Recombinant Monoclonal Antibody Overview

In addition to full-length antibodies, recombinant antibodies can also be produced in different formats. These include chimeric antibodies, Fab fragments, scFv fragments and bispecific antibodies. Besides, they can be generated in multiple expression systems, including bacteria, insect cells, yeast, and mammalian cells.

Fig 1. Recombinant antibody formats (Pic from Front. Immunol.)

Recombinant monoclonal antibodies possess some advantages and disadvantages.

• Improved reproducibility and control
• Reduced production time
• Animal-free production
• Easier isotype conversion

• High degree of technical skills required
• High cost to develop and produce

Recombinant monoclonal antibody technology comprises a group of molecular approaches useful for the cloning and production of antibodies without requiring the use of conventional tissue culture (hybridoma) methods.

Antibody phage display provides a solution for the generation of recombinant antibodies. The hallmark of the approach is the establishment of a physical link between an antibody's phenotype (i.e., that portion of the antibody protein which binds antigen, i.e., the Fab domain) and its genotype (i.e., the heavy- and light-chain DNA that encodes that particular Fab molecule). cDNA is prepared from the mRNA of the rearranged Ig genes expressed by donor B-cells, and heavy- and light-chain gene segments are amplified by the polymerase chain reaction (PCR). The heavy and light chains are cloned into one of a number of available plasmid expression vectors, resulting in a library of over 10⁸ Fab DNAs. Co-transfection of these plasmids with M13 helper phage into E. coli results in phagemid particles which carry Fab DNA on the inside and Fab protein on the outside by fusion to a phage coat protein.

In a process called panning, such 'Fab/phage display' libraries are incubated with antigen to bind Fab/phage bearing specificities of interest. Unbound Fab/phage are washed away and the bound Fab/phage are eluted and allowed to re-infect E. coli cultures for further propagation. After two to four rounds of panning, essentially all Fab/phage are specific to the antigen of interest. Phage particles bearing expressed mAb on their tips can be used directly in diagnostic assays (e.g., blood group typing) or can be genetically manipulated to express soluble antibody molecules (free of phage) that can be used in conventional (e.g., indirect Coombstype) assays.

Fig 2. Antibody phage display process (Pic from Wikipedia)

Immunoglobulin G (IgG) is a heterotetrameric molecule consisting of two heavy and two light chains, respectively, which are connected via disulfide bonds. Heavy and light chains (HC and LC) also contain intramolecular disulfide bonds for stabilization. These structural properties require a sophisticated folding apparatus as well as an oxidizing environment for the generation of disulfide bonds.

There are various expression systems available to efficiently produce recombinant antibodies and antibody fragments.

Mammalian cell lines represent the most widely used expression system for the production of recombinant antibodies. Several other hosts are being developed which are even able to produce antibodies with human-like glycosylation patterns.

E. coli is a widely used expression host for production of antibody fragments. Using high-cell density fermentation, the yield can be up to 1–2 g/L depending on the individual antibody fragment. This expression system is well established with many highly characterised expression vectors for introduction of the antibody genes into the host. Additionally the cells are inexpensive, easily grown and quickly produce small amounts of target protein for evaluation.

Yeasts, as a eukaryotic organism, has the capacity to perform post-translational modifications. In addition, they can be used even in high throughput processes and glyco-engineering enables the expression of recombinant proteins with human-like glycosylation.

Insect cells contain a better suited protein folding and secretion apparatus than prokaryotes. Their high robustness combined with less sophisticated requirements for fermentation provide some advantages compared to mammalian cells. However, the development of stable insect cell lines and process technology is not developed as far.