20.500.12556/RUL-137650 Noninvasive method for determination of immobilized protein A The pH transition method, developed for the determination of the ion-exchange group density on chromatographic stationary phase, was used for the quantification of immobilized protein A. Monolithic epoxy polyHIPE and particulate CNBr-Sepharose supports were used for immobilization. A lactate buffer was selected, having a buffer capacity peak approximately 0.5 pH units below the maximum buffer capacity of protein A. The pH transition measurements were performed at pH 4.3, where protein A exhibits maximum buffer capacity, with a lactate buffer concentration of 1 mM for protein A immobilized on polyHIPE monoliths and of 5 mM for protein A immobilized on CNBr-Sepharose. The pH transition height and full width at half maximum for the particulate support and the height for the polyHIPE matrix, showed a linear correlation with the amount of immobilized protein A determined from the absorbance difference before and after immobilization for both supports. The developed method allows a simple, non-invasive on-line determination of immobilized protein A using biological buffers, even for chromatographic columns with an amount of immobilized protein A as low as 0.25 mg. In addition, its sensitivity and duration can be easily adjusted by varying the buffer concentration and pH. direct noninvasive immobilized protein quantification protein A pH transition method polyHIPE CNBr-Sepharose direktna neinvazivna imobilizacija proteina kvantifikacija protein A metoda pH prehoda poliHIPE CNBr-sefaroza true false true Angleški jezik Slovenski jezik Članek v reviji 2022-06-24 08:40:17 2022-06-24 08:40:18 2022-09-15 04:15:35 0000-00-00 00:00:00 2022 0 0 7 str. art. 462976 Vol. 1671 24 May 2022 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 66.08:577.112 0021-9673 10.1016/j.chroma.2022.462976 103441667 1-s2.0-S0021967322001741-main.pdf 1-s2.0-S0021967322001741-main.pdf 1 0372A609895695A9A73FCEB90A1FD0F1 efa02e27b12382bd1e71c15a907598b504866087a13075fa262d972dbfff8271 e692ee08-f388-11ec-8aca-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=157684 https://www.sciencedirect.com/science/article/pii/S0021967322001741 0 515eaa75-f388-11ec-8aca-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=157680 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0