Table 1.
Subset of conditions screened to optimize the enzymatic remodeling process. Efficiency was determined by conversion of remodeled trastuzumab-3 into ADC and determination of drug-to-antibody ratio (DAR) with RP-HPLC. In all cases buffers were set at pH 7.5 and remodeling was performed at 15 mg/mL antibody concentration (100 µM) in the presence of 1% (w/w) endo SH. For full set of conditions, including other pH values (see ESI†)
| Entry | Buffer* | UDP-3 | GalNAcT | MnCl2 | AP | efficiency |
|---|---|---|---|---|---|---|
| 1 | tris | 37.5 equiv. | 5% | 10 mM | – | ≥90% |
| 2 | tris | 20 equiv. | 0.5% | 10 mM | – | 63% |
| 3 | histidine | 75%–95% | ||||
| 4 | HEPES | 63% | ||||
| 5 | tricine | 73%–93% | ||||
| 6 | histidine | 25 equiv. | 1% | 6 mM | 79% | |
| 7 | 1.5% | 88% | ||||
| 8 | 2% | 95% | ||||
| 9 | 20 equiv. | 3% | 91% | |||
| 10 | 15 equiv. | 3% | 91% | |||
| 11 | 2% | 80% | ||||
| 12 | 10 equiv. | 3% | 89% | |||
| 13 | 15 equiv. | 2% | 0.01% | 94% | ||
| 14 | 1.5% | 93% | ||||
| 15 | 10 equiv. | 3% | 97% |
A table with screening conditions to optimize conversion of antibody into azido-remodeled antibody based on variation of buffer, quantity of UDP-3, quantity of GalNAc-transferase, quantity of alkaline phosphatase and quantity of MnCl2