The use of antibodies for medical procedures has been gaining immense popularity recently. The word “antibody” is probably because of their numerous applications in vaccination, blood typing, and immunology. This article will discuss the basic concepts behind recombinant antibody expression and how they are used in medical procedures. We will also explain how these procedures work and give tips to help you get the most out of this technique.

What are Antibodies?

Antibodies (Abs) belong to the immunoglobulins protein family (Igs). Antibodies are essential immune system components, generated spontaneously by lymphocytes in response to infection by microorganisms such as bacteria or viruses. Immunoglobulin G is the most widespread antibody generated by humans (IgG). Antigens refer to the molecules that antibodies detect and attach to. The interaction between antibodies and antigens is very selective and potent.

Due to the strong affinity and specificity between antibodies and antigens, Abs have also been created as crucial research tools that are widely employed. In 1941, Albert Coons, Hugh Creech, and Norman Jones from Harvard Medical School and Harvard University published the first publication detailing the use of antibodies in scientific studies. Anti-pneumococci type III rabbit antibody tagged with a fluorescent dye was evaluated for tissue immunostaining.

Since those early days, the use of antibodies in research has expanded to encompass various tests.

In current research and therapeutic uses, monoclonals, polyclonals, and recombinant antibodies are the most common forms of antibodies. Each antibody exhibits unique benefits and drawbacks depending on its use.

Polyclonal Vs. Monoclonal Antibodies

The names “polyclonal” and “monoclonal” are derived from the immune system’s antibody production process. When a foreign antigen attaches to a B-cell lymphocyte’s surface antigen receptor, the B-lymphocyte undergoes differentiation into memory B-cells and plasma cells, releasing antibodies specific to the provoking antigenic epitope.

What are recombinant proteins? Since antigens can range in size from tiny molecules to large recombinant proteins, a given antigen could contain several epitopes. Since many B-cells can detect the same antigen, the ensuing immune response consists of antibodies released by various B-cells that target distinct epitopes on the same antigen. This type of reaction is known as polyclonal.

Essentially, a polyclonal antibody is a collection of antibodies directed against a certain antigen by several B-cell clones.

Suppose, theoretically, that only one B-cell reacted to a specific antigen. Since this cell recognizes a single epitope, it will develop, divide, and release antibodies that are unique to this epitope. This is referred to as a monoclonal response.

Essentially, a monoclonal antibody is a collection of antibodies produced by a single B-cell clone and is specific to a single antigenic epitope.

Commercial Production of Antibodies

In general, polyclonal antibodies are more accessible to generate than monoclonal antibodies. The basic workflow for producing a polyclonal antibody, which can take between two and four months, is as follows:

• Careful selection and processing of the antigen to achieve high purity.
• Choosing the host species (often a rabbit or mouse) based on the desired quantity of antibody and the species from which the antigen was extracted.
• If the antigen of interest is poorly immunogenic, an adjuvant should be used to increase the immune response.
• Immunization of an animal host with the desired antigen. The injection route (subcutaneous, intraperitoneal, intravascular, etc.) is determined by the antigen’s chemical composition and the host’s species.
• Antiserum collection once a significant immune response has been attained. Further purification processes are often required to improve the antiserum’s sensitivity and specificity.

In contrast, producing a monoclonal antibody is more arduous and specialized. The typical technique for producing a monoclonal antibody, which takes around six months (both polyclonal and monoclonal antibodies share the first four stages), is outlined below:

• Collecting B-cells that produce antibodies from the spleen or other lymphoid tissues.
• A “hybridoma” is created by the fusion of non-secreting B-cells with myeloma cells. This process immortalizes the B-cells, allowing them to withstand several in vitro passages.
• Screening/selection of successfully fused hybrids in HAT media, followed by enrichment of single clones through ELISA, immunohistochemistry, or flow cytometry.
• Cloning, expansion, and cryopreservation of monoclonal hybridomas. These cells may now be used to produce monoclonal antibodies.
• Recovering and purifying antibodies from the supernatant of a culture.

Another approach for producing monoclonal antibodies that have been taking the biotechnology industry by storm is the recent advancements in recombinant DNA technology. The process of recombinant antibody production includes cloning antibody gene libraries onto phage vectors and allowing the phages to infect a host cell line. The host cells will then generate daughter phages that display the recombinant antibodies on their surfaces. After selecting antibodies with the desired properties, the genes can be placed into an expression system, and antibodies can be generated at a large scale.

Advantages and Disadvantages of Polyclonal, Monoclonal, and Recombinant Antibodies

The decision between producing a polyclonal or monoclonal antibody depends on several criteria, including the cost, timeline, and planned application of the antibody by the end user.

The primary benefit of polyclonal antibodies is their low production cost and rapid turnaround time from antigen preparation to antibody collection. In addition to detecting several epitopes on an antigen, polyclonal antibodies have a higher overall affinity for their antigen and hence a better detection efficiency.

However, the heterogeneous character of polyclonal antibodies renders them more susceptible to batch-to-batch variation and cross-reactivity with other molecules, leading to a greater background. In general, polyclonal antibodies are better suitable for situations in which great sensitivity is desired and when antigens are present in small amounts or under non-native circumstances.

In general, monoclonal antibodies are more effective than polyclonal antibodies. For instance, homogeneity is maintained across batches since the antibodies are generated from a single B-cell clone, and cross-reactivity with other molecules is minimized because they only detect a single epitope per antigen.

Once generated, hybridomas also serve as a renewable supply of antibodies, an advantage over polyclonal antibodies, which depend on the host animal’s longevity. However, these functional gains come at a price: monoclonal antibodies are substantially more expensive to generate, need more specialized training to develop, and have a significantly longer turnaround time. When high antigen-specific specificity is required, for example, when monitoring changes in protein structure or phosphorylation status, monoclonal antibodies can be used in many ways as polyclonal antibodies.

In certain applications, monoclonal antibodies may be too specific, a limitation that can be circumvented by combining numerous monoclonal antibodies. When creating a therapeutic antibody, the specificity of monoclonal antibodies is desirable, as they are less likely to exhibit off-target binding.

The advantages and disadvantages of recombinant antibodies are identical to those of monoclonal antibodies. Like monoclonal antibodies, recombinant antibodies need substantial time and financial commitments to generate, at least initially. Once recombinant technologies are developed, antibodies can be made swiftly and without the requirement for animal hosts, which is a significant benefit of this technique. As recombinant antibody technologies continue to advance, they will replace conventional methods for producing monoclonal antibodies.

Recombinant Antibodies

What are recombinant antibodies? They are a form of monoclonal antibodies produced in vitro from synthetic gene synthesis or peptide synthesis without immunizing animals or growing hybridomas.

To comprehend the emergence of recombinant antibody technology, we must return to 1984, when Morrison SL. and Neuberger MS. cloned the Ig genes from hybridomas to alter them in vitro and express the first chimeric Abs. Morrison replaced the mouse Fc fragment with a human Fc fragment, and Neuberger made Abs in plasmacyte cell lines without hybridomas.

Any species of antibody-producing animals can be used to clone recombinant Abs. It is possible to alter the sequence once it has been cloned; this is one of the critical benefits of recombinant Abs. For instance, Fc fragments from one species can be exchanged with fragments from another. The variable chain on Fab fragments can be altered to vary their binding specificity or affinity.

Recombinant Abs offers various benefits over monoclonal monoclonals. The first is the technology’s excellent repeatability. Since the recombinant Ab gene sequence is known and has been cloned, they are more dependable and repeatable than monoclonal monoclonals. Their second advantage over monoclonal antibodies is the time required to manufacture recombinant antibodies. In contrast to hybridomas, which need several months to generate functional Abs, recombinant Abs technology often reduces production time to a few weeks.

Recently, gene synthesis, custom protein synthesis, and production of recombinant antibodies comprise animal-free processes. This is because the conditions and regulations for animal experimentation are becoming increasingly rigorous, and the production of recombinant Abs does not require animals.

Large-Scale Recombinant Antibodies Production

We can produce recombinant antibodies in various bacterial, yeast, and mammalian expression systems. Bacterial expression systems are attractive as they require fewer steps than mammalian expression systems, and they can also produce high yields of antibodies. However, bacterial expression systems are challenging to scale up and require specialized bacterial strains.

Moreover, bacterial antibodies are susceptible to proteolysis and are, therefore, of limited therapeutic value. Yeasts have been used to produce molecules for many years, and a new generation of yeast-based expression platforms has been developed in recent years. These platforms can produce high-quality antibodies with high yields at low costs.

Current limitations of recombinant antibodies

There are many advantages of antibodies produced via recombinant methods, including ease of production, reduced risk of immunogenicity, and robustness. However, antibodies produced via recombinant methods have limited therapeutic value due to several limitations.

• Low Affinity: The affinity of antibodies produced via recombinant methods is often low (10-100-fold lower than hybridoma antibodies). This low affinity is because the recombinant antibodies are produced not by human cells but by bacteria or yeast, which have different binding properties than humans. Consequently, these low-affinity antibodies do not perform as well in vivo.
• Proteolysis: Recombinant antibodies are highly susceptible to proteolysis; their therapeutic value is substantially reduced after intravenous administration due to the degradation of the molecules by macrophages. Therefore, these molecules are limited for immuno-oncology applications or for treating cancer patients with hypo-proliferative conditions, where macrophages are not a significant source of proteolysis.
• Low Quality: Another limitation of recombinant antibodies is their low purity. Typically, the purity of antibodies produced via recombinant methods ranges between 95-99%, and therefore antibodies produced through recombinant methods are unsuitable for use in diagnostic applications.

What is the importance of recombinant antibodies in custom gene synthesis?

The ability to generate fully human therapeutics will be crucial in advancing precision medicine. This is because humanized therapeutics will be less likely to cause an immune response in patients, improving treatment outcomes, reducing side effects, and reducing costs. The potential market for recombinant antibodies is expected to grow substantially in the coming years as the number of patients with cancer is predicted to rise.

Meanwhile, the cost of gene therapy will become increasingly important and accessible due to the higher prices of common cancer immunotherapies, such as checkpoint inhibitors. Custom-made therapeutic molecules are important because the global antibody market is expected to reach $13.7 billion by 2023, growing at a CAGR of 10.6% during the forecast period.

Final Thoughts – Recombinant Antibodies Are the Present and the Future

The history of the usage of antibodies in research and therapeutic contexts demonstrates the inventiveness of scientists in manipulating biological processes such as immunity for their ends. Custom antibody-based technologies have come a long way, from using full serum to the invention of recombinant Abs.

The production of polyclonal antibodies is simple and affordable, and their poly-binding capabilities make them excellent for identifying the three-dimensional conformation of proteins. However, polyclonals have an elevated risk of non-specific binding, need immunizing animals, and have significant lot-to-lot variability difficulties.

Due to their impressive history of effectiveness, polyclonal and monoclonal antibodies will continue to be employed for a considerable amount of time in various applications until recombinant Abs achieve the same degree of characterization and acceptance by the scientific community.

Recombinant chromosome and antibodies are excellent tools with great potential in research and therapeutic settings. The benefits of recombinant antibodies, such as complete control over their production, from Ab sequence to antibody/antigen binding, the engineering possibilities, and the absence of animal usage, make recombinant Abs highly desirable for research, diagnostic, and clinical uses.