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

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

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2015-01-18)
Marie-Claire Janin-Bussat, Marina Dillenbourg, Nathalie Corvaia, Alain Beck, Christine Klinguer-Hamour
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Residual Solvent - Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Isopropyl alcohol, meets USP testing specifications
Sigma-Aldrich
Acetonitrile solution, contains 0.035 % (v/v) trifluoroacetic acid, suitable for HPLC
Supelco
Calcium standard for AAS, analytical standard, 1.000 g/L Ca+2 in hydrochloric acid, traceable to BAM
Sigma-Aldrich
DL-Dithiothreitol solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Acetonitrile solution, contains 10.0% acetone, 40.0% 2-propanol, 0.05% formic acid
Sigma-Aldrich
Acetonitrile solution, contains 0.05 % (w/v) ammonium formate, 0.1 % (v/v) formic acid, 5 % (v/v) water, suitable for HPLC
Sigma-Aldrich
Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) formic acid, suitable for HPLC
Supelco
DL-Dithiothreitol solution, 1 M in H2O
Sigma-Aldrich
Isopropyl alcohol, ≥99.7%, FCC, FG
Sigma-Aldrich
Acetic acid, ≥99.5%, FCC, FG
Sigma-Aldrich
Acetic acid, natural, ≥99.5%, FG
Sigma-Aldrich
Acetic acid, for luminescence, BioUltra, ≥99.5% (GC)
Sigma-Aldrich
Acetic acid-12C2, 99.9 atom % 12C
Sigma-Aldrich
Acetic acid, glacial, ACS reagent, ≥99.7%
Sigma-Aldrich
Acetic acid, glacial, ReagentPlus®, ≥99%
Sigma-Aldrich
Acetic acid, glacial, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8%
Sigma-Aldrich
Acetic acid, glacial, puriss., 99-100%
Sigma-Aldrich
Acetic acid solution, suitable for HPLC
Sigma-Aldrich
Acetic acid, glacial, ≥99.99% trace metals basis
Sigma-Aldrich
Acetic acid, glacial, puriss., meets analytical specification of Ph. Eur., BP, USP, FCC, 99.8-100.5%
Sigma-Aldrich
2-Propanol, ACS reagent, ≥99.5%
Sigma-Aldrich
Formic acid, ACS reagent, ≥96%
Sigma-Aldrich
Acetonitrile, ReagentPlus®, 99%
Sigma-Aldrich
Formic acid solution, BioUltra, 1.0 M in H2O
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Acetonitrile, suitable for HPLC-GC, ≥99.8% (GC)
Sigma-Aldrich
2-Propanol, HPLC Plus, for HPLC, GC, and residue analysis, 99.9%