Development of an Efficient Ion-Exchange Chromatography Method for Charge Variant Analysis of Monoclonal Antibodies within the Isoelectric Point Range of 7.3–9.4

Abstract

Background: Monoclonal antibodies (mAbs) are biopharmaceuticals used as targeted therapies for immune-related diseases. These large protein molecules, produced through biotechnological processes, possess structural complexity and require stringent manufacturing controls. Variability in production or storage can lead to molecular heterogeneity and charge variants, which may affect critical product attributes such as stability, biological activity, and immunogenicity. Therefore, charge variant analysis is an essential step in quality assurance. Although ion-exchange chromatography (IEC), particularly using pH-gradient separation, is commonly employed for this purpose, it has not yet been established as a compendial standard method, and supporting data remain limited. Consequently, method development and validation are required to ensure effective quality control of mAbs, enabling broader application to various antibodies with different isoelectric points (pI).

Objectives: This study aimed to develop a charge variant analysis method using ion-exchange chromatography (IEX) for monoclonal antibodies (mAbs) with an isoelectric points (pI) ranging from 7.3 to 9.4. This study further aimed to validate the method according to international standards to ensure its suitability for the routine quality control of monoclonal antibodies marketed domestically.

Methods: This experimental study, conducted from October 2024 to July 2025, aimed to optimize and validate an analytical method for the charge variant profiling of seven monoclonal antibodies (mAbs)—including Omalizumab, Infliximab, Atezolizumab, Adalimumab, Pertuzumab, Trastuzumab, and Rituximab—with pI ranging from 7.3 to 9.4. Utilizing ion-exchange chromatography, the method development focused on achieving optimal separation conditions, followed by a rigorous validation process in accordance with regulatory standards. The method was evaluated based on critical parameters, including specificity, precision, accuracy, linearity, limit of detection (LOD), limit of quantitation (LOQ), and robustness, to ensure its suitability for the quality control of commercially available monoclonal antibodies in the domestic market.

Results: The developed method successfully characterized charge variants of mAbs with pI ranging from 7.3 to 9.4. It demonstrated high specificity and precision, with intra- and inter-day % RSD values below 5 % and no significant differences observed across instruments or analysts. Linearity was confirmed within the concentration range of 0.50–8.00 mg/ml, with accuracy between 90–110 %. The limits of quantitation was 1.00 mg/ml, while the limits of detection were 0.30 mg/ml, respectively. The method also proved robust under variations in column temperature and mobile phase stability for up to 7 days.

Conclusions: The ion-exchange chromatography method demonstrated specificity, accuracy, precision, linearity, and robustness, and was applicable for charge variant analysis of diverse mAbs with pI ranging from 7.3 to 9.4. This validated method is suitable as a standard procedure for quality control and regulatory submission of monoclonal antibody products in Thailand