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Recombinant antibodies are one of the most successful biological reagents for research, therapy, and diagnostics. Their use has become widespread due to the enhanced batch-to-batch consistency and purity obtained by recombinant antibody production methods. On this page, we answer all most frequently asked questions about recombinant antibodies and describe ProteoGenix’s platform of production and its unique guarantees.
We can start producing recombinant antibodies from hybridoma cell lines, purified antibody samples, or antibody sequences. Additionally, we can engineer your antibody by affinity maturation, humanization, bispecific antibody development, antibody-drug conjugate development, or antibody reformatting (i.e. isotype switching) to improve developability and fine-tune its reactivity.
We can express antibodies in a variety of recombinant systems including bacterial (Escherichia coli and Bacillus subtilis), yeast (Pichia pastoris and Saccharomyces cerevisiae), insect cells, and mammalian cells (Chinese hamster ovary, CHO, or Human embryonic kidney, HEK). In mammalian cells, recombinant production can be performed using transient expression or stable cell line generation approaches.
Choosing an adequate expression system depends on the format of the antibody (VHH, scFv, Fab, Fc-fusion proteins, IgG, or other full-length antibodies) and its final application. For full-length antibodies, the recombinant antibody production system of choice continues to be mammalian; especially CHO and HEK cell lines. These cell lines can perform proper protein folding and adequate glycosylation – two characteristics with a great impact on antibody’s reactivity.
With few exceptions, for most therapeutic and diagnostic applications we recommend using CHO or HEK cell lines. These cells are able to perform human-like glycosylation, minimizing potential adverse reactions to new treatments. For the production of antibody fragments (scFv, Fab, or VHH), the use of alternative systems like yeast or bacteria may be tested to achieve high and consistent yields while bypassing the need for stable cell line development.
At ProteoGenix, our team has 28+ years of experience in protein production. We are qualified to produce even the most difficult proteins while still providing the strongest guarantees. Since our inception, we have successfully produced over 500 antibodies using our proprietary XtenCHO™ cell line including full-length (IgG, IgM…) antibodies and antibody fragments (scFv, Fab, Fc, and VHH) with highly consistent results. Beyond naked antibodies, we have strong expertise in the production of therapeutic antibody formats including antibody-drug conjugates (25+ projects), and bispecific antibodies (30+ projects).
The most important advantage of partnering with us is the access to our vast range of complementary services. This including the possibility of maximizing developability (i.e. affinity, stability, etc.), fine-tuning antibody’s cross-reactivity (i.e. widening or narrowing its affinity towards different targets), performing antibody reformatting or engineering, and validating it in a target application (i.e. Western Blot, Immunohistochemistry, Immunoprecipitation, Flow Cytometry, among others). Additionally, we can carry out antibody conjugation with enzymatic or fluorescent tags for many immunoassays.
Thanks to our experience and fully-optimized protocols, we can guarantee antibody quantities at competitive prices. We also offer a vast diversity of cell lines for recombinant expression, including our high-yield proprietary system, XtenCHO™ designed for optimal antibody production.
XtenCHO™ was originally engineered from a CHO cell line. These cells were genetically modified by means of gene knockout and gene integration to improve antibody secretion and metabolism. Subsequent studies revealed that XtenCHO™ consistently outperforms other commercially available cell lines such as ExpiCHO™ and CHO-S™ by 1.5 and 9.7x, respectively. Additionally, our proprietary cell line has shown good performances when producing humanized and bispecific antibodies while maintaining an adequate protein structure and post-translational modifications.
Recombinant antibodies are monoclonal antibodies produced in vitro. This is achieved by cloning antibody-encoding genes into expression vectors followed by a process of transfection or transformation. These antibodies are quickly replacing conventional monoclonal antibodies for many applications including research, therapy, and diagnostics.
Discover the full definition of a recombinant antibody in our dedicated article: What is a recombinant antibody?
The terms monoclonal and recombinant antibodies are often used interchangeably, but in reality, they represent two different types of antibodies. Monoclonal antibodies refer to IgG molecules generated and produced in hybridoma cell lines. In contrast, recombinant antibodies can be IgG immunoglobulins or any other antibody format (scFv, Fab, IgM, etc.) produced in vitro via a recombinant expression system such as bacteria, yeast, insect, plant, and mammalian cells.
Nevertheless, both monoclonal and recombinant antibodies possess antigen-specificity given that they derive from a single plasma cell clone or a single antibody sequence, respectively. In contrast, polyclonal antibodies are exclusively generated by hyperimmunizing animal hosts and harvesting their IgG-enriched plasma for downstream purification. In their essence, polyclonal antibodies contain a mixture of monoclonal antibodies derived from different B cell lineages and consequently displaying different epitope-specificity.
For therapeutic and large-scale diagnostic applications, the use of recombinant antibodies has become the norm. The production of these molecules is more amenable to scale-up. Plus, recombinant antibodies have a superior batch-to-batch consistency in comparison to monoclonal and polyclonal antibodies. However, the use of monoclonal antibodies produced in native hybridoma cell lines continues to be widespread for research applications.
Some experts have urged researchers to replace monoclonal with recombinant antibodies in their immunoassays. Monoclonal antibodies often suffer from inconsistent production yields and low purity resulting in conflicting results. Unlike monoclonal antibodies, recombinant antibodies can be more easily produced, characterized, and validated. This ease of manipulation makes them desirable for many immunoassays including flow cytometry, immunohistochemistry, Western Blot, immunoprecipitation, among others.
Antibody sequences are required to produce recombinant antibodies. For this reason, these immunoglobulins are more amenable to engineering (bispecific antibody development, affinity maturation, antibody-drug conjugation, etc.) and reformatting (isotype switching, etc.). Moreover, since these reagents are produced in high-yield expression systems, production is easier to scale-up and batch-to-batch consistency is markedly superior to that obtained by conventional monoclonal antibody production methods.
Additionally, most high-performing recombinant antibody production systems can grow in chemically defined media devoid of animal components. This contributes to higher batch purities and an enhanced reproducibility of the production process.
To learn more about the benefits of using recombinant antibodies, read the complete article: What are the benefits of using recombinant antibodies?
Recombinant antibodies bypass most limitations of conventional monoclonal antibody production methods. Nevertheless, their implementation by research labs has lagged for some applications. This slow shift in research from monoclonal to recombinant antibodies can be tied to the difficulties in changing existing and well-established protocols for new production methods. However, these hurdles can be bypassed by the use of commercially available kits fully optimized for the transient and fast expression of recombinant antibodies.
As their definition indicates, recombinant antibodies are produced in vitro using high-yield expression systems. Most antibodies, especially full-length IgG molecules, are preferentially produced in mammalian systems based on Chinese hamster ovary (CHO) or Human embryonic kidney (HEK) cell lines. Using these cell lines, researchers can opt for transient or stable antibody production. The former is extremely useful for small-scale applications with short lead times and reasonable yields, while the latter is the method of choice for large-scale and commercial production of antibodies for therapeutic or diagnostic applications.
In a nutshell, recombinant antibody production starts with cloning antibody-encoding genes into high-expression vectors, transfecting or transforming cells, selecting positive clones, and scaling up antibody production.
Learn more about the processes of recombinant antibody production in our dedicated article: How are recombinant antibodies produced?
Mammalian expression systems such as Chinese hamster ovary (CHO), Human embryonic kidney (HEK), Baby hamster kidney (BKH), Mouse myeloma (NS0), and Per.C6 cells are the most commonly used for antibody production. The reason why these systems are so successful is tied to their ability to perform correct antibody folding and glycosylation. Moreover, since these cell lines secrete antibodies into the culture medium, purification can be more easily achieved.
These systems still present important challenges including their inability to replicate exogenous DNA vectors (i.e. plasmids). For this reason, many alternative expression systems are constantly being developed and optimized. The most popular systems for recombinant antibody production are plants (due to their low cost) and yeast (due to their enhanced flexibility), but few antibodies produced in these systems have reached commercial applications because of the challenges in transfecting plant systems and yeast’s inability to perform human-like glycosylation. Nevertheless, as many scientists continue to push the boundaries of recombinant production, these systems may soon reach commercial viability.
The terms transient and stable expression are mostly used to describe mammalian expression systems. Given that these systems are unable to replicate plasmids, DNA vectors containing antibody-encoding genes tend to be lost throughout consecutive cycles of cell division. For this reason, when mammalian cells are transfected with vectors containing only antibody genes, selection markers, and reporter genes, antibody expression is only temporarily achieved.
This is called transient expression and it is the most common process of recombinant antibody expression. The popularity of this approach is explained by its significantly shorter lead time and reasonable productivity.
For large-scale commercial applications, however, DNA vectors need to be stably integrated into the genome of the mammalian cell line. Additionally, before production can be scaled-up, a single clone needs to be selected. Monoclone selection is the most time-consuming part of the process of stable cell line generation and it can take up to 4 months.
One of the most limiting steps of the recombinant antibody production process is the transfection or transformation step. The procedure requires the permeabilization of cell membranes using physical (e.g. electroporation) or chemical (e.g. calcium phosphate) means. This results in a drop of viability that can often lead to insufficient positive clones for further screening. For this reason, many approaches to optimization start by using more effective methods of transfection.
Other steps of this process that can be readily optimized include the optimization of the gene cloning process and vector design. In addition to these changes, cell lines already in use for recombinant antibody production can be further engineered for better productivity. These approaches, however, are more time-consuming because engineering cells takes significantly longer than optimizing the transfection process. Nevertheless, the growing demand for antibodies continues to push for more cost-effective methods resulting in the generation of improved cell lines for recombinant production.
Recombinant antibodies are typically produced in mammalian systems using transient or stable expression. When transient expression is used, it typically takes between 3-4 weeks to produce at least 1 mg of purified antibody (starting with an antibody vector).
Stable expression is a more time-intensive process due to the need for integrating the antibody-encoding genes into the genome of the expression host and isolating single clones before production can be scaled-up. Starting with an expression vector, stable cell line generation can take up to 5-6 months. However, once the stable cell line is created and production is scaled-up, recombinant antibodies can be produced in only a few weeks (quantity dependent).
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