An imaged capillary isoelectric focusing (icIEF) - UV fluorescence imaging detection method is described for the direct charge heterogeneity characterization of recombinant human erythropoietin (rhEPO) drug products (DPs). rhEPO is one of the most important protein therapeutics for biopharmaceutical industry worldwide. As a heavily glycosylated protein therapeutic, its charge heterogeneity must be carefully monitored in each step of manufacturing and storage. Current charge characterization methods suffer from challenges to characterize rhEPO DPs, due to low sensitivity of the method and potential for interference from the DP's formulation. The method described herein leverages the separation power of imaged cIEF separation combined with the increased sensitivity afforded by UV fluorescence imaging detection and requires no pre-treatment of the DP sample prior to analysis. The method was evaluated initially using a simulated DP, and subsequently a mini method validation was performed using a commercial rhEPO DP sample according to the guideline set by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The limit of quantitation (LOQ) of the method is validated to be 20.3 IU/mL (or 0.10 µg/mL), which is approximately 100 times more sensitive than CZE - UV absorption detection method. To demonstrate the applicability of the method for use, 8 different commercial rhEPO DPs with concentrations ranging from 2000 IU/mL - 10,000 IU/mL were successfully evaluated. This method allows for sensitive, rapid analysis of low concentration rhEPO drug products without sample pre-treatment to provide critical charge heterogeneity information.
Keywords: Drug product (DP); Erythropoietin (EPO); Imaged capillary isoelectric focusing (icIEF); Native fluorescence detection.
Copyright © 2021. Published by Elsevier B.V.