L-(+)-Cysteine → HCID 178448357, compound

Participants

Reaction Conditions

Reaction Steps

The generation of cAC10 ADCs with two and four MMAE molecules per antibody involved a partial reduction followed by reaction with Val-Cit-MMAE. The antibody cAC10 (10 mg/ml) was partially reduced by addition of DTT to a final DTT:mAb molar ratio of 3.0 followed by incubation at 37° C. for ˜2 h. The reduction reaction was then chilled to ˜10° C. and the reduced cAC10 purified away from excess DTT via diafiltration. Following diafiltration, the thiol concentration in the partially-reduced cAC10 was determined by the DTNB (Ellman's) assay; in this manner, an average of about 2 disulfide bonds were reduced, thus exposing about 4 reduced Cys:mAb. To conjugate all of the reduced Cys, Val-Cit-cMMAE was added to a final Val-Cit-MMAE:reduced Cys molar ratio of about 1.15. The conjugation reaction was carried out in the presence of 15% v/v of DMSO and allowed to proceed at about 10° C. for about 30 min. Following the conjugation reaction, excess free Cys (2 moles of Cys per mole of Val-Cit-MMAE) was added to quench unreacted Val-Cit-MMAE to produce the Cys-Val-Cit-MMAE adduct. The Cys quenching reaction was allowed to proceed at about 10° C. for about 30 min. The Cys-quenched reaction mixture was purified and buffer-exchanged into PBS by diafiltration to obtain the partially loaded cAC10-Val-Cit-MMAE.

Provenance & Evidence