Noninvasive method for determination of immobilized protein AMravljak, Rok (Avtor) Stantič, Metka (Avtor) Bizjak, Ožbej (Avtor) Podgornik, Aleš (Avtor) direct noninvasive immobilized proteinquantificationprotein ApH transition methodpolyHIPECNBr-SepharoseThe 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.20222022-06-24 08:40:17Članek v reviji137650UDK: 66.08:577.112ISSN pri članku: 0021-9673DOI: 10.1016/j.chroma.2022.462976COBISS_ID: 103441667sl