Rprotein a sepharose fast flow column

R5 mutant antibody where Asp60, Thr63, Ser65, Ser67 and Asp70 of the 12–10H light chain were replaced with Arg, simultaneously, and A3 mutant antibody where Tyr33 and Arg52 in the heavy chain and Tyr32 in the light chain of 12–10H were mutated with Ala, simultaneously, were prepared by CHO expression system following the methodology described previously^{24 (link)}. In principle, the DNA sequences of the heavy and light chains of the antibody were subcloned into the pcDNA3.4 vector (Thermo Fisher Scientific). The vectors were transiently transfected into ExpiCHO cells (Thermo Fisher Scientific) using ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific) in accordance with the manufacturer's standard protocol. The cells were cultured for 8 days at 37 °C and 8% CO₂. The cultures were centrifuged at 400 × g for 15 min, and the supernatant was collected. Each supernatant was applied onto an rProtein A Sepharose Fast Flow column (GE Healthcare) equilibrated with PBS at pH 7.4. The fraction bound to the column was washed with the PBS and subsequently eluted with Pierce IgG Elution Buffer (Thermo Fisher Scientific). The eluted fraction was neutralized by addition of 2 M Tris–HCl (pH 8.0) and further purified by size exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) equilibrated with PBS at pH 7.4.