The biopharmaceutical industry is putting significant effort into developing fast, reliable ways to produce monoclonal antibodies with high yields. Titers have gone above the 5 g/L mark because of progress in mammalian cell culture. Platform-based methods for developing downstream processes are now well established.
They keep improving as more and more products are integrated into these platforms. Because cell culture has become more productive, bioprocess development now focuses on steps following the production bioreactor. This has renewed interest in separation methods other than chromatography. This article discusses current industrial antibody production methods, with an emphasis on downstream technologies for custom antibodies and antibody-related products, such as:
The post also talks about how these technologies might evolve in the future.
What Are Monoclonal Antibodies?
Monoclonal antibodies are a group of antibodies derived from a single B cell clone. As large molecules with complex structures, they are significantly more challenging to manufacture than small-molecule therapies like pills or traditional vaccines. Consequently, it is difficult to produce monoclonal antibodies quickly and in large quantities. The global COVID-19 outbreak made these manufacturing constraints even more apparent, necessitating rapid advancements in custom monoclonal antibody production and recombinant protein production, among others.
The Traditional Monoclonal Antibody Production
The conventional protocol for monoclonal antibody (mAb) production often begins with the formation of hybridomas. This is accomplished by fusing myeloma cells with splenocytes (such as B cells) that produce the desired antibody. These B cells originate almost exclusively from animal sources, specifically mice. Following the fusion of the cells, many clones are screened and selected according to their immunoglobulin class and antigen specificity.
Each "hit," or promising candidate hybridoma cell line, is verified, validated, and characterized using additional tests. Once validated, the clones are scaled up to facilitate downstream bioprocesses.
A Standard Method for Making Monoclonal Antibodies
The typical steps in the monoclonal antibody production process include the following:
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Immunization of mice and isolation of splenocytes: Mice are first immunized with a specific antigen, and their blood is tested for antibody production. The antibody-producing splenocytes are then harvested to create hybridomas in the lab.
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Myeloma cell preparation: Myeloma cells are immortalized cells that, when combined with splenocytes, become hybridomas with an infinite capacity for growth. These myeloma cells are prepared for the fusion process.
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Fusion: Polyethylene glycol (PEG) facilitates the fusion of myeloma cells and isolated splenocytes, leading to the formation of hybridomas.
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Screening and selection of clones: Clones are analyzed for their antigen specificity and immunoglobulin class before being selected for further study.
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Functional characterization: This step involves verifying, validating, and characterizing (using ELISA kits, for example) each potentially high-productivity colony.
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Scale-up and weaning: The goal is to increase the number of clones that yield the desired antibodies and gradually remove the selection agent(s).
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Expansion: This entails further increasing the number of clones that produce the required antibodies using equipment such as bioreactors or large flasks.
The use of monoclonal antibodies in targeted therapeutics has profoundly influenced the treatment of various diseases, and biosimilars have made these treatments more widely available. Consequently, the rest of this blog will review the progression of technologies used in the small- and large-scale production of monoclonal antibodies for therapeutic and commercial purposes.
Innovations in mABs Expression Procedures
Since its introduction in 1977, hybridoma immortalization has been employed to produce and identify mAbs. Over the years, the mAb technology first developed in rodents has been swiftly and effectively applied to advance human diagnostics and therapies.
Hybridoma Technology
Therapeutic monoclonal antibodies have been created using the mouse hybridoma technique. This method takes advantage of the natural functions of myeloma cells and B lymphocytes to create immortal hybridoma cells that produce mAbs highly selective for the target antigen. Polyethylene glycol (PEG), which acts as a fusogen, is used to fuse the B cells to the myeloma cells. The antibodies produced using this method are versatile therapeutic agents with a specificity tailored to the antigen under consideration.
However, mice often produce mAbs with low affinity or poor specificity. Other species apart from rodents are generally not used to produce mAbs because it is difficult to establish immortalized cell lines that efficiently produce antibodies using hybridoma technology.
Phage Display
To expose the exterior peptides of lysogenic bacteriophages, G. Smith invented phage display technology in 1985. To date, this approach has emerged as a leading technique for producing numerous proteins, peptides, and antibodies. It is based on the physical coupling of the phage genotype (single-stranded DNA within the virion) and phenotype (the expression of the phage coat fusion protein).
This method makes it possible to create phage display libraries with up to $10^{10}$ different phage variants. These libraries are used to test the affinity of protein-ligand interactions, characterize and recognize epitopes, select enzyme substrates, screen antibody repertoires, and facilitate the formation of analogous phage particles from an E. coli clone.
Amplification of Single B Cell
An alternative method for the generation of mAbs from immunized animals is the direct cloning of heavy chain variable (VH) and light chain variable (VL—both lambda and kappa) components from single antigen-specific antibody-secreting cells (ASPCs) through the aid of polymerase chain reaction (PCR).
However, there are challenges, such as the high cost and technical skill required to isolate ASPCs. Other limiting procedures include amplifying the cell's VL and VH genes and assembling the immunoglobulin light (IgL) and heavy (IgH) chains. To address this, a high-throughput cloning method utilizing single cells as templates was developed for the fast and scalable production of mAbs. This was accomplished by creating a contactless magnetic power transmission apparatus (MAGrahd) for the automated generation of cDNA from single-cell genes via homopolymer tailing and target-selective joint PCR for IgL and IgH genes.
Innovation in Monoclonal Antibody Engineering
Affinity Maturation
Engineering for affinity maturation of mAbs has been researched extensively to improve their ability to bind to antigens; this is likely one of the most studied areas ( Chowdhury & Wu, 2005). One way to achieve this is to create vast libraries of CDRs or complete variable domains that have been randomly mutated, and then select variants with higher affinity from the mutant pool. Antibody libraries with mutations within the variable regions are generated via three primary methods:
- Random CDR mutagenesis
- Chain shuffling
- Error-prone PCR and DNA shuffling
Focused mutagenesis is another way to create small libraries, which increases the chance of finding variants with higher affinity. Two key approaches include:
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Targeted mutagenesis: In this method, 20 different amino acids are substituted randomly into each of the six CDRs.
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Hotspot mutagenesis: In this method, hotspots are chosen based on their relationship to the structure and function of the antibody. Small libraries of about 400 clones are then created, and higher-affinity variants are selected (Ho et al., 2005).
Recent research has uncovered the role of activation-induced cytidine deaminase (AID) enzymes in somatic hypermutation and class switch recombination in Ig V domains (Muramatsu et al., 1999). There is evidence that this enzyme promotes the somatic hypermutation of transfected genes in hybridomas and fibroblasts. Therefore, AID has the potential to be utilized in upcoming research to produce modified antibodies with greater affinity and specificity.
Enhancements in the Effector Function of Monoclonal Antibodies
The effectiveness of mAbs as medicines depends on their ability to bind to antigens and trigger effector activity (Chowdhury & Wu, 2005). These activities include both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). ADCC involves the binding of an antigen-antibody complex to the Fc receptor (FcR) and the subsequent activation of nearby effector cells, such as macrophages and natural killer (NK) cells, to lyse or opsonize the target cell.
In the CDC, the antigen-antibody complex activates the complement system, resulting in the lysis or opsonization of target cells. Consequently, glycoengineering or protein engineering can be used to strengthen antibody binding to FcRs or complement factors, thereby improving therapeutic efficacy.
To further enhance effector function, mAbs can be modified to incorporate additional capabilities. For example, cytotoxic drugs or radioisotopes are conjugated to mAb components for cancer therapy. In contrast, enzymes are utilized in antibody-directed enzyme prodrug therapy (ADEPT), and cytokines are employed to stimulate an antitumor immune response. In addition, novel miniantibodies, such as the diabody (an scFv-based antibody dimer), triabody (an scFv-based antibody trimer), and tetrabody (an scFv-based antibody tetramer), were developed to facilitate the effector functions of therapeutic mAbs by improving tissue penetration and target proximity.
Advances in Cancer Treatment Through mAbs
The advent of hybridoma technology changed clinical research, biotechnology, cell biology, and immunology significantly. Prior to its introduction, antibodies were produced by repeatedly immunizing animals with a target antigen. The crude sera from these animals were then used to treat patients. However, allergic reactions were common, and administering unrefined sera often proved ineffective.
As a result of fusing immortal myeloma cells with mouse splenocytes, the hybridoma method enabled the production of high-purity mAbs against specific target antigens.
mAbs have been used extensively for diagnosing and treating cancer. Nevertheless, administering mouse monoclonal antibodies to patients can trigger a Human Anti-Mouse Antibody (HAMA) response, leading to rapid clearance and high immunogenicity. To circumvent these problems, contemporary biotechnological advances in genetic engineering have helped reduce immunogenicity, facilitated the development of antibody-drug conjugates, and enabled the creation of minimized antibody segments for cancer diagnostics.
Recent progress includes:
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Bispecific Antibodies: Partially and fully humanized bispecific antibodies help extend the half-life of mAbs and increase their clinical utility.
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Transgenic Mouse Antibodies: These possess two identical light and heavy chains (CL, CH), variable domains (VL, VH), and antigen-binding sites (CDRs) within the variable domains. The immunogenicity of an antibody is primarily caused by its constant region (Fc), which is connected by disulfide bonds (-S-S-).
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Chimeric Monoclonal Antibodies: These are made by replacing the constant IgG region of a mouse antibody with that of a human antibody.
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Humanized Antibodies: A humanized antibody is constructed by grafting the DNA of the three CDRs from the mouse variable region into a human IgG framework. It contains more than 90% human sequences.
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Fully Humanized Antibodies: Produced via phage display or transgenic mice with human Ig genes, these are entirely human in origin, making them far less likely to cause an immune response compared to chimeric or humanized versions.
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Antibody Fragments: Bispecific antibodies have two antigen-binding domains and bind to two different antigens. Shorter antibody fragments have also been developed, such as divalent and monovalent scFvs, which bind to antigens with high specificity.
The FDA in the US and health authorities in the EU have approved many mAbs for therapeutic use. These antibodies treat serious diseases such as human cancers, inflammatory responses, autoimmunity, organ transplant rejection, and hematological malignancies. Furthermore, multiple other therapeutic mAbs are in different stages of clinical testing to treat pathogens like the Zika virus, Ebola, and HIV/AIDS.
Conclusion
Since the 1960s, there have been significant changes and improvements in custom antibody services and protein production services globally. Using powerful methods like single B-cell amplification, phage display, and hybridoma technology, assisted by PCR, scientists have engineered a wide range of antibodies and fragments with diverse structures. Monoclonal antibodies are crucial because they are highly specific and vital to the immune system's response to antigens of interest.
They provide immense value in the medical field, biotechnology, cell biology, immunology, and biochemistry. Further research will help us understand the full potential of mAb development and discover new avenues of progress for the future. Do you have any questions about the large or small-scale production of monoclonal antibodies for various purposes? Do not hesitate to contact us.