Binding activity of cMAbs produced by mammalian cells with different vector systems.
(A) Selection of suitable host cells for transient production of cMAbs. The cMAb CK3 was transiently produced by 8 different mammalian host cells (∼5×106 cells each/10 cm dish) for 5 days post-transfection using a LF-2000 reagent. The undiluted supernatants including a control (supernatant from CHO-K1 cells transfected by only pIRES-DHFR vector without chimeric chains), were used for cMAb CK3 titration. The number in the parenthesis mark indicates the cMAb CK3 titer. (B) Performance of the re-engineered vector systems for cMAb CK3 production was evaluated by CHO-K1 cells transfected using a LF-LTX reagent. Five days post-transfection of the vectors (10 µg) into the cells (∼1×106 cells/10 cm dish), the undiluted supernatants were used for cMAb CK3 titration, including controls (stable cMAb CK3* at 2 µM MTX: pIRES-H3-DHFR/pIRES-L3-DHFR and culture supernatant by only pIRES-DHFR vector). (C) Binding property of 3 cMAbs produced by CHO-DG44 cells without DHFR amplification. For ELISA, the pIRES-H(L)-DHFR vectors containing chimeric H-chain (H1, H2, and H3) or L-chain (L1, L2, and L3) were cotransfected into CHO-DG44 cells (∼106 cells/10 cm dish) using a LF-2000 reagent and the master seed cells were incubated for 3 days to get sufficient amounts of cMAbs before the DHFR-amplification process. (D) Comparison of binding property of stable cMAbs with the original mouse MAbs. Affinity-purified antibodies were adjusted to the same concentration starting with 5 µg/ml.