In 1960, the concept of bispecific antibodies was proposed. Bispecific antibodies, also known as bispecific monoclonal antibodies, are artificially synthesized antibodies using genetic engineering technology. As an engineered artificial antibody, bispecific antibodies usually belong to the IgG subclass and contain an antigen binding fragment targeting the CD3 subunit. Bispecific antibodies have two specific antigen binding sites that can simultaneously bind and recognize two different antigens, or two different epitopes of an antigen. Compared with monoclonal antibodies, bispecific antibodies have an additional specific antigen binding site, thus possessing stronger specificity and targeting ability, which can more accurately target tumor cells and reduce off target toxicity. Bispecific antibodies can simultaneously perform multiple biological functions, such as recruiting immune cells, blocking signaling pathways, and directly killing tumor cells. Early bispecific antibodies were mainly prepared through chemical conjugation or cell fusion, but this method may have progressed slowly due to the random combination and difficulty in isolating the target combination. With the continuous development of genetic engineering technology, many new technology platforms have been developed, such as knots in holes (KIH), CrossMab, DVD Ig, etc. These platforms effectively solve problems such as heavy chain and light chain mismatches, and improve the uniformity and yield of bispecific antibodies.

The main methods for bispecific antibody production include chemical coupling, four source hybridoma, and genetic engineering antibody preparation. Among them, the chemical coupling method couples two intact IgG or two F (ab ') 2 antibody fragments into bispecific antibodies using chemical coupling agents such as phthalimide and dithioacylbenzoic acid. This method is simple and easy to operate, but it may damage the antigen binding site, reduce antibody activity, and the coupling agent itself also has a certain degree of carcinogenicity. The four source hybridoma method is based on the fusion of somatic cells from two different hybridoma cell lines to express the corresponding mouse IgG. Through genetic engineering technology, antibodies can be genetically modified to form bispecific antibodies. Two different monoclonal antibodies were constructed, and the Fab fragments or heavy chain and light chain variable regions of the two antibodies were cleaved separately. Through cross-linking reaction or chain recombination technology, the two fragments were combined to form a bispecific antibody. Although the complexity of genetic engineering technology is relatively high, it is currently the most commonly used method for bispecific antibody production to adjust the structure and function of antibodies. When conducting bispecific antibody design, the principle of antibody cross reactivity may be utilized, but antibody cross reactivity may cause non-specific reactions, so it will be carefully considered in the practical application of bispecific antibody design, such as bispecific antibody therapy.

Bispecific antibody purification is the process of isolating and purifying high-purity target antibodies. Two methods, centrifugation and deep filtration, are used to remove some soluble impurities. The target bispecific antibody is initially captured by affinity chromatography. For IgG like bispecific antibodies, protein A affinity chromatography is used, while for non IgG like bispecific antibodies, light chain based affinity chromatography can be used. Afterwards, the antibody is incubated under low pH conditions for a certain period of time, causing the protein structure on the surface of the virus envelope to be disrupted, thereby losing the ability to infect cells. Intermediate deep filtration is performed to further remove impurities such as host cell proteins (HCP). The purity of antibodies is improved through methods such as ion exchange chromatography, and any residual viruses are removed through nanofiltration or ultrafiltration techniques. Finally, the sample is concentrated and replaced with a suitable formulation buffer.