Dynamical analysis of antibody aggregation in the CHO cell culture with Thermo Responsive Protein A (TRPA) column

Background Aggregation of therapeutic antibody is generally occurred in its manufacturing process, and should be suppressed and removed because its potential risk for unexpected immune response [1,2]. Protein A affinity chromatography is the first purification step in the monoclonal antibody manufacturing. Although the affinity purification is a powerful technique, high affinity between protein A and antibody requires acidic condition (below pH 3.0) to elute the captured antibody molecules. Exposure to acidic condition can induce the denaturation and aggregation of antibody molecules, demonstrating the necessity of novel strategy to reduce the antibody aggregation in the affinity purification process. Here we introduced a novel affinity purification strategy, thermo responsive protein A (TRPA) resin. TRPA is an engineered protein A ligand which adopts folded structure under 10°C and unfolds at moderate temperature, above 25°C. TRPA resin can control capture and elution of antibody by changing column temperature, making it possible to elute antibody molecules without low pH condition. In this study, we applied the TRPA column to the purification of Ex3 humanized IgGlike single-chained bispecific diabody-Fc (Ex3-scDb-Fc) [3]. The bispecific diabody is the promising candidate for next-generation therapeutic antibody, whereas it shows aggregation tendency. Furthermore, we observed the time-dependent formation of antibody aggregation in the culture process of the recombinant Chinese hamster ovary (CHO) cell line with TRPA column. Materials and methods CHO Top-H cell line producing the Ex3-scDb-Fc [4] was cultivated in a 1L-glass bioreactor with working volume of 750mL serum-free medium. Viable cell densities and antibody concentrations in the medium was determined with Vi-Cell XRTM cell viability analyzer (Beckman Coulter) and by ELISA, respectively. The bispecific diabody was purified with conventional protein A (PA) column or thermo responsive protein A (TRPA) column, which were connected with AKTA prime plus (GE Healthcare). Elution of antibody was performed by acidic pH solution (pH2.7) for PA column and by raising column temperature to 45°C for TRPA column. Aggregate formation was analyzed with Superdex 200 10/30 GL column (GE Healthcare).