Stress-Induced Antibody Aggregates: Insights from Native SEC-MS with Postcolumn Denaturation

Area of Science:

• Biopharmaceutical Analysis
• Protein Chemistry
• Analytical Chemistry

Background:

• Characterizing high-molecular-weight species (HMWS) is crucial for monoclonal antibody (mAb) therapeutic development.
• Native mass spectrometry (MS) is a powerful tool for HMWS analysis, offering high accuracy and sensitivity.
• Routine implementation of native MS for HMWS is challenging due to data acquisition and interpretation complexities.

Purpose of the Study:

• To develop and evaluate a platform method for native SEC-MS characterization of HMWS in stressed mAb samples.
• To provide a practical guideline for incorporating native SEC-MS into degradation pathway studies.
• To enhance the understanding of stress-induced antibody aggregation.

Main Methods:

• Systematic evaluation of a platform method for native SEC-MS.
• Utilized conventional quadrupole time-of-flight mass spectrometry without hardware modifications.
• Applied postcolumn denaturation (PCD) to differentiate aggregate types.

Main Results:

• Successfully characterized aggregates in a model IgG4 antibody under thermal and low-pH stress.
• The developed platform method demonstrated wide accessibility and effectiveness.
• Postcolumn denaturation enabled clear identification of covalent versus noncovalent aggregates.

Conclusions:

• The developed native SEC-MS platform method is a robust and versatile tool for HMWS characterization.
• This approach facilitates the study of degradation pathways in mAb therapeutics.
• The method can improve the development of safe and effective monoclonal antibody therapies.