Introduction to Antibody Production

Antibodies are proteins that help our body fight against a range of diseases and infections. They bind to antigens with specificity and initiate a cascade of reactions to eliminate foreign organisms.

Antibodies are one component of the adaptive immune response and are produced by a subset of B cells (or B lymphocytes) called plasma cells. They consist of heavy and light chains that are involved in the recognition and binding of antigen. There are five classes of antibodies that are distinguishable through the Fc (fragment crystallizable) region: IgA, IgE, IgD, IgG and IgM.

This article explores antibody production and highlights why it is crucial for the medical and pharmaceutical industries.

Natural Antibody Production

The immune response required to develop antibodies involves a series of steps:

• Antigen Capture: Antigen presenting cells (APCs), like macrophages and dendritic cells, capture foreign molecules in a process called phagocytosis. APCs engulf the invader and break them down for presentation to B cells.
• Antigen Presentation: APCs present fragments of the invader, which constitute epitopes, to B cells. This epitope will be the blueprint for the antigen binding site within the variable region of the antibody.
• Somatic Hypermutation: B cells undergo somatic hypermutation, allowing them to code for high-affinity antibodies against the presented epitope.
• Antibody Production: Depending on the class (or isotype) of the antibody being produced, they can circulate in the bloodstream or remain bound to the surface of B cells. Antibodies preferentially bind to the corresponding epitopes on invading molecules, aiding in its elimination from the body.
• Antibody Function: Antibodies can directly disable pathogens or signal the immune system to destroy them, depending on the type of pathogen.

Recombinant Antibody Production

Researchers are now able to harness the power and process of B cells to genetically engineer antibodies against specific pathogens. Antibodies are engineered and produced with highly specific antigen binding sites within the variable regions. This characteristic bestows therapeutic utility and makes them attractive to the medical and pharmaceutical industries.

Let’s break down some of the ways recombinant antibodies are produced.

• Hybridomas: Hybridoma cells are created by fusing isolated antibody producing B cells and myeloma cells (cancer cells). The B cells are typically isolated from an animal (mouse and rabbit are common) that has been immunized against a specific antigen.
• Phage Display: This technique leverages a library of viruses (bacteriophages) displaying various binding regions of antibodies with varying affinities toward a selected antigen. The antibody fragment found within a selected phage is sequenced and cloned into an expression vector to produce a recombinant antibody.
• Cell Line Development: Desired antibody genetic sequences are computationally designed then cloned into plasmids and transfected into cell lines like the Chinese Hamster Ovary (CHO). Monoclonality is established and the cell lines are scaled up for production.

Requisite Antibody Production Steps

Prior to production, antibody targets must be identified and validated. This process typically involves animal studies and assays designed to characterize both the antigen and antibody. Below are a few important steps that are required along the antibody production process.

• Immunogen Preparation: This is an antigen preparation stage. Antigens (recombinant or native proteins) are prepared in chosen expression systems, such as mammalian, insect, or bacterial cells, to achieve a specific enhanced immune response by the inoculated host. Antigens can also be expressed by bacteriophages as part of the phage display technique.
• Immunization: Immunization plays a crucial role in presenting antigens in a suitable form to stimulate a robust immune response. This necessary step leads to the generation of antibody-secreting cells specifically targeting the chosen antigen and is a requirement for the hybridoma technique.
• Collection: Antibodies, produced in vivo or in vitro, are collected for further characterization.
• Screening: During production, screening helps identify producers with the highest titer and desired specificity.
• Isotyping: Isotyping is a critical step in antibody production, where the class (e.g., IgG or IgM) and subclass (e.g., IgG1 or IgG2a) of a monoclonal antibody are determined. It helps choose suitable purification and modification methods for the antibody molecule.
• Purification: A selective purification method is chosen based on the desired antibody characteristics. Affinity chromatography and ion exchange chromatography are popular methods. Affinity purification via ligands like protein A or G and precipitation methods are also widely used.

Monoclonal Antibody Production

Monoclonal antibodies (mAbs) are a uniform blend of antibodies that exhibit specificity and affinity towards a single epitope of a target antigen. The most widely used method for recombinant monoclonal antibody production is hybridoma technology. Monoclonal antibodies are derived from a single isolated B cell or hybridoma.

Polyclonal Antibody Production

Polyclonal antibodies (pAbs) are immunoglobulin molecules produced by different B cell lineages. They are a heterogeneous population that can recognize multiple epitopes of a specific antigen.

Polyclonal antibody applications are limited compared to monoclonal antibodies because they traditionally exhibit lower specificity, greater batch-to-batch variation during manufacturing, and cross-reactivity.

Multispecific Antibodies

Multispecific (bispecific and trispecific) antibodies, including antibody fragments, are a fast-growing area of research. These antibodies can bind multiple antigens simultaneously which is favorable for therapeutic development, particularly in immuno-oncology. Multispecifics are produced by genetically engineering and combining fragments from multiple antibodies into a single construct and expressing them via cell lines, like CHO cells. Given their complexity, multispecifics face unique challenges related to their design, purification, characterization, and assay development to test for product quality attributes as compared to traditional monoclonal antibodies.

Challenges and Future Perspectives

The growing number of approved monoclonal antibody (mAb) therapies indicates that mAbs and their derivatives will remain a central focus in biotherapeutics for the foreseeable future. However, currently, several limitations of antibody production should be addressed for its extensive applications, such as:

• Batch-to-batch variability
• Immunogenic response
• Time and cost associated with development
• Low titers attributed to inefficient scale up and GMP manufacturing
• Antibody stability during storage and transportation

Genetic engineering technologies are now used to reduce the immunogenic response against antibodies. It becomes possible to create monoclonal antibodies with improved characteristics and capabilities through innovative methods like recombinant DNA technology.

Further, nanocarrier technology and computational tools are being leveraged to improve the stability of monoclonal antibodies and for creating formulations suitable for non-traditional administration. These advancements can pave the way for non-invasive administration routes, opening new possibilities for delivering mAbs to patients.

recent-articles