title: Structural Characterization of Cross-Linked Species in Trastuzumab Emtansine (Kadcyla).
abstract: The antibody-drug conjugate, trastuzumab emtansine (Kadcyla), is produced by attachment of the antitubulin drug, DM1, to lysine amines via a heterobifunctional linker, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Following the reaction of the N-hydroxysuccinimide activated linker with antibody lysines to produce a linker-modified intermediate (Tmab-MCC), DM1 is added to yield the desired product. In addition to the expected distribution of drug-linked forms (from 0 to 8), mass spectrometry also demonstrates the presence of a second distribution shifted by about +222 Da. This series is consistent with the presence of a population containing a bound linker without DM1 ("unconjugated linker"). Extended characterization of trastuzumab emtansine was performed using capillary isoelectic focusing, CE-SDS, peptide mapping, and LC/MS following (18)O labeling of peptide digests to identify this family of product variants. These studies demonstrate that the presence of these +222 Da species is due to an unexpected reaction of the maleimide moiety in the MCC linker with antibody lysine residues to produce cross-linked species that cannot conjugate to DM1.
authors.affiliation: Department of Protein Analytical Chemistry, Genentech, Inc. , 1 DNA way, South San Francisco, California 94080-4990, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Protein Analytical Chemistry, Genentech, Inc. , 1 DNA way, South San Francisco, California 94080-4990, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Kim
authors.affiliation: Department of Protein Analytical Chemistry, Genentech, Inc. , 1 DNA way, South San Francisco, California 94080-4990, United States.
authors.forename: Laura
authors.initials: L
authors.lastname: Zheng
authors.affiliation: Department of Protein Analytical Chemistry, Genentech, Inc. , 1 DNA way, South San Francisco, California 94080-4990, United States.
authors.forename: Galahad
authors.initials: G
authors.lastname: Deperalta
authors.affiliation: Department of Protein Analytical Chemistry, Genentech, Inc. , 1 DNA way, South San Francisco, California 94080-4990, United States.
authors.forename: Fred
authors.initials: F
authors.lastname: Jacobson
mesh_terms: D000080044:Ado-Trastuzumab Emtansine; D000595:Amino Acid Sequence; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008453:Maytansine / Q000031:analogs & derivatives* / Q000737:chemistry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D008301:Maleimides; D010455:Peptides; D008453:Maytansine; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester; D008239:Lysine; D000068878:Trastuzumab; D000080044:Ado-Trastuzumab Emtansine