Establishing Multi-Dimensional LC-MS Systems for Versatile Workflows to Analyze Therapeutic Antibodies at Different...

Area of Science:

• Analytical Chemistry
• Biopharmaceutical Analysis
• Chromatography

Background:

• Multi-dimensional liquid chromatography-mass spectrometry (mD-LC-MS) is crucial for in-depth biopharmaceutical characterization.
• Assessing chromatographically resolved product variants requires streamlined and semi-automated methods.
• The complexity of mD-LC-MS can limit its accessibility in research and industry.

Purpose of the Study:

• To demystify and enhance the accessibility of mD-LC-MS technology.
• To detail a robust and user-friendly instrument platform for biopharmaceutical analysis.
• To enable seamless switching between intact, subunit, and peptide mapping workflows.

Main Methods:

• Modification of a commercial Two-Dimensional Liquid Chromatography (2D-LC) system with hardware and software extensions.
• Development of two versatile mD-LC-MS setups.
• Demonstration using a case study involving cation exchange chromatography of a bispecific antibody, including online reduction, enzymatic digestion, and mass spectrometry analysis.

Main Results:

• Both proposed mD-LC-MS setups demonstrated accuracy and reproducibility.
• The systems effectively identified and quantified product species despite complex chromatograms.
• Routine usability of mD-LC-MS for therapeutic biomolecule characterization was highlighted.

Conclusions:

• The reliability and accuracy of mD-LC-MS support its routine application in biopharmaceutical analysis.
• Detailed system descriptions and insights lower the entry barrier for this advanced technology.
• Increased accessibility benefits a broader scientific community in research and industrial settings.