Monoclonal antibody production: Process, Technologies & Steps

Monoclonal antibody production leads to substantial quantities of identical mAbs, designed to recognize and bind to specific cellular targets within the body. Biotechnological progress has led to the development of several approaches in monoclonal antibody production, meaning that there is more than one way how monoclonal antibodies are produced.This article gives an overview of the most popular and successful methods for monoclonal antibody production. From initial hybridoma production to phage display, single B-cell technology and recombinant production – we will explain monoclonal antibody production step by step.

Importance of monoclonal antibody production

The importance of monoclonal antibody production can be illustrated by a look at the plain figures: Over a hundred mAbs have been approved by the US FDA for human use in immunology (autoimmune diseases such as rheumatoid arthritis), infectious diseases (e.g. against SARS-COV-2 “coronavirus”), and oncology (e.g. immunotherapies, Antibody-Dependent Cellular Cytotoxicity (ADCC) abs and antibody-drug conjugates). 

With thousands of ongoing preclinical and clinical trials, antibody-based biopharmaceuticals are one of the best-selling classes of biomolecules in today’s market. The global antibody therapies market has rapidly expanded and is estimated to reach 638 billion US dollars in 2032 with a compound annual growth rate of 11.8% from 2023 to 2032¹. There are currently three methods in use for human monoclonal antibody production: 

There are currently three methods in use for human monoclonal antibody production: 

• hybridoma technology
• phage display technology
• single B-cell technology

Each method has its advantages and disadvantages and is chosen based on the specific requirements on monoclonal antibody production. In the next section, we will take a closer look at the technologies. To learn more about antibody production in general, read our articles: antibody production and in vitro antibody production.

In brief – the difference between polyclonal antibodies and monoclonal antibodies

One of the key differences between monoclonal antibodies and polyclonal antibodies is given away by their names – by the suffixes “mono-” and “poly-”, to be precise. While polyclonal antibodies are a set of genetically varying antibodies, mAbs have been cultured from genetically identical cells.
While groups of polyclonal and monoclonal antibodies may both be directed towards the same antigen, pAbs will bind to different epitopes on that antigen. The genetically identical mAbs, on the other hand, will bind to one specific epitope, depending on the exact antibody candidate that has been opted for in mAb development.

Monoclonal antibody production: Technologies in the production process

In the following chapters, we will provide an overview of technologies that have proven viable approaches in monoclonal antibody production.

Hybridoma technique in the production of monoclonal antibodies

MAbs were initially generated in mice using hybridoma technology. Invented by Georges Kohler and Cesar Milstein in 1975², this involved immunizing animals with the antigen of interest, followed by the fusion of specific B lymphocytes (most commonly obtained from the spleen of the animal) with immortalized myeloma cells. This results in the generation of hybrid cells — hybridomas —cloned to obtain stable monoclonal cell lines. A large-scale production culture to produce the desired quantity of the antibody can be achieved after selecting antibody-secreting clones of interest. Smaller-scale cell culture production involves cell culture flasks, and scaling up necessitates the use of bioreactors. The type of bioreactor depends on the cell type, monoclonal antibody titre required, and associated production cost. 

Hybridoma technology has the advantage of producing highly pure and specific antibodies using a reproducible and scalable method, with unlimited production of mAbs possible using the in vitro method. The challenges associated with hybridoma technology are the time-consuming nature of the method and the high costs and effort required for production. Other considerations are the potential immunogenic responses that may arise due to the mouse origin of the antibodies and the low viable efficiency of cells, with more than 99% of cells dying during the cell fusion step³. 

Advances in hybridoma technology have involved chimerization and humanization. Chimerization consists of replacing the constant segment of mouse protein with a segment of human IgG, achieved by transfecting mouse myeloma cells with chimeric genes. Here, selected leads identified after the screening step in hybridoma technology are used for chimeric or humanized monoclonal antibody production. By doing so, the chimeric antibodies reduce immunogenicity — meaning side effects due to being recognized as foreign by the patient’s immune system.
Humanization goes a step further in overcoming in vivo tolerance in humans as the only mouse-specific sequences are the complementary determining regions of the variable regions — which define the antigen-binding functionality. Many antibodies approved by the United States Food and Drug Administration are generated by hybridoma technology and employ either chimeric or humanized versions⁴. An example of a therapeutic chimeric antibody is Remicade (infliximab), used to treat rheumatoid arthritis (RA). The first humanized mAb was daclizumab, which is an anti-CD25 mAb approved for preventing transplant rejection.

Phage display in mAb production

Initially developed in 1990, phage display technology is a powerful method used to generate mAbs⁵. Here, a collection of ab fragment-displaying phages — known as the combinatorial antibody phage library — is used to screen and identify the antigen of interest. The first steps in the process involve cloning antibody gene fragments into vectors. Filamentous phage and phagemids are employed as vectors. M13 is an example of a filamentous phage that encodes all the genes for assembling structural viral proteins, and the ab of interest can be displayed on the surface of the phage by fusion with the phage coat proteins. Phagemids, on the other hand, require helper phage to produce functional virion phage particles. 

In both cases, a phage is generated using vectors to transform the E. coli host cell. This is followed by a selection step known as biopanning, where the phage library is exposed to the target antigen by removing non-binders via washing steps. Elution steps then involve either lowering the pH or competitive elution. Typically, multiple rounds of biopanning are performed to ensure the development of full-length human antibody constructs with strong, specific interactions with the target epitope. The resulting construct is then introduced into suitable mammalian cells that produce the desired antibody therapies.

The advantages of phage technology include the availability of commercially available phage libraries and the possibility of redesigning natural complementarity-determining regions — the antibody loops that make up the antigen binding site — for improved specificity and affinity. The disadvantage of the technology is that it can be more expensive than generating hybridomas after animal immunization, although the subsequent selection steps are quicker and cheaper with phage display technology⁶. The first therapeutic phage display antibody produced was adalimumab (Humira®), which was approved by the U.S. Food and Drugs Administration in 2002 for severe rheumatoid arthritis. 

Single B-cell technologies in the production of monoclonal antibodies

Single B-cell antibody technology represents another significant method to produce monoclonal antibodies, which has enabled the isolation and rapid production of highly specific mAbs from individual B-cells. This technology has been particularly instrumental in infectious diseases and cancer for developing neutralizing antibodies.

The first step in the technology involves the screening and isolation of the antibody-secreting cells from peripheral blood or lymphoid tissue samples, which can be performed in a random or antigen-specific manner. The random approach includes micromanipulation, laser capture microdissection and fluorescence-activated cell sorting (FACS). Conversely, the antigen-specific approach necessitates an extensive workflow to sort antigen-specific B-cells from a wider pool of B-cells. Techniques used include antigen-coated magnetic beads, fluorochrome-labelled antigens via multi-parameter FACS, the hemolytic plaque assay and a fluorescent foci method⁷.

Following isolation, antibody amino acids are amplified using reverse transcription-PCR (RT-PCR). Promising genes are then cloned and expressed in mammalian cell lines to produce a first set of antibody candidates to screen and characterize their expression properties. The hits are then introduced into mammalian cell cultures to produce fully human mAbs.The advantage of single B-cell antibody technology is that compared to hybridoma technology, it is highly efficient in obtaining specific mAbs combined with there being no need to euthanize animals. In addition, native mAbs with natural cognate VH and VL pairing are preserved⁶. The disadvantages of the technique are the high price of the associated equipment (e.g. single-cell sorting devices), the fact that RT-PCR procedures are challenging, and monoclonal antibodies targeting B-cell markers are not available for all species⁶.

Production steps in monoclonal antibody development

The production of monoclonal antibodies involves several key steps: immunization, hybridoma production, screening and cloning, and monoclonal antibody purification. Let’s take a closer look at these steps based on the production of monoclonal antibodies from hybridomas.

1. Immunization: Injection of an animal with the antigen of interest. The animal’s immune system will then recognize the antigen as foreign and produce an immune response. This immune response will result in the production of B-cells that produce polyclonal antibodies against the antigen.
2. Cell fusion: B-cells are extracted from the animal’s spleen and fused with myeloma cells to create hybridoma cells after immunization. Myeloma cells are a type of cancerous B-cell that can be grown indefinitely in the lab. The hybridoma cells created through fusion will have the ability to produce large amounts of antibodies.
3. Screening: Hybridoma cells are screened to identify the cells that produce the desired antibody. This characterization is done using a technique called enzyme-linked immunosorbent assay (ELISA). ELISA involves coating a plate with the antigen of interest and then adding the hybridoma cells. If a hybridoma cell produces the desired antibody, its receptors will bind to the antigen on the plate, and the antibody can be detected using a secondary antibody.
4. Cloning: Once the hybridoma cells that produce the desired antibody are identified, they are cloned. Cloning involves isolating a single cell and allowing it to divide and grow into a population of identical cells.

Monoclonal antibody purification: This final step in monoclonal antibody production involves separating the mAbs from other proteins and reagents in the cell culture. This is typically done using a combination of chromatography techniques. At evitria, for instance, we offer techniques like affinity chromatography using protein A and other resins, as well as specialized columns and protein polishing methods, such as size exclusion chromatography and ion exchange chromatography. After that, the mAbs are ready for the formulation of the antibody treatment of interest.

Challenges & Limitations of the monoclonal antibody production process

Despite their numerous applications and advantages, monoclonal antibodies have challenges and limitations. Critical steps in monoclonal antibody development must be managed carefully to achieve high-quality immunoglobulins in the desired quantity. 

Here are some of the significant challenges and limitations associated with the production and use of monoclonal antibodies:

• Production challenges: The production of monoclonal antibodies can be complex and expensive, requiring specialized equipment and expertise. In addition, the production process may be time-consuming, taking several months to generate a suitable monoclonal antibody.
• Immunogenicity: Monoclonal antibodies can elicit an immune response in the patient, developing anti-drug antibodies (ADAs). ADAs can reduce the efficacy of the monoclonal antibody and may cause adverse effects.
• Specificity and cross-reactivity: While monoclonal antibodies are highly specific to their target antigen, they may also cross-react with other antigens, leading to false positive results or unintended side effects.
• Tumor heterogeneity: In cancer therapy, monoclonal antibodies may not be effective against all tumor cells due to the heterogeneity of the tumor microenvironment and the presence of resistant cell populations.
• Cost: Monoclonal antibody therapies can be expensive, limiting access for some patients and healthcare systems. 

For this reason, various organizations, including academic institutions, biotechnology and pharmaceutical companies, prefer to externalize mAb production to develop diagnostics and therapeutics. 

To learn more about the topic, you can read the article Manufacturers of Monoclonal Antibodies

Recombinant monoclonal antibody production

Recombinant monoclonal antibody production is an alternative method for producing monoclonal antibodies that involves genetic engineering monoclonal antibodies and transcribing genes to create highly specific and functional recombinant antibodies. Unlike traditional monoclonal antibody production methods, recombinant monoclonal antibody production does not require the use of animals for antibody production, and it can produce large quantities of highly specific antibodies in a shorter time frame.

The process of recombinant antibody production involves the isolation and identification of the variable regions of the antibody genes, which are responsible for the antibody’s antigen-binding specificity. These variable regions are then inserted into expression vectors, which are used to produce large quantities of recombinant antibodies in mammalian or bacterial cell culture systems.

One of the significant advantages of recombinant antibody production is the ability to engineer and modify antibodies to enhance their therapeutic properties, such as increased binding affinity, improved pharmacokinetics, and reduced immunogenicity. 

In addition to these advantages, recombinant antibody production can reduce the variability associated with traditional monoclonal antibody production methods, as recombinant antibodies are produced using a standardised and controlled manufacturing process. This can lead to more consistent and reliable antibody products, which can be important for clinical applications.evitria excels at the custom manufacture of recombinant antibodies. Earlier this year, evitria contributed to a publication in JID Innovations by Numab Therapeutics AG in Switzerland and Kaken Pharmaceutical in Tokyo to produce therapeutic antibodies for atopic dermatitis (AD)⁸. AD is a T-helper 2 cell–driven chronic skin disease characterized by systemic inflammation, barrier dysfunction, and persistent itching symptoms. Treatment of AD involves inhibition of IL-4/IL-13 signalling with dupilumab. However, clinical responses are slow in many patients and remain modest, as some symptoms are dependent on IL-31, which is only partially reduced by IL-4/IL-13 inhibition. Using recombinant antibody technology, evitria employed published sequence data to produce the antibody dupilumab (an anti-IL-4R antibody) and an anti-IL31 antibody — BMS-981164. The study compared the concomitant use of dupilumab and BMS-981164 with a bispecific tetravalent antibody. The tested tetravalent antibody (NM26-2198) demonstrated comparable potency to the combination therapy and reduced the troubling itching symptoms, which reduced inflammation and improved the quality of life for patients

Recombinant antibody expression with evitria

Evitria offers a service for the production of recombinant monoclonal antibodies using our CHO cell expression system. We provide customized solutions for the production of high-quality antibodies in large quantities. As one of the leading recombinant antibody expression service providers, we have generated over 20,000 antibodies for customers globally. With our expertise and experience in the field of antibody engineering, we find customized solutions to meet your needs in custom antibody production.

References

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4. Parray, H. A. et al. Hybridoma technology is a versatile method for the isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives. International Immunopharmacology vol. 85 Preprint at https://doi.org/10.1016/j.intimp.2020.106639 (2020).

5. McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, (1990).

6. Moraes, J. Z. et al. Hybridoma technology: is it still useful? Current Research in Immunology vol. 2 Preprint at https://doi.org/10.1016/j.crimmu.2021.03.002 (2021).

7. Tiller, T. Single B cell antibody technologies. New Biotechnology vol. 28 Preprint at https://doi.org/10.1016/j.nbt.2011.03.014 (2011).8. Tietz, J. et al. A Bispecific, Tetravalent Antibody Targeting Inflammatory and Pruritogenic Pathways in Atopic Dermatitis. JID Innov4, (2024).