IMPROVED ION-EXCHANGED HPLC METHOD IN mAb USING pH GRADIENT AND ITS COMPARISON WITH cIEF.

Ion-exchange chromatography (IEX) and capillary isoelectric focusing (cIEF) are two common methods used for molecular charge heterogeneity analysis of therapeutic proteins. The two methods are complementary to each other and each method has its own advantage and disadvantages. For example, a sample injected on an IEX column can be easily fraction collected but, sometimes, it lacks the ability to provide good separation of charge variants, such as those present in monoclonal antibody (mAb) samples. While cIEF has much better resolution than IEX, the separated peaks cannot be collected. Recently, pIsep, a new ion chromatographic technique developed by CryoBioPhysica, has been successfully applied to analyze charged heterogeneities in mAbs. The pIsep separation utilizes an external pH gradient and the charged variants are resolved based on pI differences. In this study, an acidic variant co-eluted with main peak on traditional salt-gradient cation-exchange chromatography (CEX) is separated by pIsep and the fraction was collected for further characterization. The collected fractions were used to characterize the correlation between CEX and cIEF peaks and the results from both methods are consistent. [ABSTRACT FROM PUBLISHER]