Process Design for Bispecific Antibodies

Abstract Bispecific antibodies provide an opportunity to target two different antigens with a single therapeutic. Applications range from combining two drug activities into one molecule to unique modes of action like the recruiting of immune cells to tumors. However, this advancement in antibody engineering brings new process challenges for cell line development, assembly, purification, and analytics. Creation of bispecific antibody from four individual amino acid sequences (different light and heavy chains) is a process which must be optimized to minimize burden on downstream processing. Knob-and-hole mutations in the Fc region ensure correct hetero-dimerization of heavy chains, however to create bispecific with correct pairing of light chains with heavy chains, two upstream strategies exist. We discuss assembly of bispecific antibody from separately-expressed half-antibodies, as well as an alternative domain-crossover approach called CrossMab, which is produced in a single production fermentation. Bispecific antibodies from both approaches may present product-related variants which must be eliminated in the purification process. Removal of product-related variants requires new purification development approaches, because their structures can resemble that of the bispecific antibody. Case studies from two different bispecific antibody formats will be discussed.