Antibodies are indispensable tools in biomedicine. Their high specificity and low off-target effects make them amenable to wide-ranging applications in biotechnology, such as diagnostics, therapeutics, and research.
This guide is designed to provide you with a comprehensive understanding of the fundamental aspects involved in antibody production techniques. We will also introduce you to dedicated antibody expression services, making this guide a practical resource for your work.
Antibody structure and function
Antibody production plays a critical function in the immune system’s defense mechanism in response to the presence of foreign substances, such as pathogens or antigens. These large, Y-shaped proteins belong to the immunoglobulin superfamily and are responsible for recognizing and binding to specific antigens, marking them for destruction or neutralization.
Antibodies are glycoproteins produced by B lymphocytes and consist of two structural units: heavy and light chains. The variable region is the N-terminal end, which consists of approximately one hundred amino acids of the light and heavy chains and is responsible for antigen recognition.
In mammalian cells, there are five main classes — or isotypes — of antibodies. Classes are defined by the type of mammalian Ig heavy chains, α, δ, ε, γ and μ, which are found in IgA, IgD, IgE, IgG antibodies, and IgM antibodies, respectively.
Read more on: What is an antibody?
History of antibody production
The potential use of antibodies was first recognized in 1890 by Dr Emil von Behring and Shibasabura Kitasato. They demonstrated that serum transfer from animals immunized against diphtheria could be used to cure other animals suffering from the same disease. This was an example of the powerful applications of polyclonal antibodies.
This landmark discovery paved the way for the development of antibody drugs and vaccines, a testament to the transformative power of antibody production. It was later, in 1897, that Paul Ehrlich described his “side-chain model”, which laid the foundation for work by others to define the specific nature of the binding site of the antibody to an antigen.
In the 1970s, Köhler and Milstein published their findings on how to generate large quantities of antibodies (secreted by immortalized hybridomas) of a predefined specificity.
This marked a big advancement in immunology, as monoclonal antibodies showed less cross-reactivity than polyclonal antibodies, an important advantage in the production of such therapies.
The first monoclonal antibody designed for therapeutic use in humans was approved by the U.S. Food and Drug Administration (FDA) in 1986. Orthoclone OKT3® (muromonab-CD3) targeted the CD3 receptors on the surface of T-cells and was used to reduce acute rejection in organ transplant patients.
Since then, monoclonal antibody development has helped to target specific mutations and defects in protein structure and protein expression in many diseases, including cancer, chronic inflammatory diseases, infectious diseases, and cardiovascular diseases.
New developments in recombinant engineering technologies and genetic sequencing made it possible to produce chimeric, humanized and fully human monoclonal antibodies at a rapid rate. Today, the antibody market is one of the fastest growing markets in medicine, with products like recombinant or bispecific antibodies opening entirely new possibilities for the life sciences sector.
Types of antibody production
Different types of antibodies continue to dominate biotherapeutics, diagnostics and research today.
Polyclonal antibody production
Polyclonal antibodies are a heterogeneous mix of antibodies produced by different B cell clones in the body. Polyclonal antibody production involves injecting a target antigen into experimental animals (including rabbits, goats, horses, and hamsters), stimulating their immune system to produce a diverse array of antibodies as an antibody response.
This immune response results in a mixture of antibodies, each targeting different epitopes on the antigen. After a certain period of time, blood is collected from the animal, and serum containing these polyclonal antibodies is isolated and further processed.
Polyclonal antibodies can be produced quickly, cheaply, and on a large scale. They can also be highly specific and sensitive for detecting low-quantity proteins, which lends them well to diagnostic and research applications (e.g. capture antibody in sandwich ELISA). However, they are prone to batch-to-batch variability and have a higher propensity for cross-reactivity due to the recognition of multiple epitopes in the immunogen sequence.
Read more on polyclonal antibody production
Monoclonal antibody production
Monoclonal antibody production entails generating identical antibodies, each designed to bind to a single epitope on an antigen with high affinity. There are different technologies on the market feasible for monoclonal antibody production.
A summary of the steps to the antibody production process employing the hybridoma method includes purification and formation of an immunogen, immunization of animal cells, isolation of B lymphocytes, cell fusion, cloning screening and monoclonal antibody production.
Learn more on monoclonal antibody production and the process steps: Steps in antibody production
Monoclonal antibodies can be produced by in vivo and in vitro methods. In vivo methods of production of monoclonal antibodies involve injecting hybridoma cells into the intraperitoneal cavity of mice. This results in the accumulation of ascites fluid that contains the antibodies.
Hybridoma-based strategies have the advantage of being well-characterized and of low complexity. However, they suffer from disadvantages such as the need for experimental animals (typically mice) and the low efficiency of B lymphocyte-myeloma cell fusion step.1
In Europe, the ascites method was banned in 1998 and followed in 2020 by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) recommendations for the use of non-animal methods when alternative methods are available to prevent distress caused to animals2.
The in vitro method involves the culture of hybridoma cells. This method precludes the need for animal use and laboratory personnel experienced in animal handling, as well as conformity to frameworks for research ethics. The drawbacks of the in vitro method are some hybridomas do not grow well or are lost in culture as well as are more costly3.
The ethical concerns and the desire for alternative methods have driven the exploration of in vitro techniques and recombinant antibody production.
Recombinant antibody production
Recombinant antibody production employs genetic engineering to create specific antibodies. First, antibody genes are isolated and cloned into expression vectors. These vectors are then introduced into host cells, such as bacteria or mammalian cells. The host cells produce the recombinant antibodies, which can be harvested and purified.
The process of recombinant antibody production overcomes the limitations of hybridoma technology, which can undergo genetic drift that can lead to batch-to-batch variability. Recombinant technology also circumvents the need for costly long-term storage involving liquid nitrogen tanks, which carry the risk of cell lines dying or losing the ability to secrete antibodies.
Recombinant antibody discovery platforms
Recombinant antibody discovery platforms include antibody phage display and the single B-cell antibody technology.
Phage display
Phage display enables researchers to recognize recombinant monoclonal antibodies against antigens faster and without the help of assays involving animal immunization. Here, genetically engineered bacteriophages display antibody fragments, enabling targeted antibody selection and optimization without animal immunization.
The first step is to construct a phage display library by cloning antibody gene fragments into vectors. Antibody sequencing information is first acquired from B-cells from the spleen, peripheral blood mononuclear cells (PBMCs) and bone marrow.
Different libraries exist, including immune libraries derived from human donors who have received vaccination or are suffering from a disease. Universal libraries are generated from natural naïve human antibodies. The antibody — in the single-chain variable fragment (scFv) or antigen-binding fragment (Fab) forms —is fused to a phage coat protein, which is expressed on the surface of the filamentous phage.
Antibodies from the phage libraries are selected by assessing binding to the immobilized antigen and subsequent washing and elution steps. This biopanning technique has multiple rounds consisting of repetition of the above steps to ensure the enrichment of specific phage binders. Finally, the collected phage can be identified by DNA sequencing.
Single B-cell antibody technology
This powerful technique can generate monoclonal antibodies from humans and immunized animals. Following isolation, B cells [from splenocytes and Human peripheral blood mononuclear cells (PBMCs)] are sorted using FACS. Extraction of mRNA is undertaken from B cells that are either lysed directly or cultured to obtain RNA.
The construction of complementary DNA (cDNA) from the single B cells allows the analysis of expressed IgH and IgL chain genes. The variable regions are cloned into vectors for recombinant production of antibodies. Screening of antibodies allows the determination of the reactivity profile and biophysical characteristics of antibodies or fragments.
The choice of the ideal antibody production method depends on various factors, as each technique has unique advantages and disadvantages. Below, we will examine some of them in more detail.
Summary of advantages and disadvantages of monoclonal antibody generation strategies
Adapted from1
| Technology | Pros | Cons |
| Original hybridoma | – Preserves the native pairing of variable and constant regions gene combination – Antibodies undergo in vivo affinity maturation | – Hybridoma cell lines may be genetically unstable – High technical expertise is needed |
| Antibody phage display | – Animal host is not required. – Screening of large number of clones increases chances of generating effective mAb | – Antibody formats are limited to ScFv and Fab, which lack initiation of effector functions – Phage display library construction can be expensive |
| Single B cell | – High efficiency in obtaining specific mAbs, compared to hybridoma technology – Isolation of native mAbs with the preservation of natural cognate VH and VL pairing | – Antibodies targeting B cell markers are not available for all species, which are needed for cell sorting – RT-PCR procedures might be -challenging |
Read more on recombinant antibody production
Recombinant expression systems
Bacterial cell expression
While microbial cells like yeast and bacteria are not able to process glycosylation as needed for full-length mAb production, they have proven very valuable in the production of smaller biologics, including single-domain antibodies and non-glycosylated antibody fragments. E. coli is a popular host for the expression of commercially successful antibody-fragment products like Lucentis and Cimzia.
E. coli can easily take up foreign DNA material, mainly plasmids, and provides high-level protein expression and scalable production. Moreover, antibody fragments expressed in E. coli hosts are also suitable for phage display. Advantages over yeast or mammalian cells are their fast growth ability, uncomplicated gene manipulation, and inexpensive cultivation.
Read more: Antibody production in bacteria
Mammalian cell expression
Most cells used in antibody production today are mammalian cells due to their ability to perform protein folding and post-translational modifications necessary to guarantee efficient and secure therapeutic antibodies. Two of the most commonly used host cells are Chinese hamster ovary (CHO) cells and human embryonic kidney cells (HEK cells). It is comparably easy to grow both cell types in suspension cultures, and they have shown high efficiency in the antibody production process.
CHO cells have proven to be one of the most valuable mammalian cell lines in the research and production of antibodies. At evitria, CHO cells are used because their outstanding qualities make them the best choice in recombinant antibody production. Through the improvement of cell culture conditions and post-translational modifications, it became possible to produce antibodies more safely with fewer risks of contamination.
Read more: Antibody production in CHO cells
Steps following antibody production
Whether antibodies are produced using in vivo or in vitro procedures, purification and characterization of antibodies remain vital process steps.
Antibody purification
After the antibody has been produced, antibody purification is needed to isolate it from the serum or culture supernatant of hybridoma cells. Different chromatographic methods, ranging in complexity, are used. The chosen method depends on the desired specificity of the produced antibody.
Read more: Antibody purification
Antibody characterization
Antibody characterization is achieved through different methods throughout production and purification. The specificity of an antibody is identified through screening batches for their binding activity, often using ELISA. The concentration of antibodies is assessed through titering. Isotyping describes the process of determining a monoclonal antibody’s class and identity.
Read more: Antibody characterization
Challenges in Antibody Production
Antibody production poses several challenges to ensure efficient and reliable production. Variability in antibody yields and quality, arising from cell line heterogeneity, culture conditions, and purification techniques, needs to be managed to ensure consistency in manufacture. Optimizing production timelines and costs is another challenge, requiring a balance between high yields, quality, and cost-effectiveness.
Scaling up production from laboratory to industrial scale presents its own difficulties, demanding careful optimization of culture conditions and purification methods. Ethical considerations surrounding animal use in antibody production drive the need for alternative methods without compromising quantity or quality.
Optimization of Antibody Production
Antibody production challenges can be effectively addressed using recombinant antibody production techniques, particularly by employing Chinese Hamster Ovarian (CHO) cells. Recombinant antibody production with CHO cells offers a scalable and reliable platform for efficient, stable and high-throughput antibody production with low batch-to-batch variability.
It enables fast production timelines, from laboratory to industrial scale, optimizing culture conditions and purification methods. Moreover, it addresses ethical considerations by providing an alternative to animal-based methods without compromising quantity or quality.
However, the choice of production technology depends on the specific needs of the antibody. Different antibody formats and characteristics may require alternative approaches. Researchers must carefully evaluate the requirements and select the appropriate production technology to ensure optimal results. Continual advancement in innovative strategies and technologies, particularly in recombinant antibody production using CHO cells, will play a crucial role in overcoming these challenges and driving the field forward.
Antibody production with evitria AG
As a producer of recombinant antibodies, we at evitria AG understand the importance of addressing these challenges and are committed to providing high-quality recombinant antibodies to meet the diverse needs of the scientific community.
Through continuous innovation and collaboration, we strive to contribute to advancements in antibody production and support research and development in various fields.
- 1.Moraes JZ, Hamaguchi B, Braggion C, et al. Hybridoma technology: is it still useful? Current Research in Immunology. Published online 2021:32-40. doi:10.1016/j.crimmu.2021.03.002
- 2.EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). European Commission. Published 2020. https://joint-research-centre.ec.europa.eu/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam_en
- 3.Monoclonal Antibody Production. National Academies Press; 1999. doi:10.17226/9450
