The production of bispecific molecules through controlled Fab-Arm-Exchange (cFAE) is a multistep process. First, two parental monoclonal antibodies (mAbs) are expressed by independent Chinese hamster ovary (CHO) cell lines and purified by Protein A chromatography. Second, parental mAbs are subjected to a mild reducing agent, resulting in reduction of inter-heavy chain disulfide bonds at the hinge region and separation of parental mAb Fab-arms (heavy chain-light chain pair). Next, each Fab-arm combines with the complementary Fab-arm forming a heterodimer bispecific antibody. Last, the reformation of disulfide bonds between Fab-arms is achieved upon removal of the reducing agent. During the manufacturing process of parental monoclonal antibodies, there is potential for the formation of high molecular weight species (HMWS) consisting of self-associated mAbs. Following Protein A chromatography, the HMWS may impact cFAE efficiency and biological activity of the bispecific antibody. A Statistical Design of Experiment (DOE) study was executed at small scale to assess the hypothesis that HMWS does not participate in heterodimer bispecific formation consequently placing added strain on polishing chromatography processes to remove HMWS among other impurities downstream of the cFAE.

Effect of homodimer IgG4 bispecific antibody aggregates on controlled Fab-Arm-Exchange (cFAE)

2019 Spring National Meeting

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