Isoelectric focusing of cross-linked monoclonal antibody heterodimers, homodimers and derivatized monoclonal antibodies.

Abstract

Anti-tumor monoclonal antibodies were cross-linked to the anti-CD3 T-cell antibody OKT3 by the use of the heterobifunctional cross-linker succinimidyl-3-(2-pyridyldithio)propionate. Derivatized monoclonal antibodies, heterodimers, and homodimers were resolved by analytical isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Isoelectric points of the derivatized antibodies were lower than native antibodies, consistent with lysine derivatization. Antibodies derivatized with 2-iminothiolane were equivalent or slightly higher in pI compared with native antibodies. Heterodimers focused in microheterogeneous bands between the pI extremes of the parent derivatized antibodies. The isoelectric points of homodimers were lower than those of parental antibodies and could be distinguished from heterodimers. Reduced and alkylated heterodimers were resolved into their constituent antibodies by isoelectric focusing.