18.11.2021 duration: 9 min

Recombinant antibodies: next level antibody technology

Recombinant antibodies (rAbs) are antibody fragments expressed by using recombinant antibody coding genes. They overcome polyclonal and hybridoma-based monoclonal antibodies weaknesses in terms of quality and reproducibility.

Since they offer many advantages in medicine and research, recombinant antibodies are considered the next level in antibody science. 

Recombinant antibodies are produced in cell lines, or in vitro, unlike traditional monoclonal antibodies (mAbs), which rely heavily on laboratory animals. Moreover, recombinant monoclonal antibodies have a very well-defined molecular composition, while traditional mAbs are more prone to quality and reproducibility issues.

Recombinant antibodies are used in medicine as well as in life science. They are therapeutic treatments for many diseases, including cancer or autoimmune disorders.

This overview will illuminate the basics of rAbs. From the application in medicine and science to recombinant antibody production and the advantages of recombinant Abs in contrast to traditional monoclonal antibodies, this article will teach you all you need to know on this topic. But first, a general introduction to antibodies. 

What are antibodies?

Antibodies (Abs) are proteins that belong to the class of immunoglobulins. They are naturally produced by certain types of blood cells of vertebrate animals and are part of the adaptive immune system. Humans produce several types of antibodies, the so-called immunoglobulins G (IgG), with IgG1 being the most common among them.

Antibodies are generally Y-shaped molecules consisting of heavy chains and light chains that bind to their target, or antigen, with a variable region. The antigen binding sites are located at the two tips of the “Y” and they mediate a very strong and specific binding interaction between antibody and antigen, similar to a receptor. The base of the “Y” is termed constant region (in contrast to the variable domain), although it’s sequence varies between species.

Antigens are usually parts of bacteria, microorganisms, or surface constituents of viruses. Antibodies bind to such pathogens, covering their surfaces, thus inactivating them and marking them as foreign to the immune system.

The high specificity of the antibody-antigen binding process led to the development of antibodies as valuable tools for diagnostic and therapeutic medicine and biological research. Nowadays, polyclonal, monoclonal and recombinant antibodies are available, and they come with their individual profile of pros and cons, depending on the specific application.

Polyclonal vs. monoclonal antibodies

Polyclonal vs. monoclonal antibodies - evitria

When a pathogen enters the human body and is exposed to the immune system, it is attacked and subsequently digested. The resulting fragments are presented to B cells, which in turn produce antibodies that are specific to a single feature of the pathogen’s constituents, or epitopes. 

Each B cell produces one specific antibody against an epitope. Naturally, when an animal is immunized against a pathogen, a plethora of individual B cells will produce different antibodies against many epitopes of the antigen. Such a family of antibodies is termed polyclonal antibodies. If a single B cell is cloned, the resulting cell population will produce a single antibody against one epitope – these are monoclonal antibodies.

Whether polyclonal or monoclonal antibodies are advantageous, is entirely dependent on the application.

Polyclonal antibodies

Polyclonal antibody - evitria

To trigger polyclonal antibody production, animals are injected with the antigen of interest. Further, repeat injections after initial animal immunization may be beneficial in order to increase the Ab titer in the blood. 

Next, the polyclonal antibodies are harvested as solution in the serum by bleeding the animal and removing all blood cells. The polyclonal antibodies may be subjected to further purification by removing serum proteins. The choice of animal depends on the desired amounts of Abs, their isotypes and immune response. Common animals for Ab production are rabbits, mice, horses, goats and llamas.

Key advantages of this process are the relatively low time and capital investment to obtain antibodies, but the use of animals poses ethical questions. Another disadvantage is the batch-to-batch variability, since they are obtained from different individual animals.

Polyclonal antibodies are especially well suited for immunoprecipitation, co-IP and ChIP applications.

Monoclonal antibodies

Moloclonal antibody - evitria

Monoclonal antibodies are one level more sophisticated and more refined than polyclonal antibodies.

Monoclonal antibodies (mAbs) are produced by single, individual B cells, unlike polyclonals which stem from a population of diverse B cells. The isolation of such a B cell for large scale production of Abs is next to impossible and in addition, B cells have a relatively short expected life time.

A major breakthrough was the development of the hybridoma technology: B cells are harvested from immunized animals and then fused with myeloma cells. The resulting hybridoma cells carry the ability for antigen production of the B cell and the immortality of the myeloma cell.

Hybridoma cells are then selected for specificity and promising candidates are further cultured and injected into a host animal’s abdomen. Subsequently, the desired monoclonal antibodies are harvested from the culture supernatant or ascites liquid.

This process is more time-consuming and sophisticated than the production of polyclonal antibodies, but many applications in medicine (e.g., therapeutic antibodies) require the high purity and specificity to minimize adverse side effects.

Recombinant antibodies – the next level

Recombinant antibodies, in short rAbs, are considered the next level of antibodies, beyond polyclonal and monoclonal antibodies. Over time, it became clear that novel technologies and applications in medicine and research could not perform satisfactorily with polyclonal’s and even monoclonal’s weaknesses. 

Polyclonal antibodies are expected to show broader off-target binding due to animal use, but even mAbs were found to have some variability. Investigations showed that hybridoma cell lines tend to mutate and hence produce variations of the crucial antigen binding site.

Advances in recombinant technology facilitated several breakthroughs, leading to the advent of recombinant antibody production on a large scale. Recombinant technology allows the design and cloning of custom tailored genes into mammalian cells, which in turn produce antibodies in unprecedented quality and reproducibility

Transient antibody expression in CHO (in vitro) even circumvents the questionable use of laboratory animals in contrast to in vivo antibody generation. If you make the Abs using a synthetic or human Ab library, then that eliminates the use of animals.

Thus the ability to generate recombinant antibodies overcomes most draw-backs of both polyclonal and monoclonal antibodies.

What are recombinant antibodies?

Recombinant antibodies (rAbs) show mono-specific binding to a single epitope, just like a monoclonal antibody, but their production begins at the genomic level and is entirely in vitro. Using molecular biology techniques, synthetic genes coding for the antibody of interest are designed and introduced into cell lines, thus avoiding the use of animals or hybridoma cells. 

Traditional monoclonal antibody production relies on the successful immunization of an animal to trigger the formation of antibody producing B cells. In contrast, recombinant antibody production circumvents immunization of animals, therefore expanding the scope of accessible antigens to even highly toxic materials. 

Recombinant antibody production

Recombinant antibody production relies on a process called antibody expression. The recombinant antibodies have to be produced in expression systems developed from mammalian cell lines rather than E. coli cells.

How are recombinant antibodies made?

Recombinant antibody production

Recombinant antibody production commences with the isolation of promising genetic material (nucleic acids) coding for antibody candidates or the use of a library of genes with randomized antigen binding site sequences for antibody engineering.

Promising genes are introduced into expression vectors that display the associated antibodies on their surface (antibody phage display library technique). In the so-called panning technique, such a bacteriophage library is exposed to an immobilized antigen, the weak binders can be washed away from strong binders, which adhere to the antigen unless special reagents are used. 

Repetition of this selection process with increasingly stringent conditions leaves only the strongest and most specific antibodies in the antibody library.

This in vitro process allows the manipulation of the genes to create new antibodies, reduce immunogenicity or even only select for antibody fragments (e.g., scfv or fab fragments).

Next, the most promising antibody genes are cloned into suitable cell lines that function as expression platforms. Since antibodies are rather complex proteins that undergo modifications after expression, higher cells (eukaryotic cells) are usually needed. Bacterial cells such as escherichia coli would not yield the desired antibody products. 

One example would be yeast cells, but best quality antibodies are produced with CHO (chinese hamster ovary) cells and HEK (human embryonic kidney) cells. As a result of methodology research and ongoing mammalian cell culture optimizations, the yields of human antibodies in these expression systems could be improved to over 12 g / liter, without the use of animals​1​.

Are animals needed to make recombinant antibodies?

One method for producing large numbers of identical antibodies is the hybridoma technology, which starts by injecting a mouse with an antigen to provoke an immune response.

In recombinant antibody production, no animals are needed. When using a synthetic or human Ab library, there is no need for an immunization of an animal. No mice or hamsters are used in the recombinant antibody production! It does not require any animals in the production process as cell lines cultured in a lab are used as the base material. 

Who can make recombinant antibodies?

Specialists in recombinant antibody production offer the necessary capacities and know-how for a fast and qualitative expression of antibodies. Therefore, many companies outsource their production to such a specialist.

To make a monoclonal antibody, researchers first have to identify the right antigen to attack. Some important determining factors for functional antibodies include:

  • effector functions
  • immune response 
  • long-term efficacy

In terms of cancer treatment, the process requires the isolation and characterization of human recombinant antibodies directed against the respective tumor cell. Recombinant antibodies have become the favored type, as they are easier and faster to produce than other types of monoclonal antibodies.​

Recombinant antibody advantages

Recombinant antibody technology circumnavigates the many disadvantages of conventional monoclonal antibody production, while offering several inherent advances in addition to the general expansion of the scope of applications.

1. Scalability

Once a recombinant antibody is established and hence the optimal genetic sequence is known, the incorporation of the gene into host cells is uncomplicated.

Further, the use of well-established expression platforms facilitates the upscaling of the necessary manufacturing processes, which are comparatively lean.

2. In vitro process – animal-free

Being an in vitro process entirely, recombinant antibody production is an agile technology that can switch between individual assignments on short notice. In contrast, the immunization of animals and subsequent generation of stable hybridoma cell lines is more time consuming.

Additionally, in vitro processes are more economical since they do not require as many resources or produce as much waste as animal using methods.

3. High-throughput production

The agility of recombinant technology allows the fast production of diverse antibodies. Conventional methods relying on animals and hybridoma cell lines show much more inertia and are not suitable for high-throughput at all.

4. Best quality

Since the underlying genetic material is easily optimized through the phage display method, recombinant antibodies can be designed to exhibit superior high affinity, sensitivity and specificity over traditional monoclonal antibodies.

5. Consistency and batch-to-batch reproducibility 

Hybridoma cell lines are prone to spontaneous mutations, thus leading to potential consistency issues between batches. Polyclonal antibodies are known for lower reproducibility, since each batch is derived from individual animals.

Production of recombinant antibodies relies on entirely defined and well-controlled genetic sequences and thus yields highly consistent antibody products. This process leads to very good reproducibility and validation between batches.

Interested in recombinant antibodies?

    Application of recombinant antibodies

    Recombinant antibodies are used in numerous applications in medicine and life sciences, such as:

    • Biologic therapeutics against cancer, autoimmune diseases, infections
    • Tracers in imaging methods (e.g., PET scans)
    • Diagnostic tools in lateral flow test kits, ELISA, and other immunoassays
    • Basic science tools in molecular biology (e.g., Western blots, immunofluorescence, flow cytometry, immunohistochemistry)

    Despite their profound impact on medicine and life sciences, traditional monoclonal antibodies (mAbs) have several serious disadvantages. Antibodies generated through the use of recombinant technology overcome those issues of traditional mAbs.

    Traditional antibody production suffers from longer development and production time scales, whereas recombinant antibody technology is faster and more reliable.

    Why are recombinant antibodies important?

    The scope of recombinant antibody applications is tremendous. The recent pandemic outbreak of SARS-CoV-2 virus had a big impact on everybody’s life. The strategies to fight the pandemic rely heavily on recombinant antibodies. 

    One example is the development of therapeutic antibodies that neutralize virus particles in infected patients. Recombinant antibodies that bind to surface proteins of SARS-CoV-2 are used in lateral flow test kits (“antigen tests”) to detect acute infections.

    A recent publication of Canadian scientists in the Journal of Molecular Biology​​2​ highlights the possibility for the synthesis of highly sensitive and specific reporter assays against SARS-CoV-2 virus particles using recombinant antibodies.

    They created a conjugate reporter system consisting of two halves of a luminescence enzyme. Each half is attached to an antibody via a peptide linker. The antibodies were developed to have affinity to regions of viral surface proteins using recombinant and phage display technologies.

    In the presence of the viral surface proteins, the antibodies bind to them, thus enabling the attached enzyme halves to reconstitute to a functional enzyme. The reporter system then emits light. This publication illuminates how rAbs can be rapidly developed into tools for important applications.​

    The benefits of afucosylation

    Natural antibodies not only bind their respective antigen, they highlight it to the immune system as harmful and mark it for neutralization. This effect is called antibody dependent cellular cytotoxicity (ADCC) and is of high significance in the development of therapeutic antibodies in oncology.

    The underlying signalling process involves the recognition of a chain of carbohydrates on the antibodies. Research has shown that the nature of the carbohydrates influences the severity of the ADCC and that afucosylated antibodies (i.e., lacking fucose in the carbohydrate chain) have a particularly positive effect on ADCC.

    Therefore, the ability to effect afucosylation of recombinant antibodies adds a whole new dimension to the scope of antibody technology. In addition to being able to develop highly specific antibodies to virtually any antigen through recombinant antibody technology, afucosylation allows the fine tuning of the strength of the immune system’s response to the antigen.

    This fact opens the door to developing more potent and more tolerable therapeutic antibodies.

    1. 1.
      Frenzel A, Hust M, Schirrmann T. Expression of Recombinant Antibodies. Front Immunol. Published online 2013. doi:10.3389/fimmu.2013.00217
    2. 2.
      Fellouse FA, Miersch S, Chen C, Michnick SW. Structure-based Design of a Specific, Homogeneous Luminescence Enzyme Reporter Assay for SARS-CoV-2. Journal of Molecular Biology. Published online June 2021:166983. doi:10.1016/j.jmb.2021.166983
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