An automated homogeneous method for quantifying polysorbate using fluorescence polarization.
An automated fluorescence polarization (FP) assay has been developed for the quantitation of polysorbate in bioprocess samples. Using the lipophilic probe 5-dodecanoylaminofluorescein (DAF), polysorbate concentrations above the critical micelle concentration can be quantified by the FP increase that results when DAF inserts into the detergent micelles. The specificity, accuracy, and precision of this assay were defined for samples obtained from vaccine purification processes. Spike recoveries were 98-106% for purified products and 110-120% for crude process intermediates. The coefficients of variation for intra- and interassay precision were less than 9 and 14%, respectively. Because of the operational simplicity of the assay, all of the assay steps from sample preparation to data reduction were automated on a Tecan liquid-handling workstation. The combination of a rapid assay and an automated format makes this method well suited to the routine analysis of samples from trial purification processes which are carried out during the development of a vaccine or therapeutic protein. This method should be adaptable for the quantitation of other detergents into which DAF will insert.
Wenger
Marc D
MD
Department of Biologics Development and Engineering, Merck Research Laboratories, Merck & Co., Inc., WP42A-20, West Point, PA 19486, USA.
Bowman
Amy M
AM
Thorsteinsson
Marc V
MV
Little
Kristine K
KK
Wang
Leslie
L
Zhong
Jinglin
J
Lee
Ann L
AL
DePhillips
Peter
P
D001331:Automation; D003902:Detergents; D019793:Fluorescein; D005454:Fluorescence Polarization / Q000379:methods*; D007850:Lauric Acids; D008722:Methods; D008823:Micelles; D011136:Polysorbates / Q000032:analysis*; D014612:Vaccines / Q000032:analysis
D003902:Detergents; D007850:Lauric Acids; D008823:Micelles; D011136:Polysorbates; D014612:Vaccines; D019793:Fluorescein