization can be chemical (e.g. Calcium Phosphate, Lipids, etc.) or physical (e.g. electroporation) and needs to be optimized for each specific expression system and cell line.
Positive clones are often isolated by using metabolic or antibiotic selection markers. The process of selection is rarely used in transient expression systems due to being time-consuming and thus potentially limiting production yields. In this way, it is often more productive to increase transfection efficiency than to select/enrich positive clones within transient pools.
Metabolic systems of selection are more frequently used with mammalian cell lines for stable production. The two most common ones are the dihydrofolate reductase (DHFR) system and the glutamine synthetase (GS) system.
In contrast to transient systems, single stable clones need to be isolated before production can be scaled up. The process of single clone isolation can be achieved by several methods including the limiting dilution method (the most conventional approach) or the VIPS™ (Verified In-Situ Plate Seeding). Although the limiting dilution method is the most conventional approach to single clone selection, it is more labor-intensive and is limited by the fact it is only able to select the most abundant clones.
These two limitations are compensated by the high throughput approach used in the VIPS™ method.
Medium-scale recombinant production is typically carried out in 3L flasks (1L culture medium) with satisfactory results when using transient systems. However, commercial large-scale production needs to be carried out in large bioreactors (several liters of culture medium) and typically reserved for stable production.
Scaling-up to these large quantities requires that the recombinant cells are first acclimatized to the culture medium used for production (typically different from the medium used for transfection and clone selection). Afterward, once the cells reach the appropriate density, they can be subsequently transferred to greater volumes of culture medium.
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