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. 2023 Feb 21;9(3):476–487. doi: 10.1021/acscentsci.2c01437

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© 2023 The Authors. Published by American Chemical Society

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Chemical construction of a full antibody to generate Fc_CD20-(Fab_HER2)₂SynAb 1. A Mono-PD method for the construction of a full antibody Fc_CD20-(Fab_HER2)₂ SynAb 1. Fc_CD205 is sequentially reduced and rebridged with Br₂PD-BCN 4. The resulting Fc_CD20-(BCN)₂6 is then reacted with Fab_HER2-Tz 7 to generate Fc_CD20-(Fab_HER2)₂ SynAb 1 after SEC purification. B Br₂PD-Tz 3 used for SynAb synthesis. C Br₂PD-BCN 4 used for SynAb synthesis. D SDS-PAGE analysis of Fc_CD20-(Fab_HER2)₂ SynAb 1 formation. Lane 1: Ladder. Lane 2: Fab_HER28. Lane 3: Fc_CD205. Lane 4: Crude Fc_CD20-(Fab_HER2)₂ SynAb 1. E UV trace of SEC purification of Fc_CD20-(Fab_HER2)₂ SynAb 1. F SDS-PAGE analysis of SEC purification of SynAb 1. Lane 1: Ladder. Lane 2–3: Aggregates. Lane 4–6: SynAb 1. Lane 7–8: BCN-Fc_CD20-Fab_HER2. Lane 11–14: Fab_HER28. G LC-MS analysis of Fc_CD205. Observed mass: 49868 Da. H LC-MS analysis of Fab_HER2-Tz 7. Expected mass: 48334 Da. Observed mass: 48321 Da. I, J LC-MS analysis of Fc_CD20-(BCN)₂6. Expected mass: 50873 Da (Fc_CD20-(BCN)₂6) and 25438 Da (disulfide scrambled Fc_CD20-(BCN)₂6). Observed mass: 50874 and 25437 Da. K, L LC-MS analysis of Fc_CD20-(Fab_HER2)₂ SynAb 1. Expected mass: 147486 Da (Fc_CD20-(Fab_HER2)₂1) and 73744 Da (disulfide scrambled Fc_CD20-(Fab_HER2)₂1). Observed mass: 147491 Da, 148246 Da (Δ = 755 Da) and 73745 Da.