Recombinant antibody expression, the process of producing antibodies with defined sequences, is a crucial aspect of biotechnology. Employing recombinant antibody expression strategies ensures high batch-to-batch consistency and reproducibility and maintains high specificity. Whether it be for biomarker detection, life science research, or therapeutics, recombinant antibodies play a significant role. Recognizing this, evitria partners with biotechnology businesses to provide recombinant antibody expression services for custom-made products. This blog aims to provide a comprehensive overview of the main process steps involved in recombinant antibody expression, highlighting their importance and application in biotechnology.
Recombinant antibody expression – a definition
Recombinant antibody expression describes the process of producing antibodies using recombinant DNA technology. This involves genomic techniques including cloning the genes that encode the antibody of interest and then expressing them in a suitable host organism, such as bacteria, yeast, or mammalian cell lines.
Engineering antibodies using recombinant technology enables the development of antibodies into different isotypes, but also different formats, each with defined characteristics, such as bispecific antibodies. Recombinant monoclonal antibodies provide advantages in homogenous specificity and high affinity, as well as defined effector functions. For this reason, recombinant antibody engineering has revolutionized the development of novel diagnostics, therapeutics, and research tools.
Comparison of recombinant antibodies with polyclonal and monoclonal antibodies
Recombinant antibodies, also known as engineered antibodies, are immunoglobulins produced through genetic engineering techniques. These techniques involve combining genetic material encoding the heavy and light chains of an antibody to create a functional antibody molecule that binds to a specific epitope.
Differences between polyclonal and recombinant antibodies
In contrast to the polyclonal and monoclonal antibody production process, recombinant antibody production produces antibodies using in vitro rather than in vivo techniques, which do not necessitate immunization of animals or hybridoma cultivation.
Polyclonal antibodies are a heterogeneous mix of antibodies with different affinities and specificities. The advantages of the inexpensiveness of the process and high specificity in detecting low-quantity proteins result in polyclonal antibodies still remaining extensively used as analytical tools for biomedical research, food safety assessments1 and treatment of acute rejection in renal transplantation – rabbit anti-thymocyte globulin (rATG)2. The disadvantage of polyclonal antibodies is that they can display variable performance between different batches. Recombinant antibodies, on the other hand, have advantages over polyclonal antibodies because of their defined antibody sequences and specificity.
Differences between monoclonal and recombinant antibodies
Monoclonal antibodies are derived from a single B cell parent clone, and antibodies bind highly specifically to a single epitope. The disadvantage of monoclonal antibodies produced using hybridoma cell lines is they undergo genetic drift over time, which can give rise to variants and introduce lot-to-lot variability. Recombinant antibodies circumvent this problem by allowing sequence-specific antibodies to be expressed and produced in the expression host. This highly controlled and monitored process ensures high reproducibility. This is one of the reasons monoclonal antibodies are often converted to recombinant antibodies by amplifying and sequencing cDNAs encoding the VH and VL domains from a hybridoma cell line.
Development of recombinant antibody technology
Chimeric antibodies were the first recombinant antibodies originally developed by Morrison and Neuberger in 19843. The chimeric antibody design consisted of light and heavy chains of mouse variable regions combined with human constant regions in an effort to produce less of an immune response in humans compared with mouse monoclonals. Mammalian lymphoid cells were used to produce the chimeric antibody.
The first recombinant chimeric antibody, abciximab, was approved by the FDA for cardiovascular disease in 1994 and used the same murine hybridoma process with the replacement of the constant region with a human constant domain. Abciximab targeted the glycoprotein (GP) IIb/IIIa receptor of human platelets and inhibited platelet aggregation. The recombinant chimeric strategy led to the development of humanized antibodies — where all mouse antibody regions are replaced with human genomic sequences except for the complementarity-determining regions (that directly bind to the antigen). The success of humanized antibodies in therapeutic use consequently led to the discovery of fully human antibodies in 1990 by Sir Gregory Winter and George Smith using antibody phage display technology.
Main steps in recombinant antibody expression
Recombinant antibody expression usually involves the following stages, which can vary depending on the ultimate objective of the project:
- Selection of appropriate host expression system
Recombinant proteins can be produced by protein synthesis machinery from prokaryotic or eukaryotic cellular systems. The choice of host cell—for example, bacterial, yeast, insect, or mammalian cell lines—is determined by the functional activity required, protein type, and desired yield. - Designing and optimizing the expression vector
Following the design of the target and expression vectors, a cloning scheme, such as a parallelization strategy, is employed to enable high throughput cloning and expression. - Defining the strategy of the gene delivery
Numerous techniques exist to introduce an antibody gene of interest into cells via either stable or transient transfection. Transient transfection and stable transfection can both be employed. Methods include chemical transfection (e.g. polyethyleneimine), using electric current to alter membrane permeability (electroporation), or Recombinant Viral Transduction, allowing stable transgene integration and continuous expression for difficult-to-transfect cells. - Optimizing the culture conditions
Healthy cell growth and transfection susceptibility rely on various components in the supernatant, such as glucose, nutrients, serum, and vitamins. Additional media components, such as hormones and growth factors, have been shown to lead to optimizations in antibody expression. - Determining the purification approach
Purification of recombinant antibodies can be achieved manually or through chromatography systems (e.g. flow cytometry, ELISA, protein A purification, or Western blotting), depending on the application and purity/concentration needs.
What is needed in recombinant antibody expression?
What is needed in recombinant antibody expression?
Whether for small- or large-scale production, producing recombinant proteins necessitates trained labour and a fully equipped biotech laboratory, which includes access to sequencing technologies, cloning reagents, molecular biology equipment, cell culture incubators and consumables, microscopes, immunoassays, protein purification and analysis tools. Scaling up the production of the manufacturing process would require using a bioreactor.
Expression systems for recombinant antibodies
The choice of expression system for recombinant protein expression depends on various factors, such as the size and complexity of the antibody, the desired yield and quality of the final product, and available resources.
Bacteria, including Escherichia coli (E. coli), are often used to express recombinant antibodies since they are easily manipulated and have a fast growth rate. Indeed, there are more than 85 approved E. coli-produced protein therapeutics in the US/EU, such as Caplacizumab (Cablivi®), Brolucizumab (Beovu®), and Tebentafusp (Kimmtrak®)4. However, protein aggregation into inclusion bodies can hamper recombinant protein expression in bacteria. If this occurs, proteins must be resolubilized and refolded into functional forms.
Mammalian cell lines, in particular CHO cells or HEK293 cells, are thus the often preferred and most commonly used expression system for recombinant antibody production because they easily allow post-translational modifications and can produce antibodies with human-like glycosylation patterns.
HEK293 cells (human embryonic kidney cells) are often chosen as hosts for protein expression because of their quick transfection rate, easy handling, and high protein production. Additionally, they are simple to propagate and sustain and are compatible with various transfection techniques. However, they are restricted to research environments due to various limitations, such as their propensity to clump together.
CHO cells (Chinese hamster ovary cells) are popular host cells that exhibit strong growth in suspension cultures, adapt effortlessly to serum-free media, and generate and secrete recombinant antibodies with higher specificity on the multi-gram scale. Due to their origin in hamsters, these cells are less prone to human viral contaminants yet can still perform glycosylation compatible with humans. They are able to produce a great variety of recombinant proteins, from fab fragments like scFv (single-chain variable fragments) to full-length antibodies.
Reformatting antibodies?
Recombinant antibodies are necessary for several reasons. They offer versatility in antibody formats and engineering, allowing for the generation of tailored antibodies with specific properties. Furthermore, recombinant antibody systems can generate fully human or humanized antibodies, reducing the risk of immunogenicity in therapeutic applications. Antibody fragments, a type of recombinant antibody, are smaller and have unique advantages, such as improved tissue penetration. They are particularly useful in applications where full-size antibodies may not be optimal.
Recombinant antibody technology enables the production of bispecific antibodies, which can simultaneously bind to two different antigens. As therapeutic antibodies, they have promising applications in targeted drug delivery and modulating immune responses. Proper formation of disulfide bonds, crucial for antibody stability, is ensured through recombinant antibody expression. This improves antibody quality and reduces aggregation.
Production of recombinant antibodies at evitria
In the publication by Melo et al. in Frontiers in Immunology, researchers from the University of Groningen detailed their collaboration with evitria to produce a bispecific antibody that enhances the anti-tumour activity of T cells. By targeting both CD27 and epidermal growth factor (EGFR), the bispecific antibody, CD27xEGFR, could specifically target carcinomas, which commonly overexpress EGFR. Concomitantly targeting co-stimulation of CD27 by crosslinking enhanced T-cell activation in terms of proliferation to target tumour cells. Potential toxicity was reduced in the bispecific antibody design by introducing LALAPG point mutations to yield an Fc-silent human IgG1, thus reducing FcR-mediated antibody effector functions5.
At evitria, we specialize in recombinant antibody expression services based on CHO cells (Chinese Hamster Ovary cells), which allows us to produce high-throughput, high-quality antibodies with outstanding speed.
Since we are focused on antibody and protein expression, we can support partners worldwide with specialized knowledge in various questions and challenges associated with different projects — from the first pilot study to the delivery of different antibody products.
References
- Campbell, K. et al. Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology. Anal Chem 79, (2007). ↩︎
- FDA. Thymoglobulin. Approved blood products https://www.fda.gov/vaccines-blood-biologics/approved-blood-products/thymoglobulin (2024). ↩︎
- Morrison, S. L., Johnson, M. J., Herzenberg, L. A. & Oi, V. T. Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci U S A 81, (1984). ↩︎
- Rashid, M. H. Full-length recombinant antibodies from Escherichia coli: production, characterization, effector function (Fc) engineering, and clinical evaluation. mAbs vol. 14 Preprint at https://doi.org/10.1080/19420862.2022.2111748 (2022). ↩︎
- Melo, V. et al. EGFR-selective activation of CD27 co-stimulatory signaling by a bispecific antibody enhances anti-tumor activity of T cells. Front Immunol 14, (2023). ↩︎