Antibodies are glycoproteins produced by the host body as an immune response to neutralize pathogen attacks. They bind to the molecular structure¹ (known as antigen) of the pathogen through their Fab (monovalent antibody fragment) domains and initiate an effector function through their Fc domain, such as activation of natural killer cells or phagocytosis.

The remarkable specificity and affinity of antibodies for their target antigens have made them an attractive tool for therapeutic applications. Monoclonal antibodies, derived from a single clone of B cells², exhibit monospecificity, meaning they recognize and bind to a single epitope on a specific antigen. This precision makes monoclonal antibodies invaluable in targeted therapies and diagnostics. In contrast, polyclonal antibodies³ are derived from a heterogeneous pool of serum antibodies, each recognizing different epitopes of the antigen. These antibodies are generated by immunizing animals with the desired antigen, eliciting an immune response that produces a diverse array of antibodies from various B cell lineages.

While both monoclonal and polyclonal antibodies have been utilized in therapeutic and biomedical applications, monoclonal antibodies are often preferred in clinical settings. This preference stems from their high specificity, homogeneity, and reproducibility, which can be achieved through cell culture techniques. However, novel techniques are also being developed and tried by researchers to increase the production of therapeutic antibodies, which we will further explore in the article.

Traditional monoclonal antibody production

A monoclonal antibody was produced in mice using hybridoma technology for the first time in 1975. Köhler and Milstein developed the technique⁴ to produce unique specific monoclonal antibodies by fusing B-lymphocytes from the spleen with the myeloma cell line, lacking the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) gene. In this basic process, the resulting hybridoma cells are then cultured in vitro in a specific medium having hypoxanthine-aminopterin-thymidine, which helps in the selection of only hybridoma clones.

Initially, the hybridoma culture produces a mix of antibodies from various primary B-lymphocyte clones, resulting in a polyclonal antibody mixture. The individual cells are then separated into each into a separate well using the dilution technique. The well is then screened for desired antibody activity. After the selection and screening of specific clones producing specific antibodies against a specific antigen, the selected hybridomas are grown, recloned, and retested. The successful hybridomas and the monoclonal antibodies are then produced and preserved in liquid nitrogen.

Köhler and Milstein were awarded the Nobel prize for the development of this innovative hybridoma technology as it was a breakthrough in the biotechnology field. However, more studies are required to overcome the limitations associated with the technique⁵, such as:

• Time-consuming and expensive technology
• Higher risk of contaminated cultures
• Lower cell viability
• Production of unstable fused cells
• It is not feasible to produce antibodies against small peptides and fragment antigens

This requires improvement in the hybridoma technology approach or the introduction of some new technologies that can help overcome these limitations for the production of monoclonal antibodies at scale.

Advanced technologies for monoclonal antibody production

Other than hybridoma technology, some newer techniques have been developed for the production of monoclonal antibodies. It includes:

Recombinant DNA technology

This is an effective approach to preparing immortalized human and animal cells for cell fusion⁶ with the goal of monoclonal antibody production. Methods such as DNA micro-injection, spheroplast fusion, and calcium phosphate-mediated DNA precipitation assist in the introduction of viral genes into primary cells, facilitating the establishment of differentiated cell lines.

The microinjection technique uses a small, fine, bore glass needle or microcapillary pipette to introduce the desired DNA fragment in the nucleus or cytoplasm.

During the spheroplast fusion, the cell walls of the host organisms are removed through enzymatic digestion, and this is followed by their fusion in polyethylene glycol.

The calcium phosphate-mediated DNA precipitation works on the principle of forming a complex with the negatively charged DNA, which is then internalized by cells, leading to the expression of the desired DNA fragment.

The recombinant DNA technique is particularly effective in achieving high expression levels of desired or inserted genes.

Phage display technology

Among the many in vitro antibody selection techniques, phage display is the oldest and most widely used approach. It was first developed by George P. Smith in 1985. It involves amplifying the VH and VL segments by converting the isolated mRNA from the B-cells (obtained from human blood) into cDNA using the PCR technique.

The amplified segments are then cloned in a vector alongside the bacteriophage PIII protein. The generated construct is employed to infect E. coli, resulting in the formation of a library comprising around 10^10 cells with the aid of a helper phage. This is followed by the release of bacteriophage from E. coli with VH and VL segments as part of its coat and the selection of specific VH and VL segments against a specific antigen.

This developed library finds extensive applications in biotechnology and biomedical fields due to its faster procedures compared to hybridoma technology, the absence of immunization requirements, and the capability to produce antibodies for toxic antigens.

Single B cell technology

This method involves isolating single cells from PBMCs (Peripheral Blood Mononuclear Cells) or lymphoid tissues using techniques such as fluorescence-activated cell sorting or laser capture microdissection and cloning each Ig heavy and light chain using reverse transcription-polymerase chain reaction (RT-PCR) technique. This is followed by cloning the expressed genes in mammalian cell lines to obtain recombinant monoclonal antibodies (mAbs) that are screened and evaluated using cell-based microarray chip systems and microengraving approaches.

Transgenic animals

The technology was introduced in 1994 to obtain complete human monoclonal antibodies⁷ . During the process, two transgenic mouse lines are modified by replacing their endogenous Ig genes with human immunoglobulin (Ig) genes. So, after the immunization, the transgenic mice will produce fully human antibodies. The first FDA-approved monoclonal antibody generated using the technology was panitumumab. It’s an anti-epidermal growth factor receptor (EGFR) used in the treatment of metastatic colorectal cancer (mCRC).

Cell-free protein synthesis

It is one of the most advanced approaches for monoclonal antibody production without involving living cells and the need for transfection. In this method, cells are lysed and washed to obtain all the essential components for⁸ elongation, initiation, and transcription. Then, the protein synthesis reaction is initiated by adding energy substrates, DNA, nucleotides, amino acids, salts, and other cofactors to the prepared cell lysate (generally CHO cell lysate). It’s an effective approach that allows rapid production and screening of hundreds of antibody candidates in one go. This also saves time by eliminating cloning steps.

The future of monoclonal antibody production

There is a need for effective production techniques that can scale up the extended applications of monoclonal antibodies for therapeutic uses in cancer and immunological disorders while increasing efficiency and reducing side effects. While hybridoma technology stands as one of the most widely utilized approaches in monoclonal antibody production, it necessitates additional research to improve cell viability, stability, and fusion efficiency and streamline the labor-intensive process.

Currently, methods like transgenic animals, phage display, and single B cells are gaining popularity for human antibody production. The integration of single B-cells with next-generation sequencing techniquesis considered a potent tool for the future in clinical and pharmaceutical settings. Further, there is a need for high-throughput screening systems to identify viruses and toxins and assess proteins' ability to neutralize or block receptors. Moreover, AI (artificial intelligence)-driven algorithms¹⁰ and bioinformatics solutions¹¹ are being explored to catalyze research aimed at enhancing monoclonal antibody development.

References

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