Format

Send to

Choose Destination
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Feb 15;981-982:9-13. doi: 10.1016/j.jchromb.2014.12.017. Epub 2014 Dec 24.

Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis.

Author information

1
Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France.
2
Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France. Electronic address: alain.beck@pierre-fabre.com.
3
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.

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.

KEYWORDS:

Antibody drug conjugates; Brentuximab vedotin; Drug-loaded peptide mapping; IdeS; Liquid chromatography mass spectrometry; Payload positional isomers

PMID:
25596378
DOI:
10.1016/j.jchromb.2014.12.017
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center