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 online 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.(c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )