title: Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis.
abstract: Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. Because ADCs combine the monoclonal antibody specificity with the high toxicity of a drug, they can selectively kill tumor cells while minimizing toxicity to normal cells. Most of the current ADCs in clinical trials are controlled, but heterogeneous mixtures of isomers and isoforms. Very few protocols on ADC characterization at the peptide level have been published to date. Here, we report on the improvement of an ADC peptide mapping protocol to characterize the drug-loaded peptides by LC-MS analysis. These methods were developed on brentuximab vedotin (Adcetris), a commercial ADC with an average of four drugs linked to interchain cysteine residues of its antibody component. Because of the drug hydrophobicity, all the steps of this protocol including enzymatic digestion were improved to maintain the hydrophobic drug-loaded peptides in solution, allowing their unambiguous identification by LC-MS. For the first time, the payloads positional isomers observed by RP-HPLC after IdeS-digestion and reduction of the ADC were also characterized.
authors.affiliation: Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Janin-Bussat
authors.affiliation: Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France.
authors.forename: Marina
authors.initials: M
authors.lastname: Dillenbourg
authors.affiliation: Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Corvaia
authors.affiliation: Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France. Electronic address: alain.beck@pierre-fabre.com.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France. Electronic address: christine.klinguer.hamour@pierre-fabre.com.
authors.forename: Christine
authors.initials: C
authors.lastname: Klinguer-Hamour
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000737:chemistry*; D018796:Immunoconjugates / Q000737:chemistry*; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*
chemical_list: D018796:Immunoconjugates; D010446:Peptide Fragments; D003545:Cysteine
keywords: Antibody drug conjugates; Brentuximab vedotin; Drug-loaded peptide mapping; IdeS; Liquid chromatography mass spectrometry; Payload positional isomers