A roadmap for IgG-like bispecific antibody purification

IgG-like bispecific antibodies (bsAbs) can be broadly divided into two categories based on their formats: asymmetric ones and symmetric ones. Production of asymmetric bsAbs usually involves 4 different chains (H1+H2+L1+L2), and various by-products can be formed due to chain mispairing, unbalanced chain expression and incomplete assembly. For symmetric bsAbs, bispecificity is usually achieved through adding an extra functional domain to light or heavy chain in a symmetric manner. In this case, chain extension leads to an increased chance of inter-molecular domain swapping, resulting in an elevated content of aggregates. Thus, impurities associated with bsAb production are highly diverse. Furthermore, some of these impurities (e.g., homodimers) share close similarity with the desired product. Consequently, purification of bsAbs represents greater challenges than that of regular monospecifc antibodies. In general, it is unrealistic to develop a platform approach. Here we introduce a roadmap for bsAb purification based on relevant information in the literature. For each type of commonly observed product related impurities, the map provides one or more paths that leads to its removal. As the roadmap contains information for removing various bsAb-specific impurities, it can be used as a general guide for bsAb purification.