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.

A Concise, Modular Antibody–Oligonucleotide Conjugation Strategy Based on Disuccinimidyl Ester Activation Chemistry

“…For example, the heterobifunctional crosslinker succinimidyl‐4‐( N ‐maleimidomethyl) cyclohexane‐1‐carboxylate (SMCC) is used to directly modify surface lysine residues, and is then incubated with thiol‐containing oligonucleotide followed by buffer exchange. Although widely used, this approach is prone to side reactions of the maleimide moiety of the linker with reduced cysteine or proximal lysine residues on the antibody, producing intra‐ and intermolecular crosslinked species that degrade antibody activity and specificity. Even if the desired functional conjugates are obtained, the resulting species are known to have limited stability due to retro‐Michael‐type or thiol–maleimide exchange reaction decomposition.…”

Porphyrin modified trastuzumab improves efficacy of HER2 targeted photodynamic therapy of gastric cancer

“…S1), as depicted in Figure a , and coupled it to trastuzumab (Fig. b ) . The high purity of conjugate ensures that, upon systemic administration and irradiation, the typical unspecific toxicity resulting from free porphyrin impurities will not be observed .…”

Capillary Electrophoresis: Trends and Recent Advances

“…A similar approach was used for the analysis of the ADC trastuzumab emtansine. 172 Here, various analytical techniques including MS, imaging cIEF, and CGE were used for structural characterization and probing protein interactions.…”