A method for preparing a conjugate comprising a cell-binding agent (CBA) conjugated to a cytotoxic compound with a linking group, the method comprising reacting a modified cytotoxic compound with a modified CBA at a pH of about 4 to about 9, wherein: a) the modified CBA comprises a residue of a bifunctional crosslinking agent bonded to the CBA, and the residue comprises the linking group and a thiolreactive group； and b) the modified cytotoxic compound comprises a thiol group, and is produced by reacting an imine-containing cytotoxic compound bearing the thiol group with an imine reactive reagent, and wherein the imine-containing cytotoxic compound is represented by one of the compounds in the abstract image. The disclose also relates to methods of preparing similar conjugates using a variety of methods.
