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Updated: Jan 19, 2026

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Quantifying protein aggregation kinetics using electrospray differential mobility analysis.

Kaleb J Duelge1, Jeremie Parot2, Vincent A Hackley2

  • 1National Institute of Standards and Technology, 100 Bureau Dr., MS 8520, Gaithersburg, MD, 20899-8520, United States; University of Maryland, College Park, United States.

Journal of Pharmaceutical and Biomedical Analysis
|September 11, 2019
PubMed
Summary
This summary is machine-generated.

This study quantifies protein aggregation kinetics using electrospray differential mobility analysis (ES-DMA) and asymmetrical-flow field-flow fractionation (AF4). ES-DMA offers a sensitive, low-volume alternative for analyzing protein stability in bioprocessing.

Keywords:
Aggregation kineticsDifferential mobilityProtein aggregation

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Last Updated: Jan 19, 2026

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Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

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Area of Science:

  • Biophysical Chemistry
  • Protein Science
  • Analytical Chemistry

Background:

  • Protein aggregation is a significant challenge in biopharmaceutical development, potentially causing adverse clinical outcomes.
  • Accurate quantification of protein aggregation kinetics is crucial for ensuring therapeutic efficacy and safety.

Purpose of the Study:

  • To quantify thermally-induced protein aggregation kinetics for bovine serum albumin (BSA) and α-chymotrypsinogen A (α-chymo).
  • To compare the performance of electrospray differential mobility analysis (ES-DMA) with asymmetrical-flow field-flow fractionation (AF4) for protein aggregation analysis.
  • To evaluate a new methodology for adapting solutions for ES-DMA compatibility.

Main Methods:

  • Utilized electrospray differential mobility analysis (ES-DMA), an aerosol-based technique, to measure protein aggregation kinetics.
  • Employed asymmetrical-flow field-flow fractionation (AF4) with UV detection as an orthogonal validation method.
  • Investigated protein aggregation over varying concentrations and temperatures, analyzing monomer and dimer populations.

Main Results:

  • Both ES-DMA and AF4 successfully resolved monomer and dimer protein populations, enabling quantification of monomer loss.
  • BSA and α-chymo exhibited second and first-order aggregation kinetics, respectively, indicating different rate-limiting steps.
  • ES-DMA demonstrated comparable or slightly faster kinetic measurements than AF4, with a significantly lower sample volume requirement (μg mL⁻¹ vs. mg mL⁻¹).

Conclusions:

  • ES-DMA is a viable and sensitive technique for analyzing protein aggregation kinetics, particularly for small sample volumes.
  • Orthogonal measurement strategies, like comparing ES-DMA and AF4, are essential for robust kinetic analysis.
  • ES-DMA presents a promising alternative to traditional methods like size exclusion chromatography for screening protein therapeutic stability.