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SDS-PAGE01:27

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Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed  polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...
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Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain
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Stress-Induced Antibody Aggregates: Insights from Native SEC-MS with Postcolumn Denaturation.

Daniil Ivanov1, Christine Lee1, Dennis Delgado1

  • 1Sanofi R&D, Framingham, Massachusetts 01701, United States.

Journal of the American Society for Mass Spectrometry
|December 9, 2025
PubMed
Summary

This study presents a platform method using native SEC-MS to characterize high-molecular-weight species (HMWS) in stressed monoclonal antibody (mAb) therapeutics. The method effectively distinguishes between covalent and noncovalent aggregates, aiding in degradation pathway studies.

Keywords:
HMWSPCDSEC-MSaggregatesdegradation pathwaysmonoclonal antibodynative MSstress studies

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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.