Dual conjugation process for preparing antibody-drug conjugates

An antibody-drug conjugate comprising an antibody and therapeutic moieties conjugated to cysteine residues in the antibody via a cleavable or non-cleavable linker, wherein said therapeutic moieties are conjugated to one or more engineered cysteines and to one or more reduced interchain cysteines of the antibody, wherein said antibody has an engineered cysteine at heavy chain position 41, or light chain position 40 or 41 according to the Kabat numbering system, or a combination thereof, wherein said antibody-drug conjugate has an average DAR of from 2.0 to 6.0;

• and wherein said antibody-drug conjugate was made by a process that comprises:
• (a) selectively reducing the cysteine-engineered antibody 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 the 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) to form said antibody-drug conjugate.