An Integrated Workflow for Peptide Mapping of Monoclonal Antibodies

Monoclonal antibodies (mAbs) represent one of the fastest growing classes of drugs
in the pharmaceutical industry. As a protein drug, the manufacturing of an mAb from
initial expression to commercialization is a highly complex process, requiring careful
characterization of the mAb in each step. Peptide mapping, among the many analytical
methods used for mAb characterization, is an essential technique that provides primary
sequence confirmation of the mAb as well as the identification and quantitation
of post‑translational modifications (PTMs) such as deamidation, oxidation, and
Peptide mapping of an mAb using an LC/MS/MS approach involves the enzymatic
cleavage of a purified mAb into peptides, tandem mass spectrometry analysis, and
data interpretation. Sample preparation typically involves multiple steps including
denaturation, reduction, alkylation, and enzymatic digestion. Manual sample preparation
for peptide mapping can be labor-intensive, and it is susceptible to limited scalability and
This application note presents a high-throughput workflow that enables simultaneous,
highly reproducible sample digestion and sample cleanup. This was accomplished using
an Agilent AssayMAP Bravo liquid-handling robot, sensitive and accurate spectrum
acquisition with an Agilent 1290 Infinity II LC system coupled to an Agilent 6550 Q-TOF,
and automated data analysis using Agilent MassHunter BioConfirm software.

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