Polyclonal antibody production

Polyclonal antibodies represent a heterogeneous mix of antibodies, with each antibody recognizing different epitopes of a particular antigen.

Polyclonal antibody production typically starts with immunizing an animal with the target antigen to stimulate an immune response, involving the production of antigen-specific antibodies by the animal's B cells (Fig. 4). Immunizations of the same antigen are repeated at intervals of several weeks to increase the number and affinity of antigen-specific antibodies within the animal. The resulting immune-sera (a blood portion containing the antibodies) can be used in its crude form, or the antibodies can be isolated by affinity purification.

Polyclonal antibodies consist of a mixture of antibodies representing the natural immune response to an antigen. So, they can produce a strong signal against the target antigen in their relevant application and are not biased against a single epitope. However, the disadvantages to their use are that they are limited in supply, and batch-to-batch variation is higher than with monoclonal antibodies. Polyclonal antibodies can also exhibit cross-reactivity and lack of specificity because of a higher risk of binding to other proteins with similar sequences. These issues are often addressed by cross-adsorbing (ie, further purifying) the polyclonal antibody mixture to remove antibodies with unwanted binding characteristics, often against similar antigens in particular species.

Figure 4. A typical process of polyclonal antibody production.

Monoclonal antibody production using hybridoma technology

In contrast to polyclonal antibodies, a monoclonal antibody is derived from a single B cell parent clone and will only recognize a single epitope per antigen (Fig. 5). In the hybridoma production method, B cells are immortalized by fusion with hybridoma cells, allowing for the long-term production of immunoglobulins (Ig).

The antibody-producing hybridoma cells are cloned by isolation and cultivated using tissue culture techniques. Antibodies secreted by the cells into the culture media can be harvested and used either in their crude form or purified by affinity purification. Hybridomas may have unique clone names (eg, MJFF5 (68-7) or EPR19759) to identify the exact clone. Unlike polyclonal antibodies, monoclonal antibodies are homogenous with defined specificity to one epitope.

Monoclonal antibodies produced using hybridoma cell lines are prone to experiencing genetic drift over time. An antibody produced using the same cell line several years later may have slight variations from the antibody's original version. Therefore, to preserve the antibody for confirmed long-term supply and maintain product quality, hybridoma-derived antibodies are converted to the recombinant format.

Figure 5. The difference in the specificity of polyclonal antibodies and monoclonal antibodies. Monoclonal antibody production via the hybridoma method starts with the same immunization protocol used for polyclonal antibodies. After immunization, antibody-producing cells (B cells/plasma cells) are harvested from the spleen and fused with immortal tumor cells to become hybridomas screened for antibody production and performance (Fig. 6).

The antibody-producing hybridoma cells are cloned by isolation and cultivated using tissue culture techniques. Antibodies secreted by the cells into the culture media can be harvested and used either in their crude form or purified by affinity purification. Hybridomas may have unique clone names (eg, MJFF5 (68-7) or EPR19759) to identify the exact clone. Unlike polyclonal antibodies, monoclonal antibodies are homogenous with defined specificity to one epitope.

Figure 6. Monoclonal antibody production using hybridomas. As they homogeneously detect a single epitope on an antigen, monoclonal antibodies are less likely to cross-react with other proteins and display less batch-to-batch variation than polyclonal antibodies. Monoclonal antibodies can be well characterized and defined to meet specificity and sensitivity criteria.

Monoclonal antibodies produced using hybridoma cell lines are prone to experiencing genetic drift over time. An antibody produced using the same cell line several years later may have slight variations from the antibody's original version. Therefore, to preserve the antibody for confirmed long-term supply and maintain product quality, hybridoma-derived antibodies are converted to the recombinant format.

Recombinant monoclonal antibody production

A recombinant antibody is an antibody generated in vitro using synthetic genes. The encoding sequences can be carefully controlled, allowing for optimized expression (higher antibody yields) and improved reproducibility over antibodies produced from hybridomas. Compared to traditional monoclonal and polyclonal antibodies, recombinant antibodies offer long-term, secured supply with a minimal batch-to-batch variation. Also, as the antibody-encoding sequence is known and defined, it can be further engineered and manipulated for its intended use. For example, the sequence can be modified to improve antibody binding characteristics, include tags, or incorporate an FC fragment from an alternative species.

Recombinant antibodies are produced by cloning the antibody-coding genes into a high-yield mammalian expression vector. The resulting vectors are then introduced into expression hosts to manufacture functional antibodies. Mammalian cell lines, such as HEK 293 or CHO-K1, are typically used as expression hosts to preserve the correct post-translational modifications (such as glycosylation).

Multiple methods are available to derive the antibody-coding genes, including hybridoma technology, phage display, B cell cloning, and Next-Generation Sequencing (NGS). Here we describe recombinant antibody production processes based on hybridoma and phage display technologies.

Recombinant monoclonal antibody production from existing hybridomas

Existing hybridoma-based monoclonal antibodies can be converted to recombinant monoclonal antibodies to ensure enhanced consistency and specificity. Recombinant conversion is achieved by obtaining the sequence of the antibody-producing genes from the hybridoma and expressing them in a mammalian cell line. We've outlined the process below.

1. Hybridomas are produced using the same procedure described above in Figure 6.

2. A particular hybridoma's mRNA is isolated, converted to cDNA, and then amplified using PCR to identify the antibody-encoding sequence to allow in vitro recombinant production.

3. The antibody-encoding sequence is sequenced and cloned into an expression vector.

4. The genes are then expressed in a mammalian cell line (eg, HEK 293).

5. The resulting recombinant antibody is validated to ensure the performance matches the hybridoma version.

Recombinant monoclonal antibody production by phage display

In vitro  phage display technology provides the ability to discover recombinant monoclonal antibodies against targets more rapidly and without animal immunization (in the case of naïve libraries). It involves generating an antibody library, selecting antibodies binding to the target/antigen of interest, and affinity maturation (Groff et al., 2015).

The phage display process includes the following steps (Fig. 7):

1. Target proteins or peptides can be captured on an ELISA plate or coated onto magnetic beads. These antigen-coated surfaces are used to screen the phage display library (with bacteriophage expressing an antigen-specific antibody domain, such as scFv or Fab, sticking to this coated surface).
2. Plates are washed to remove non-specific binders.
3. The specific phage display binders are subsequently eluted and transduced into bacterial cells for amplification.
4. Additional rounds of panning/screening are processed to enrich the presence of antigen-specific bacteriophage. Typically 2-3 rounds are performed.
5. Target specificity is confirmed through binding assays, such as an ELISA. DNA from positive binders is then isolated and sequenced.
6. The antibody-encoding sequence is engineered into a mammalian or IgG expression vector for large-scale manufacture of the recombinant monoclonal antibody.
7. The antibody is subsequently validated in relevant assays, such as western blot, immunofluorescence, flow cytometry, and immunohistochemistry​.

Figure 7. An example of the phage display process (Steps 1–5).