title: Trisulfide modification impacts the reduction step in antibody-drug conjugation process.
abstract: Antibody-drug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic drugs covalently bound to native heavy-heavy and heavy-light interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for drug linkage. In practice, the amount of TCEP required to achieve the desired drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for conjugation. Antibodies with higher levels of trisulfide bonds require a greater TCEP:antibody molar ratio to achieve the targeted drug-to-antibody ratio.
authors.affiliation: Department of Protein Analytical Chemistry, Genentech, Inc., 1 DNA way, South San Francisco, CA 94080-4990, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Cumnock
authors.forename: Timothy
authors.initials: T
authors.lastname: Tully
authors.forename: Christopher
authors.initials: C
authors.lastname: Cornell
authors.forename: Matthew
authors.initials: M
authors.lastname: Hutchinson
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Gorrell
authors.forename: Ken
authors.initials: K
authors.lastname: Skidmore
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Fredric
authors.initials: F
authors.lastname: Jacobson
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D013440:Sulfides / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D004364:Pharmaceutical Preparations; D013440:Sulfides