Acid pH promotes bispecific antibody formation by the redox procedure

Bispecific antibodies (BsAbs), are potential theranostics. Chemical procedures of preparation of BsAbs, in which two monospecific antibodies are split into half molecules and heterodimerized, continue to attract attention in view of their simplicity. Poor dissociation of antibodies with reduced inter-heavy chain disulfides into half molecules under neutral conditions however restricts the BsAbs formation. In this study, we report that the heterodimerization of antibodies can be improved leading to over 6-fold increase in the yield of BsAbs, by carrying out the redox procedure at pH 4.0. In view of improvement in heterodimerization, BsAbs could be conveniently prepared starting from partially purified ion-exchange fraction of the antiserum and purified by twin affinity chromatography on antigen supports. The UV, CD, intrinsic and extrinsic fluorescence spectral analysis of BsAbs prepared by the modified redox procedure were comparable with the native IgG, which suggest the absence of significant acid-pH-induced damage. ThT binding studies and native size exclusion chromatography ruled out amyloid fibril formation.