Rapid Purification Of Bispecific Mouse Antibodies By Differential Protein A Binding

Validation of bispecific antibodies (BsAb), which greatly enhance the engagement of therapeutic targets, can require development of in vivo studies that often require mouse surrogate Abs. However, preparation of highly pure BsAb samples involves multiple steps, which presents a significant hurdle for preparation of diverse sets of BsAbs. Here, we describe a one-step method for generating highly pure BsAbs. We identify two mutations in the mouse IgG2 Fc region; one which eliminates protein A binding and one which enhances protein A binding by 8-fold. A bispecific antibody generated from parental mAbs each harboring one of these mutations can be efficiently purified from the residual parental molecules in one step using protein A affinity chromatography. The structural basis of the effects of these mutations was determined by X-ray crystallography. Finally, while a mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one arm retained the ability to bind protein A, also could interact with FcRn. Pharmacokinetic analysis showed that in vitro FcRn binding was consistent with serum lifetime of these molecules. The results describe a rapid method for generating large panels of multispecific antibodies which are amenable to mouse studies.