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. 2023 Jul 24;15(11):1636–1647. doi: 10.1038/s41557-023-01280-4

Fig. 2. Generation of biotinylated bsAbs and the FabHER2–sialidase conjugate 11 with pyridazinediones.

Fig. 2

a, Generation of FabHER2–Sia–biotin 11. FabHER2–BCN 10 was reacted with Sia–Tet–N3 8 and DBCO–biotin 5 to generate FabHER2–Sia–biotin 11 after monomeric avidin agarose purification. b, SDS–PAGE analysis of FabHER2–Sia–biotin 11. Lanes 1 and 5: ladder. Lane 2: crude FabHER2–Sia–biotin 11. Lane 3: FabHER2. Lane 4: Sia–Tet–N3 8. Lane 6: non-bound fraction of monomeric avidin agarose purification. Lane 7: bound fraction of purification; FabHER2–Sia–biotin 11. c, LC-MS analysis of FabHER2–Sia–biotin 11. Expected mass: 95,873 Da. Observed mass: 95,845 Da and 95,891 Da (Δ = 46 Da, formic acid, MS adduct). d, Generation of FabHER2–FabCD3–biotin 12. FabHER2–BCN 10 was reacted with FabCD3–Tet–N3 14 to form FabHER2–FabCD3–N3 13. This construct was then reacted with DBCO–biotin 5 to generate FabHER2–FabCD3–biotin 12 after SEC purification. e, SDS–PAGE analysis of FabHER2–FabCD3–N3 13. Lane 1: ladder. Lane 2: crude FabHER2–FabCD3–N3 13. Lane 3: purified FabHER2–FabCD3–N3 13. f, Ultraviolet (UV) trace of SEC purification of FabHER2–FabCD3–N3 13. g, LC-MS analysis of FabHER2–FabCD3–N3 13. Expected mass: 96,496 Da. Observed mass: 96,506 Da. h, LC-MS analysis of FabHER2–FabCD3–biotin 12. Expected mass: 97,246 Da. Observed mass: 97,257 Da. i, Generation of FabHER2–(biotin)–FabCD20–biotin 17. FabHER2–Tet–N3 15 was reacted with DBCO–biotin 5 for 1 h to afford FabHER2–Tet–biotin 16, followed by in situ addition of BCN–PEG–BCN 2 to generate FabHER2–(biotin)–BCN 18 over a further 15 h. After removal of excess small molecule, this was reacted with FabCD20–Tet–N3 19 and DBCO–biotin 5 in situ to generate FabHER2–(biotin)–FabCD20–biotin 17 after SEC purification. j, UV trace of SEC purification of FabHER2–(biotin)–FabCD20–biotin 17. k, LC-MS analysis of FabHER2–(biotin)–FabCD20–biotin 17. Expected mass: 98,734 Da. Observed mass: 98,722 Da and 99,181 Da (biotin–FabHER2–FabHER2–biotin 20, expected mass: 99,193 Da). Generation of most Fab conjugates was carried out two or three times, yielding similar results. Each protein–protein construct was generated a single time unless otherwise stated.