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:
- 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.
| Method | Principle | Origin | Advantages | Limitations |
|---|---|---|---|---|
| Hybridoma (since 1975) | Immunization of an animal, fusion of B lymphocytes with immortalized myeloma cells, cloning of stable lines | Murine, chimerized or humanized in later steps | Highly pure and specific antibodies, reproducible and scalable, unlimited production in vitro | Time-consuming, high cost and effort, immunogenicity from murine origin, over 99% cell loss during fusion |
| Phage display (since 1990) | Combinatorial phage library screened against the antigen by biopanning, construct expressed in mammalian cells | Fully human constructs | Commercial libraries available, CDRs can be redesigned for affinity and specificity, selection steps quick and inexpensive | More expensive upfront than generating hybridomas |
| Single B-cell | Isolation of antibody-secreting cells from blood or lymphoid tissue, RT-PCR amplification, expression in mammalian cell lines | Fully human, native VH/VL pairing preserved | Highly efficient, no animal euthanasia required, native cognate pairing preserved | Costly equipment, demanding RT-PCR, B-cell marker antibodies unavailable for some species |
Hybridoma technique
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.
In summary, hybridoma production runs through five stages, each of which requires careful management:
- Injection of a pathogen or antigen and subsequent animal immunization
- Harvest of spleen cells and extraction of B lymphocytes
- Fusion with myeloma cells and selection of hybridoma cell lines
- Antibody isolation
- Continuous validation of hybridoma cell culture and antibody characterization, for example by ELISA
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.
A further constraint is genetic stability. The obtained hybridoma cells must be continuously validated because they are prone to spontaneous genetic mutations. These mutations can cause the cells to secrete undesired antibody variants or lose antibody expression capability entirely, a risk that increases over time and with successive passage cycles.
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.
Beyond cost, phage display carries two further constraints. A fundamental first antibody library can be obtained from blood donations from human donors, but relying on a single donor introduces a bias to the library due to individual immune system variability. The method also requires relatively large amounts of antigen, immobilized on suitable surfaces for in vitro selection, which can be a limiting factor when working with difficult target classes such as membrane proteins.
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.
In practice, the workflow introduces its own bottlenecks: design of suitable primers for RT-PCR sequencing, antigen reagent design for labeling, and the configuration of sorting parameters for flow cytometry. Together with the cost of single-cell sorting equipment, this limits accessibility, particularly for smaller research groups.
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.
- 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.
- 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.
- 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.
- 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.
- 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 in monoclonal antibody production
Monoclonal antibody production is a complex, sensitive process that involves several intermingled aspects of biochemistry, molecular biology, and cell biology. With over 100 mAbs approved by the FDA and more than 1,500 clinical trials ongoing in the United States alone, the development challenges that accompany each method are well documented and must be actively managed.
Antigen immunogenicity
When using animals to generate neutralizing antibodies, the antigen necessarily must provoke a sufficient immune response to raise antibody-producing B cells. Some antigens are inherently less immunogenic, especially naturally occurring proteins such as receptors and enzymes. Suitable approaches include switching to a more responsive host species, or redesigning the antigen with added structural features to increase immunogenicity.
A related challenge arises with antigens that are inherently toxic to the organism: attempted immunization can lead to severe harmful effects in the host animal. Switching to alternative species may help, but in vivo antibody generation remains fundamentally problematic for highly toxic antigens. In vitro methods such as phage display offer a route around this constraint.
Immunogenicity in patients
Using antibodies from non-human animals carries the risk of immunogenicity in patients: the patient’s immune system may recognize the antibody as foreign, generating anti-drug antibodies (ADAs). ADAs can reduce the efficacy of the therapeutic and cause adverse effects.
This is why chimerization and humanization steps are required during development for hybridoma-derived mAbs, and why fully human production methods, namely phage display, single B-cell technology, and recombinant production, have become increasingly preferred.
Specificity, cross-reactivity, and tumor heterogeneity
Monoclonal antibodies are highly specific to their target antigen, but can still cross-react with structurally similar antigens, leading to false positive results or unintended side effects. In oncology, mAbs may not be effective against all tumor cells due to the heterogeneity of the tumor microenvironment and the presence of resistant cell populations. This limitation has driven interest in bispecific and multispecific antibody formats.
Cost and production complexity
The production of monoclonal antibodies can be complex and expensive, requiring specialized equipment and expertise. Traditional timelines often run to six months or more, and that cost carries through to the finished therapeutic, which can limit 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 specialized service providers.
For a closer look at who supplies mAbs at scale, see 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 — most commonly CHO cells.
Key advantages of recombinant antibody production include:
- Sequence-level engineering: Antibodies can be modified to enhance binding affinity, improve pharmacokinetics, and reduce immunogenicity
- Reduced variability: Recombinant antibodies are produced via a standardized, controlled manufacturing process — eliminating the mutation risk inherent in hybridoma cell lines
- Advanced formats: Bispecific antibodies, Fc-silenced antibodies, and afucosylated formats are accessible only through recombinant engineering
- Speed: evitria’s CHO-based transient expression platform delivers purified recombinant antibodies in as little as 4 weeks from sequence
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 produces recombinant monoclonal antibodies in CHO cells, from sequence to purified material. Over 15+ years and more than 120,000 transfections, we have built our process around reproducibility: defined sequences, consistent batches, and no hybridoma drift. Antibodies are produced for preclinical research, at scales from screening quantities to gram amounts.
FAQs on monoclonal antibody production
Monoclonal antibody production typically begins with immunizing an animal with a specific antigen. The animal’s immune system generates B cells that produce antibodies against the antigen. These B cells are fused with myeloma cells to create hybridomas, which are capable of both producing antibodies and dividing indefinitely. The resulting hybridomas are screened using ELISA to identify the cells producing the desired antibody, then cloned and expanded. The final product is purified using chromatography techniques. Alternatively, phage display, single B-cell technology, or recombinant production can be used — the latter without any animals.
There are four main methods: hybridoma technology (fusion of B cells with myeloma cells), phage display technology (screening antibody fragment libraries displayed on bacteriophages), single B-cell technology (isolating and sequencing individual antigen-specific B cells), and recombinant antibody production (genetic engineering of antibody sequences for cell culture expression without animals).
The main development challenges include: generating a sufficient immune response against poorly immunogenic antigens; managing immunogenicity risk in patients (requiring chimerization or humanization for hybridoma-derived mAbs); the ongoing mutation risk in hybridoma cell lines; the technical demands of phage display library construction and biopanning; and the cost and complexity of single B-cell sorting workflows. Recombinant production methods address several of these challenges by moving the process entirely in vitro.
Fully human antibodies are produced using phage display technology (screening a library derived from human donor B cells), single B-cell technology (isolating and cloning antibody genes from individual human B cells), or recombinant antibody production (engineering defined human antibody sequences for CHO cell expression).
The key steps are:
1. immunization (generating an immune response in an animal or identifying sequences in vitro)
2. cell fusion or gene cloning
3. screening (ELISA or equivalent) to identify the desired antibody
4. cloning to establish a stable producing cell line
5. purification using chromatography to yield pharmaceutical-grade antibody
Traditional hybridoma-based production typically takes 6 to 12 months. At evitria, recombinant monoclonal antibodies are delivered within 5 weeks from sequence to purified antibody.
The steps are:
1. immunization with the target antigen; extraction of B cells and cell fusion with myeloma cells
2. screening hybridomas using ELISA
3. cloning of selected hybridoma cells
4. large-scale culture
5. purification by chromatography
For recombinant production, the immunization and fusion steps are replaced by sequence design, gene synthesis, vector cloning, and transient transfection into CHO cells.
Sources
1. Global Market Insights. Antibody Therapy Market – By Type [Monoclonal Antibodies (MAbs) {Oncology, Autoimmune Disease, Infectious Disease}, Antibody-Drug Conjugates (ADCs)], By End-Use (Hospitals, Specialty Centers) – Global Forecast, 2023 – 2032. https://www.gminsights.com/industry-analysis/antibody-therapy-market (2023).
2. Köhler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, (1975).
3. Mitra, S. & Tomar, P. C. Hybridoma technology; advancements, clinical significance, and future aspects. Journal of Genetic Engineering and Biotechnology vol. 19 Preprint at https://doi.org/10.1186/s43141-021-00264-6 (2021).
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).