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

Fig. 3. Synthesis of bsAb–Sia conjugates: FabHER2–FabCD20–Sia–biotin 21 and FabHER2–FabCD3–Sia–biotin CiTE 24.

Fig. 3

a, Method for the synthesis of bsAb–Sia conjugates. FabX–FabY–N3 is prepared as outlined before. This is then either SEC-purified (for maximum final purity) or taken forward without purification to be reacted with BCN–PEG–BCN 2 to generate FabX–FabY–BCN. Sia–Tet–N3 8 and DBCO–biotin 5 are then added and reacted in situ to form FabX–FabY–Sia–biotin, which is then isolated after SEC purification. b, SDS–PAGE of FabHER2–FabCD20–Sia–biotin 21 formation. Lane 1: ladder. Lane 2: FabHER2–FabCD20–Sia–biotin 21 heated at 95 °C for 5 min. Lane 3: unheated FabHER2–FabCD20–Sia–biotin 21. Lane 4: FabHER2–FabCD20–N3 22 + Sia–Tet–N3 8 (no BCN–PEG–BCN 2 was added, thus no reaction was possible). c, UV trace of SEC purification of FabHER2–FabCD20–Sia–biotin 21. d, SDS–PAGE of SEC purification of FabHER2–FabCD20–Sia–biotin 21. Lane 1: ladder. Lanes 2–3: aggregates. Lanes 4–9: FabHER2–FabCD20–Sia–biotin 21. Lane 10: FabHER2–FabCD20–BCN 23. Lanes 11–12: Sia–Tet–N3 8. e, LC-MS analysis of FabHER2–FabCD20–Sia–biotin 21. Expected mass: 144,532 Da. Observed mass: 144,553 Da. f, SDS–PAGE of FabHER2–FabCD3–Sia–biotin CiTE 24 formation. Lane 1: ladder. Lane 2: crude FabHER2–FabCD3–Sia–biotin CiTE 24. Lane 3: crude FabHER2–FabCD3–N3 13. g, UV trace of SEC purification of FabHER2–FabCD3–Sia–biotin CiTE 24. h, SDS–PAGE of SEC purification of FabHER2–FabCD3–Sia–biotin CiTE 24. Lane 1: ladder. Lane 2: crude FabHER2–FabCD3–Sia–biotin 24. Lane 3–4: aggregates. Lanes 5–7: purified FabHER2–FabCD3–Sia–biotin CiTE 24 (+ FabHER2–FabCD3–FabHER2 25 impurity). Lanes 8–11: FabHER2–FabCD3–N3 13. Lanes 12–13: Sia–Tet–N3 8. i, LC-MS analysis of impure FabHER2–FabCD3–Sia–biotin CiTE 24. Expected mass: 144,799 Da. Observed mass: 144,791 and 144,644 Da (FabHER2–FabCD3–FabHER2 trispecific antibody 25 impurity, expected mass: 144,632 Da). j, UV trace of SEC purification of FabHER2–FabCD3–Sia–biotin CiTE 24 generated from SEC-purified FabHER2–FabCD3–N3 13. k, LC-MS analysis of FabHER2–FabCD3–N3 13. Expected mass: 96,493 Da. Observed mass: 96,506 Da. l, LC-MS analysis of FabHER2–FabCD3–BCN 26. Expected mass: 97,065 Da. Observed mass: 97,081 Da. m, LC-MS analysis of pure FabHER2–FabCD3–Sia–biotin CiTE 24. Expected mass: 144,799 Da. Observed mass: 144,825 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.