Fully automated peptide mapping multi-attribute method by liquid chromatography-mass spectrometry with robotic liquid...

Area of Science:

• Biopharmaceutical Analysis
• Analytical Chemistry
• Mass Spectrometry

Background:

• Multi-attribute methods (MAM) using liquid chromatography (LC)-tandem mass spectrometry are valuable for simultaneous monitoring of product quality attributes.
• Current manual sample preparation for MAM is labor-intensive and introduces variability, hindering widespread adoption in quality control (QC).

Purpose of the Study:

• To develop a robust, high-throughput, and universally adoptable automated workflow for MAM sample preparation.
• To improve reproducibility and reduce hands-on time for peptide mapping in biopharmaceutical analysis.

Main Methods:

• Development of a fully automated peptide mapping procedure utilizing a robotic liquid handling system.
• Automation of a microdialysis step for efficient desalting prior to proteolytic digestion.
• Validation of the automated procedure against manual methods for protein sequence coverage, digestion completeness, and PTM quantification.

Main Results:

• The automated procedure demonstrated comparable results to manual methods in protein sequence coverage, digestion completeness, and post-translational modification (PTM) quantification.
• The workflow is hands-free, using readily available commercial components, and significantly reduces analyst hands-on time.
• A high-throughput data analysis workflow using Protein Metrics was also presented.

Conclusions:

• The developed automated sample preparation workflow offers a practical and reproducible solution for MAM.
• This automation can facilitate the broader implementation of MAM in biopharmaceutical QC laboratories.
• The use of commercial, off-the-shelf components lowers barriers to adoption across different laboratories.