What makes building a bispecific antibody harder than building a conventional one?

Why

A conventional IgG has one heavy and one light chain, so assembly has one answer. Brinkmann and Kontermann point out that free pairing of two heavy chains and two light chains in one cell can give 16 combinations, representing 10 different molecules, of which only one is the intended bispecific. The rest are non-functional or monospecific.

Engineering solves the two halves of the problem separately. Heavy-chain pairing is steered by altering the interface between the two upper constant domains. The knobs-into-holes design puts a large side chain on one chain and complementary small ones on the other, and the version with an added disulfide, S354C with T366W against Y349C, T366S, L368A and Y407V, gave more than 95 percent heterodimer.

Charge pairs achieve the same by electrostatic repulsion between like chains. Light-chain pairing is handled with a common light chain shared by both arms, with engineered charge pairs in the arms, or by domain swaps. Analytics then become the work.

Homodimers, half molecules and mispaired species must be measured, not assumed absent, and each format brings its own stability and manufacturability questions.