Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-CAD/ELSD and the fluorescence micelle assay

Photo-Oxidation of Therapeutic Protein Formulations: From Radical Formation to Analytical Techniques

“…The NPN assay is not able to directly distinguish degraded from intact PS, so this micelle-based method would only detect changes affecting micelle formation, which could miss the loss of PS. NPN shows a different response to different species present in PS [422]. NPN has been shown to be more sensitive to PS polyester than to the main component of PS, the monoester [423].…”

The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation

“…Fluorescent micelle assay is another PS content assay, which has the potential for automation and high-throughput application [ 13 ]. However, it cannot be used for degradation investigation, because certain degraded PS species could still form micelles as interference factors, which results in over estimation of PS content by the fluorescent micelle assay [ 14 ].…”

Novel High-Throughput Assay for Polysorbate Quantification in Biopharmaceutical Products by Using the Fluorescent Dye DiI

“…One commonly used method in pharmaceutical industry is the fluorescence micelle-based assay (FMA), which uses the partitioning of the fluorescent hydrophobic dye N-phenyl-1naphthylamine (NPN) into surfactant micelles. [7][8][9] NPN exhibits a low-fluorescence quantum yield in aqueous environments, which increases in more hydrophobic environments such as the core of the micelles. The NPN assay was originally intended for critical micelle concentration (CMC) determination, and it has also been used as a method to determine the content of PS in biopharmaceuticals.…”

“…The NPN assay was originally intended for critical micelle concentration (CMC) determination, and it has also been used as a method to determine the content of PS in biopharmaceuticals. [9][10][11] The FMA has nevertheless the following drawbacks: (1) It is an indirect method that relies on the formation of micelles rather than directly the PS content and, therefore, (2) only detects changes affecting the micelle formation, (3) NPN can interact with other hydrophobic components within biopharmaceutical formulations (e.g., proteins and silicone oil droplets), (4) the NPN assay requires an high-pressure liquid chromatography system, 2,9-11 and (5) different PSs display different contributions of autofluorescence because of impurities and degradants at the wavelength used to excite NPN (350 nm), which might lead to errors in the quantification. 2,12 To the best of our knowledge, up-todate publications report data only in the absence of protein.…”

“…2,12 To the best of our knowledge, up-todate publications report data only in the absence of protein. [8][9][10][11] Lippold et al 9 furthermore pointed out that the NPN response is not uniform across the different PS esterified species, thus misestimating PS content. The hydrophobic probe, 4,4 0 -dianilino-1,1 0binaphthyl-5,5 0 -disulfonic acid dipotassium salt, is known to bind to solvent-accessible nonpolar regions of proteins.…”