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An automated homogeneous method for quantifying polysorbate using fluorescence polarization.

Marc D Wenger1, Amy M Bowman, Marc V Thorsteinsson

  • 1Department of Biologics Development and Engineering, Merck Research Laboratories, Merck & Co., Inc., WP42A-20, West Point, PA 19486, USA.

Analytical Biochemistry
|January 15, 2005
PubMed
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A new automated fluorescence polarization (FP) assay accurately quantifies polysorbate in bioprocess samples using the DAF probe. This rapid, automated method is ideal for routine analysis during vaccine and therapeutic protein development.

Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Biopharmaceutical Analysis

Background:

  • Polysorbate is a critical excipient in many biopharmaceuticals, requiring accurate quantification during manufacturing.
  • Existing methods for polysorbate quantitation can be time-consuming or lack automation.
  • The critical micelle concentration (CMC) is an important parameter for polysorbate behavior in formulations.

Purpose of the Study:

  • To develop and validate an automated fluorescence polarization (FP) assay for quantifying polysorbate in bioprocess samples.
  • To assess the assay's specificity, accuracy, and precision for vaccine purification processes.
  • To demonstrate the method's suitability for routine analysis in biopharmaceutical development.

Main Methods:

  • Utilized the lipophilic probe 5-dodecanoylaminofluorescein (DAF) to detect polysorbate micelles.

Related Experiment Videos

  • Quantified polysorbate by measuring the increase in FP signal upon DAF insertion into detergent micelles.
  • Automated all assay steps, including sample preparation and data reduction, using a Tecan liquid-handling workstation.

    • Main Results:

    • Achieved spike recoveries of 98-106% for purified products and 110-120% for crude intermediates.
    • Demonstrated high precision with coefficients of variation <9% for intra-assay and <14% for inter-assay.
    • The assay effectively quantifies polysorbate concentrations above the critical micelle concentration.

    Conclusions:

    • The developed automated FP assay provides a rapid, specific, accurate, and precise method for polysorbate quantitation.
    • This automated approach is well-suited for routine analysis in biopharmaceutical development, particularly during vaccine and therapeutic protein purification.
    • The method shows potential for adaptation to quantify other detergents compatible with the DAF probe.