Fig. 5. Controlled synthesis of trastuzumab–DOX conjugates and localization of the payload. (a) General outline of the two-step process using in situ disulfide bond modification with NGMs followed by copper-catalysed Huisgen 1,3-dipolar cycloaddition. (b) Trastuzumab–DOX DAR series. (M) Molecular marker. (1) Untreated antibody. (2) Reduced antibody. (3) Fragmentation control of trastuzumab prepared by treatment with 2 eq. TCEP (4) Trastuzumab–DOX conjugate prepared with compound 6 (compare Fig. 3c, lane 3). (5) DAR 1.1: reaction on ice for 1 h followed by 90 min click reaction. (6) DAR 2.0: as DAR 1.1 but with addition of 10 eq. benzeneselenol after 30 min and 16 h click reaction. (7) DAR 3.1: reaction at 37 °C for 2 h followed by 90 min click reaction. (8) DAR 4.0: reaction at 37 °C for 2 h followed by 16 h click reaction. (c) Isolated yields and DAR from conjugation reactions, overall yield after SEC purification. (d) CE-SDS analysis of the trastuzumab–DOX DAR series; peak area given as percentage of total peak area. It was assumed for the calculation of correctly bridged disulphide bonds (in contrast to the population of the fragments) that the H and L chains contained 0, the HL fragment 1, the HH fragment 2 and the HHL fragment 3 correct bridges. DAR 0 material was generated by treatment of trastuzumab as outlined for DAR 3.1 material but without addition of reducing agent. (e) Binding activity of the trastuzumab DAR 1 to 4 series to HER2 compared to unmodified trastuzumab. (f) Localization of DOX in DAR 2.0 material. (M) Molecular marker. (1) Untreated antibody. (2) Trastuzumab bridged with compound 15. (3) Trastuzumab–DOX with a DAR of 2.0 after the click reaction. (4) DAR 2.0 material after pepsin digest. (5) DAR 2.0 material after papain digest. (6) Control digest of unmodified trastuzumab with pepsin. (7) Control digest of pepsin digested trastuzumab with papain.