Validatibility of a capillary isoelectric focusing method for impurity quantitation

A strategy is presented for examining the validatability of a capillary isoelectric focusing (cIEF) method, intended for quantitation of product-related impurities in a protein drug substance, according to guidelines published by the International Conference on Harmonization (ICH). The results of this study demonstrate the suitability of cIEF as an analytical method for the quantitation of two product-related impurities in a protein drug substance: a monodeamidated degradation product and an aggregated form of the parent molecule. A range of impurity levels was generated by spiking the isolated impurity species, into a representative production lot of the drug substance. Six impurity spike levels (0.5-12% impurity for deamidated species and 0.5-8% impurity for aggregated species) were analyzed in triplicate. Measurement of impurity peak area percent in the spiked samples provided the data for computing specificity, accuracy, precision, linearity and limit of quantitation (LOQ) for the impurities. Accuracy, defined as the agreement of peak area percent for impurity species with the theoretical impurity percentage from the spike ratio, was 85-96% for the deamidated species and 73-97% for the aggregated species. A linear relationship was found between the measured area percent and the theoretical percent impurity for both impurity species (coefficient of determination, r2=0.9994 for deamidated species and =0.9827 for aggregated species). Precision (repeatability) studies demonstrated a low relative standard deviation (RSD) value (<6%) at all spike levels for both impurity species. Intermediate precision and reproducibility were evaluated by simulating many of the multivariable testing conditions expected during the life cycle of an analytical method, such as multiple equipment and laboratories. Repeated analyses of the drug substance under these varied conditions, yielded RSD values of <20%, for both impurity species. The LOQ, defined as the lowest impurity level where both accuracy and precision were achieved, was assigned at the 0.5% impurity level for both impurity species. This work illustrates a successful strategy in applying the ICH validation guidelines for impurity analytical methods to a cIEF method. Moreover, the data demonstrate the ability of cIEF to be used reliably as an analytical method for impurity quantitation.