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Background Aggregation of therapeutic antibodies could be generated at different steps of the manufacturing process, posing the problem for quality control of produced antibodies. It has been well known that secreted antibodies from recombinant mammalian cells into culture medium can aggregate due to the physicochemical stresses such as media pH and osmolality, cultivation temperature [1,2]. The antibody aggregation during the cell culture process is difficult to suppress because the cell culture conditions for antibody production are generally optimized for cell culture and growth and not for suppressing the aggregate formation. Here we show the novel strategy to suppress the antibody aggregation; application of chemical chaperone to the cell culture process. It is well established that an addition of some cosolutes serves as chemical chaperone to suppress the protein aggregation. Trehalose, nonreducing sugar formed from two glucose units with a-1,1 linkage, is known as an effective chemical chaperone. In this study, we investigated the anti-aggregation effect of trehalose in the culture process of recombinant Chinese hamster ovary cell (CHO) line producing Ex3-humanized IgG-like bispecific single-chained diabody with Fc (Ex3scDb-Fc). Ex3-scDb-Fc shows the remarkable anti-tumor activity based on anti-EGFR and anti-CD3 bispecificity [3]. However, our in-house results showed that Ex3scDb-Fc shows aggregation tendency, demonstrating the necessity of developing a bioprocess for suppressing the aggregation of the bispecific diabody.