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Related Experiment Video

Updated: Jun 22, 2025

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High-Throughput Fluorometric Assay For Quantifying Polysorbate In Biopharmaceutical Products Using Micelle Activated

Ximeng Y Dow1, Qi Gao2, John L Sperduto3

  • 1Analytical Research & Development, MRL, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ, 07033, USA. ximeng.dow@merck.com.

Pharmaceutical Research
|July 2, 2024
PubMed
Summary

A new high-throughput fluorometric assay quantifies polysorbates (PSs) in biopharmaceuticals. This method, using N-Phenyl-1-Naphthylamine (NPN), offers a sensitive and accurate alternative for PS analysis.

Keywords:
N-Phenyl-1-Naphthylamine (NPN)analytical target profile (ATP)fluorescence micelle assayhigh-throughputpolysorbate quantification

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Area of Science:

  • Biopharmaceutical analysis
  • Analytical chemistry
  • Protein-drug interactions

Background:

  • Polysorbates (PSs) are critical excipients in protein-based biopharmaceuticals, influencing drug stability and efficacy.
  • Accurate quantification of PSs is essential for biopharmaceutical quality control and formulation development.
  • Existing analytical methods for PS quantification can be time-consuming and complex.

Purpose of the Study:

  • To develop a novel, high-throughput fluorometric assay for quantifying polysorbates in biopharmaceutical products.
  • To diversify the analytical methods available for polysorbate analysis.
  • To establish an assay compatible with automated workflows for enhanced efficiency.

Main Methods:

  • Utilized N-Phenyl-1-Naphthylamine (NPN) fluorescence, activated by polysorbate micelles.
  • Optimized assay parameters (wavelengths, concentrations, incubation time) based on an analytical target profile.
  • Employed Nuclear Magnetic Resonance (NMR) spectroscopy to investigate protein-PS interactions and potential interference.

Main Results:

  • Successfully developed and optimized a microplate-compatible fluorometric assay for polysorbate quantification.
  • Demonstrated the assay's specificity, accuracy, and precision through a case study.
  • NMR revealed concentration-dependent interactions between proteins and polysorbate 80 (PS80), with significant interactions observed above 75 mg/mL protein concentration.

Conclusions:

  • A robust, high-throughput fluorometric assay for polysorbate quantification in various biopharmaceutical stages (in-process, drug substance, drug product) has been established.
  • This assay serves as a valuable tool for polysorbate monitoring and control.
  • The developed method complements existing techniques like liquid chromatography with charged aerosol detection.