Page 74 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 4
Thus, a knob-in-hole antibody with the same four mutations as described above was expressed bearing a single C-terminal G4Y tag (Tras[KiH-HC]G4Y, Figure 4A). The antibody showed three distinct peaks on HPLC corresponding to the two different heavy chains and the light chain. Similar to our earlier approach, SPOCQ was first performed of Tras[KiH-HC]G4Y with BCN– lissamine, and subsequent HPLC analysis showed >95% conversion of the G4Y-HC within 20 minutes (Figure 4C), significantly faster than the 90 minutes for the dual labelling of symmetrical Tras[HC]G4Y as reported earlier.22
Analogous to Tras[KiH-HC]ST, the knob-HC was found to have a molecular weight of 24221 Da (calc. Mw: 24221 Da), and 24349 Da for the hole-HC containing the G4Y moiety (calc. Mw: 24350 Da) (Figure 4D). After SPOCQ, the molecular weight of the hole-HC increased with 879 Da to 25229 Da (calc. Mw: 25229 Da). This corresponds to the addition of 14 Da during the oxidation by mTyr and addition of 865 Da by BCN–lissamine.
Figure 4. (A) Schematic representation of Tras[KiH-HC]G4Y. (B) Scheme of the SPOCQ reaction of an oxidized tyrosine side chain functionality with BCN. (C) Graphical depiction of the time-resolved HPLC analysis of SPOCQ between Tras[KiH-HC]G4Y and BCN–lissamine. (D) Mass spectra of Tras[KiH-HC]G4Y (top) and its SPOCQ product with BCN–lissamine (bottom). (E) Mass spectrum of Tras[KiH-HC]UCHT1 as formed by SPOCQ.
Finally, we explored the suitability of Tras[KiH-HC]G4Y for the generation of protein-protein conjugates. SDS-page (SI) and LC-MS analysis showed the formation of trastuzumab-UCHT1 conjugates by SPOCQ conjugation of Tras[KiH-HC]G4Y (Figure 4E). Two masses were found by LC- MS: the first being 52351 Da (calc. Mw: 52351 Da), corresponding to the hole-HC plus 14 Da for
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