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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

Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).

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Chemical generation of bispecific Fc_CD20-(Fab_HER2)-Fab_CD3SynAb 13. A Bis-PD method for the construction of bispecific Fc_CD20-(Fab_HER2)-Fab_CD3 SynAb 13. Fc_CD205 is sequentially reduced and rebridged with bis-PD(Tz)₂9. The resulting Fc_CD20-(Tz)₂10 is then reacted with Fab_HER2-BCN 11 to generate Fc_CD20-(Fab_HER2)-Tz 12 after SEC purification. Fc_CD20-(Fab_HER2)-Tz 12 is reacted further with Fab_CD3-BCN 15 to generate bispecific Fc_CD20-(Fab_HER2)-Fab_CD3 SynAb 13 after SEC purification. B The bis-PD(Tz)₂9 used for bispecific SynAb synthesis. C Br₂PD-BCN 4 used for bispecific SynAb synthesis. D UV trace of SEC purification of Fc_CD20-(Fab_HER2)-Tz 12. E SDS-PAGE analysis of Fc_CD20-(Fab_HER2)-Tz 12 intermediate formation and SEC purification. Lane 1: Ladder. Lane 2: Anti-HER2 mAb (trastuzumab). Lane 3: Fc_CD20-(Tz)₂10. Lane 4: Fab_HER2-BCN 11. Lane 5: Crude Fc_CD20-(Fab_HER2)-Tz 12. Lane 6–7 and 13: Fc_CD20-(Fab_HER2)₂2 byproduct/larger species. Lane 8–9: Purified Fc_CD20-(Fab_HER2)-Tz 12. Lane 10–12: Left-over Fab and Fc species. F LC-MS analysis of Fc_CD205. Observed mass: 49868 Da. G LC-MS analysis of Fc_CD20-(Tz)₂10. Expected mass: 51246 Da. Observed mass: 51250 Da. H LC-MS analysis of Fab_HER2-BCN 11. Expected mass: 48141 Da. Observed mass: 48141 Da. I LC-MS analysis of Fab_CD3-BCN 15. Expected mass: 47948 Da. Observed mass: 47950 Da. J LC-MS analysis of Fc_CD20-(Fab_HER2)-Tz 12. Expected mass: 99359 Da. Observed mass: 99367 Da. K UV trace of SEC purification of bispecific Fc_CD20-(Fab_HER2)-Fab_CD3 SynAb 13. L SDS-PAGE analysis of SEC purification of Fc_CD20-(Fab_HER2)-Fab_CD3 SynAb 13. Lane 1: Ladder. Lane 2: Anti-HER2 mAb (trastuzumab). Lane 3: Fc_CD20-(Tz)₂10. Lane 4: Fab_HER2-BCN 11. Lane 5: Fc_CD20-(Fab_HER2)-Tz 12 intermediate. Lane 6–8: Large species. Lane 9–10: Fc_CD20-(Fab_HER2)-Fab_CD3 SynAb 13. Lane 11–12: Left-over Fc_CD20-(Fab_HER2)-Tz 12. M LC-MS analysis of bispecific Fc_CD20-(Fab_HER2)-Fab_CD3 SynAb 13. Expected mass: 147284 and 147398 Da. Observed mass: 147292 and 147396 Da.