Abstract
Cation-exchange chromatography (CEX) is widely used for analysis of charge heterogeneity in monoclonal antibodies (mAbs) in the biopharmaceutical industry. Charge variant separation is typically achieved by either alteration of ionic strength or pH, and detection of the various species is performed using diode array detectors (DAD) at 280 nm. In this study, we investigate the suitability of fluorescence detector (FLD) as a sensitive alternative to traditional UV280 detection-based charge variant analysis. The objective of this work is to compare the performance of charge variants detection through cation exchange chromatography between FLD and DAD detectors. This was demonstrated for a mass spectroscopy compatible, pH based CEX method utilizing volatile ammonium acetate salt. The stability of FLD detection was tested as per method validation criteria set in the ICH Q2-(R1). LODFLD and LOQFLD were 59.07 and 59.64 times lower in comparison to LODUV and LOQUV, respectively (LODFLD: 0.13 µg, LOQFLD: 0.39 µg). The LinearityFLD was obtained in the range of 1–200 µg. Accuracy calculated as recovery value was between 93.38% and 103.35% and highest RSD value obtained for robustness was 2.54% (area under the curve). The application of FLD detection based CEX as a Process Analytical Technology (PAT) enabler in mAb biopharmaceutical analysis was demonstrated through direct charge variant profiling of CHO cell harvest supernatant (without pre purification through Pro-A). The method was also applied to and found suitable for biosimilarity assessment of drug products. While the present study is focused on analysis of trastuzumab biosimilars, the proposed method is expected to be applicable to any mAb product after suitable gradient modification.
Published Version
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