Method Development and Qualification of pH-Based CEX UPLC Method for Monoclonal Antibodies.

Post-translational modifications ("PTMs") in monoclonal antibodies (mAbs) contribute to charge variant distribution, which will affect biological efficacy and safety. For the characterization of mAbs, charge variants are used as a critical quality attributes for product quality, stability consistency and effectiveness. Charge variants in mAbs are characterized by a time-consuming and a multistep process starting from cation/anion exchange chromatography, acidic/basic fractions collection and subsequent reverse phase (RP) liquid chromatography, coupled with mass spectrometry (MS) analysis. Hence, an alternative characterization approach that would be highly selective for ion exchange chromatography-based charge variant analysis, which is compatible with on-line MS detection, is needed in the biopharma industry. Against this backdrop, multiple studies are being conducted to develop a simple straight on-line charge variant analysis method. In this regard, we apply the current study, which aims to develop a charge variant analytical method, based on volatile buffers with low ionic strength that can be used for on-line MS detection of charge variants of mAbs. This would enable the detection on "PTMs" using low ionic strength mobile phase compatible with MS. Hence, fruitful data can be obtained with a single chromatography run without any test sample preparation, eliminating the need for multiple steps of analysis, time-consuming process and multiple sample preparation steps. Thus, Charge Variant Analysis-MS technique will allow the characterization of charge-related PTMs on the intact protein stage. In this regard, this study is about development of a method having combination of chromatography and volatile mobile phase for mass spectrometry detection of mAbs being analyzed in native form. The method is qualified considering pharmacopeia guidelines because the ultimate aim is to transfer this method for Quality Control (QC) release testing of a monoclonal antibody, which is critical for batch release and the regulatory point of view. Acidic and basic variants have been separated with high resolution peak profile. Furthermore, there was no matrix interference and good separation selectivity in terms of specificity was obtained using this method. The experimental data suggested for the linearity of the method are 2.4 mg/mL to 3.6 mg/mL with % RSD below 2.0%. Additionally, Limit of Quantitation is found to be 0.15 mg/mL, which is 5% of loading amount. Consistently, the data show that the method is precise under the same operating conditions with a short time interval. Overall a simple, accurate, robust and precise pH gradient cation exchange chromatography method was developed and qualified for the characterization of a therapeutic native mAb. Additionally, this method can be used to claim a biosimilar product profile of an in-house product compare to an innovator.