Monoclonal antibodies are recognized for their safety, high specificity and binding affinity, and structural stability. Given their advantageous properties and structure, they are useful for a multitude of applications like therapy, diagnostics (in vitro and medical devices), and research (basic, clinical, environmental, etc.). Monoclonal antibody production is a complex process starting with discovery and concluding in large/medium/small scale production. On this page, we cover all the essential steps of the monoclonal antibody production process and the associated challenges and opportunities. We also provide answers to the most frequently asked questions about ProteoGenix’s monoclonal antibody discovery and production platforms.
Thanks to our expertise and optimized protocols we can use a very wide range of antigens for antibody discovery including small molecules, peptides, cells, DNA, and recombinant proteins.
We specialize in mouse hybridoma and phage display technologies (no species or format restriction). Our protocols are fully optimized for both technologies. Additionally, we have developed custom workflows that take into account the needs of our clients, project complexity, and desired timelines for completion, as well as, antigen complexity and final antibody application. Given our expertise in antibody development for different applications, we ensure all your projects start with a preliminary evaluation stage where an optimized approach to antibody discovery and production is proposed.
Our integrated capacities enable us to produce a wide range of antigens including proteins, peptides, DNA, and whole cells. Importantly, before antigen production, we invest significant efforts in designing a relevant antigen based on your needs and requirements. Our in-house antigen production capabilities with our optimized antigen design ensure you save precious time and achieve the best results as quickly as possible.
Yes, we work on a fee-for-service basis meaning you get full ownership over all antibodies produced by us and are charged the full price only if you’re satisfied with the results.
Our monoclonal antibody production processes are optimized to minimize risks, protect your investment, and establish our strong commitment to delivering the highest quality product.
In particular, our hybridoma development process is designed to optimize and validate each antibody to a specific application (i.e. Western Blot, Immunoprecipitation, Immunohistochemistry, Immunofluorescence, etc.). Besides in-house validation, researchers also have the opportunity to test hybridoma-generated antibodies at their own facilities using their samples, protocol, and assay format, before they purchase them.
Our phage display antibody generation services also come with important guarantees. In particular, researchers always receive 3 different binders in every project; otherwise, they are exempt from paying the full fee.a
Monoclonal antibodies can be defined as antibodies originated from a single plasma cell clone. As a consequence, these antibodies share the same structure, format, and antigen specificity.
Monoclonal antibodies are classified according to different isotypes and subtypes. However, they all share a similar canonical Y-shaped structure consisting of two heavy chains and two light chains each containing variable and constant domains responsible for antigen interaction and immune system engagement, respectively.
Within the constant domain, there is a hinge region that can be cleaved by papain, further dividing the antibody into two Fab fragments (containing the variable domain and part of the constant domain) and a single crystallizable fragment (Fc containing the remaining parts of the constant domain).
The Fab fragments of monoclonal antibodies are responsible for interacting with different antigens. In contrast, the Fc fragment is known to be coated by glycans and to exert important effector functions.
Effector functions are a vital part of the humoral immune response as they form a bridge between innate and adaptive immunity. Many of these functions of monoclonal antibodies are still poorly understood.
However, the best well-known effector processes tied to the Fc fragment include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).
Monoclonal antibodies can be divided into different classes or isotypes according to the type of heavy chain constant region, distribution, and function in the immune response. In general, mammalians have 5 different isotypes of antibodies including IgG, IgM, IgD, IgA, and IgE. From these antibody isotypes, the IgG is the most relevant and most abundant type since it plays a major role in the immune response. For this reason, most antibodies used in research, therapy, and diagnostics are IgG immunoglobulins.
In vertebrates, monoclonal antibody synthesis is a complex process. Antibodies are synthesized by B cells in response to foreign molecules known as antigens.
However, the production of fully mature and high-affinity antibodies is typically mediated by T cells. When T and B cells recognize the same antigen, a process of linked recognition takes place, further triggering affinity maturation and isotype switching from the pentameric IgM to the most functional IgG/IgA/IgE format, depending on the location of the site of contact and nature of the antigen.
Monoclonal antibodies are prized reagents for a multitude of applications. Due to their specificity, selectivity, high binding affinity, and low toxicity/immunogenicity, monoclonal antibodies are useful for research (clinical, environmental, basic, etc.), therapy, and diagnostics including in vitro tests, medical devices, and medical imaging.
For therapeutic applications, monoclonal antibodies can be used in their “naked” form or serve as a carrier by being conjugated to a small molecule or drug. For diagnostic and research applications, monoclonal antibodies are frequently conjugated with enzymatic or fluorescent tags for visual detection of targets.
Discover all the different applications of monoclonal antibodies on our dedicated page: What are the applications of monoclonal antibodies?
Therapeutic monoclonal antibodies are considered one of the safest classes of biotherapeutics due to their high selectivity or, in other words, the ability to target specific cells or tissues with little off-target binding. Thus, their toxicity is limited to specific targets, maximizing their therapeutic efficiency. As proof of their safety, every year dozens of new antibodies are approved for clinical use, a number that is very significant within the drug approval scenario.
Side reactions to monoclonal antibody therapies are known to occur. However, these reactions manifest only in a minority of patients and they can often be predicted by studying each patient’s genetic and clinical background.
To learn more about antibody safety, read the complete article: Are monoclonal antibodies safe?
From a therapeutic point of view, the use of monoclonal antibodies is advantageous due to their dual role as antigen-binding molecules and chemical triggers of the immune response.
These properties have proven efficiency in the fight against cancer (which requires that the immune cells participate in the elimination of the cancerous cells) and autoimmune diseases (which require the suppression of the patient’s auto-antibody responses). Moreover, as antibodies can easily be designed to tackle several antigens, oligoclonal therapies against infectious diseases and natural venoms can easily be developed.
From a diagnostic and research point of view, monoclonal antibodies are prized from their high affinity, stability, and selectivity. These properties make them desirable tools to detect specific molecules even in complex samples. Moreover, given their ability to block protein interactions, antibodies are also advantageous and cost-effective reagents for the study of disease pathways and to elucidate the function of other proteins.
To discover more advantages of monoclonal antibodies over other reagents, visit the complete article: What are the benefits of using monoclonal antibodies?
The monoclonal antibody production process is a flexible process that differs according to the final application of these reagents. Therapeutic monoclonal antibody production is significantly more complex than the production of diagnostic or research antibodies.
The first stage of production is the discovery stage and it can be performed using several different technologies (i.e. hybridoma, phage display, etc.). After discovery, biotherapeutic antibody leads may be further engineered by humanization (if originated in hybridomas) or affinity maturation (after humanization or directly after discovery by phage display).
Lastly, antibodies are produced natively in hybridoma cell lines (suspension or ascites method) or recombinantly expressed in mammalian, microbial, or insect systems. Research and diagnostic antibodies may be further conjugated to enzymatic or fluorescent tags before utilization in different types of assays.
In vitro monoclonal antibody discovery and production was first achieved by Kohler and Milstein’s hybridoma technology in 1975. The cell lines originated from the fusion of antibody-secreting plasma cells and immortal myeloma cells, displayed a hybrid genotype and phenotype allowing them to survive under laboratory conditions for long periods. The prolonged survival period of these cell lines allowed researchers to screen hybridomas for antibody binding affinity.
With the advent of in vitro technologies, antibody discovery has evolved to other methods including antibody display (originally achieved by phage display), transgenic mice-based hybridoma production (expressing human antibodies), and single-cell based technologies. Today, mouse hybridoma and phage display technologies are two of the most important antibody discovery technologies for a multitude of applications.
For many decades, hybridomas remained the preferred method for antibody discovery. However, the technology poses important constraints. Hybridomas are restricted to a reduced number of species because the technique requires the development of compatible myeloma partners. Although human hybridomas have been generated before, they are typically unstable. Moreover, rabbit hybridoma generation is a patent-protected technology limiting its applicability. Thus, hybridomas are typically developed in mice or other closely related rodent species – creating murine antibodies.
These antibodies are incompatible with therapeutic applications, for this reason, they need to be humanized to reduce the risk of causing Human Anti-Mouse Antibody (HAMA) response, an allergic reaction known to reduce therapeutic efficiency. Moreover, toxic and non-immunogenic antigens cannot be used in hybridoma-based antibody generation.
The phage display technology emerged as a suitable alternative. Phage display is independent of species and antigen nature, for this reason, it is suitable to generate human antibodies for therapeutic applications or diagnostic antibodies (typically rabbit given their high specificity). Moreover, the phage display technology can be used to screen vast repertoires of camelid species (VHH antibody format) for a multitude of applications.
Hybridomas are the preferred technology for diagnostic antibody generation. The advantages of this technology for diagnostics stem from the naturally higher sensitivity/binding affinity of antibodies generated in vivo in comparison to those obtained from in vitro applications (i.e. phage display). Moreover, hybridoma generation is a robust and fully mature technology, allowing researchers to reduce the costs of their custom antibody generation projects.
The phage display technology is a suitable alternative in some situations: (i) when antibodies with different antigen-specificity need to be generated; (ii) when developing fast diagnostic tools; (iii) when the cross-reactivity of a specific antibody needs to be adapted; (iv) when developing antibodies against toxic antigens.
Many molecules can be used as antigens for antibody discovery including peptides, proteins, small molecules, DNA, RNA, and whole cells. In vivo antibody discovery methods (i.e. hybridomas) require antigens to be antigenic, immunogenic (able to generate an immune response), and non-toxic.
In contrast, in vitro antibody discovery methods such as phage display are more flexible and compatible even with non-immunogenic and toxic antigens. Other structural considerations must also be taken into account depending on the intended application of the antibody. For instance, if the target antigen is denatured in a specific application (e.g. Western Blot), it is more advantageous to generate a peptide antibody instead of using a protein or cells which retain the native structure of the antigen.
On the contrary, if the target epitope is located in a region with a complex structure, it is more advantageous to use proteins or whole cells for antibody discovery. Lastly, genetic immunization approaches are advantageous when the antigen cannot be produced with high purity and yield in recombinant systems.
Initially, monoclonal antibodies were produced natively in hybridomas either by growing them in suspension (in the presence or absence of animal serum) or through the ascites methods.
With the advent of recombinant technologies, antibody production shifted from native generation in hybridomas to cloning and expression in advanced systems such as mammalian (Chinese hamster ovary – CHO cells; human embryonic kidney – HEK cells; among others), yeast (Pichia pastoris, Saccharomyces cerevisiae, etc.), bacterial (Escherichia coli, Bacillus subtilis, etc.), and insect systems.
To learn more about common antibody production processes, read the full article: How are monoclonal antibodies produced?
The choice of an adequate monoclonal antibody production system depends on its intended use. For therapeutic applications, monoclonal antibodies need to be produced in systems able to perform human-like glycosylation as glycans influence the effector functions of antibodies and thus their therapeutic efficacy. In these cases, CHO and HEK cell lines are the preferred systems for monoclonal antibody production.
For other applications like research and diagnostics, where only small or medium-scale quantities are needed and glycans are not considered vital, monoclonal antibodies can be expressed in high-performing and simple systems such as bacterial and yeast.
Lead times for monoclonal antibody generation vary significantly depending on the final application. The most complex antibody production projects are the ones requiring large-scale commercial manufacture, such as biotherapeutic and diagnostic antibodies for in vitro tests and medical devices. These antibodies are typically produced in recombinant mammalian systems via stable cell line generation approaches. Given the technical complexity of this process, stable cell line generation starts at 5-6 months. The process ensures that antibody-encoding genes are stably integrated into the genome and that these cell lines maintain their high productivity even after multiple freeze-thaw cycles.
Antibodies for research applications can also be produced in recombinant mammalian systems but they are seldom produced in stable cell lines. On the contrary, research antibodies are conventionally produced using transient expression processes (cells lose their ability to express antibodies after several growth cycles) or microbial transformation approaches (as glycans on the Fc fragment are often not essential for most research applications).
For a detailed description of antibody production times and stages, read the full article: How long does it take to produce monoclonal antibodies?
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