Dual conjugation process for preparing antibody-drug conjugates

A process for preparing an antibody-drug conjugate, which comprises an antibody and therapeutic moieties conjugated to the antibody, the process comprising the steps of:

• (a) selectively reducing a cysteine-engineered antibody comprising reacting an antibody comprising one or more engineered cysteines at positions selected from heavy chain 40, 41, and 89 according to the Kabat numbering system, heavy chain 152, 153, 155, and 171 according to the Eu numbering system, light chain 40 and 41 according to the Kabat numbering system, and light chain 165 and 168 according to the Eu numbering system, with a compound according to formula (I), (II), (III), (IV), (V), (VI) or (VII)
• or a salt thereof;
• (b) further reducing the selectively reduced antibody of step (a) with an interchain disulfide bond reducing agent to form one or more reduced interchain cysteines;
• and
• (c) conjugating therapeutic moieties via cleavable or non-cleavable linkers to the one or more engineered cysteines and to at least one reduced interchain cysteine of the further reduced antibody of step (b).