Bispecific antibodies (bsAbs) can simultaneously bind to two different types of antigens or two different epitopes on the same antigen, and can be designed in a variety of structural forms, which can be divided into two categories: IgG-like and non-IgG-like. The former retains the structure of two Fab arms and one Fc region of the traditional mAb, but the two Fab sites bind different antigens, which are usually produced by the quadroma, or hybrid hybridoma, but this method relies on random chance to form available bsAbs, results in low efficiency. Another method for making IgG-like bsAbs, called “knobs into hole” (KiH), relies on introducing mutations of large amino acids in the heavy chain of one mAb, and small amino acid mutations in another heavy chain. This allows for better binding of the heavy chain of interest (and its corresponding light chain) and allows for more reliable production of bsAbs. Non-IgG-like bsAbs completely lack the Fc region, so the design strategy is relatively simple. This includes chemically linked Fabs, as well as various types of bivalent and trivalent single-chain variable fragments (ScFvs). There are also some fusion proteins that mimic the variable domains of two antibodies. Among these, new formats also include bispecific T cell engagers (BiTEs), tetravalent antiparallel constructs (TandAbs), and VH-only Bi-Nanobodies. Common features of various non-IgG-like bsAbs are low molecular weight and thus high tumor tissue permeability, but relatively short half-life.

Through different structural designs and mechanisms of action, the therapeutic advantages of bsAbs include: mediating immune cells to kill tumor cells; dual targeting immune checkpoints, exerting unique or overlapping functions, effectively preventing drug resistance ; enhanced specificity, and reduced off-target toxicity; effectively reduce treatment costs, as studies have shown that the therapeutic effect of BiTE can be 100-1,000 times that of conventional antibodies, and the dose can be as low as 1/2000 of the original dose, thus significantly reduce drug treatment costs. Compared with combination therapy, the cost of bsAb is also much lower than the cost of combining two single drugs.

E. coli is a common choice for scFv expression because the bacteria grow rapidly on inexpensive media and can produce large amounts of protein. However, expressed scFv molecules may be misfolded or form inclusion bodies, requiring additional downstream process steps to solubilize and refold the protein. These problems can be overcome using mammalian cell lines, such as CHO, because eukaryotic cells have advanced protein folding processes and the ability to undergo complex post-translational modifications, which can stably express proteins to high titers, and have good process scalability. For similar reasons, IgG-like bsAbs are mainly expressed in mammalian cell lines. But for asymmetric heterodimer structures, specific protein/cell engineering is required, such as the reported KiH and CrossMab techniques, to facilitate correct pairing and mitigate the challenges posed by impurities, such as free heavy chains, light chains, homodimers, mispaired antibodies, improving product manufacturability.

The composition of cell culture media has been shown to affect product quality, so media design should be considered as a means of controlling the levels of aggregates and variants in cell culture harvests. At the same time, adjustment of cell culture temperature has also been shown to control the formation of half-antibodies and aggregates. In terms of culture methods, studies have shown that compared with traditional fed-batch culture, perfusion culture can improve bsAb quality and productivity by reducing the residence time of products in the bioreactor, preventing accumulation and concentration increase, and reducing the physical or chemical degradation of bsAb molecules.

In the downstream process, due to the certain structural similarity between mAb and bsAb, many bsAb purification methods are developed based on the mature purification process of mAbs, such as affinity, charge, size, hydrophobicity and mixed mode-based chromatography steps, but also apply other strategies to overcome the unique challenges bsAbs pose, including aggregates, fragment formation, and mispaired products. Protein A affinity chromatography is still one of the most commonly used affinity purification methods in bsAb downstream processes. In addition, in the design of bsAb, by introducing a point mutation in a heavy chain to change the binding affinity of Protein A, the homologous and heterologous dimers can be separated through differential Protein A affinity chromatography. Protein A affinity chromatography has also been shown to be effective in separating half-antibodies from target products by pH gradient elution. The same strategy has also been explored for Protein G. For fragment-based bsAbs, you can choose immobilized metal affinity chromatography (IMAC) or affinity ligands that can bind to the CH1 region or Kappa or Lambda chains, such as Protein L affinity chromatography. Ion-exchange chromatography is usually used as a purification step, combined with specific operating conditions, it can also be used to remove mis-paired products and fragments. Mixed-mode chromatography by combining and utilizing multiple fundamental separation techniques shows the potential to further enhance the capabilities of downstream purification platforms, and different types of such resins have been shown to remove mis-paired products, fragments, and aggregates. Tangential flow filtration is often used for concentration and buffer exchange purposes in the process, but some studies have also shown that bsAb aggregates can be removed well by selecting a specific pore size, such as 300kD.