Department of Chemical Engineering, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, 110016, India.
Department of Chemical Engineering, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, 110016, India.
Talanta. 2024 Aug 15;276:126232. doi: 10.1016/j.talanta.2024.126232. Epub 2024 May 10.
Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60-90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 μg to 160 μg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.
在生物制药开发中，对异质性进行稳健监测对于生产安全有效的生物治疗产品至关重要。多属性监测 (MAM) 已成为监测 mAb 异质性（如脱酰胺、唾液酸化、糖基化和氧化）的有效工具。在 mAb 开发过程中，传统的生物制药分析依赖于使用一维方法，使用非挥发性溶剂监测效价和基于电荷的异质性，而不与质谱 (MS) 直接耦合。这种方法需要通过 ProA 分析 mAb 收获物来估计效价，然后对纯化样品进行单独的阳离子交换色谱 (CEX) 分析，以估计基于电荷的异质性。由于需要进行馏分收集和缓冲液交换步骤，因此这可能需要长达 60-90 分钟。在这项工作中，开发了一种基于二维液相色谱 (2DLC) 和质谱的方法，其中第一维为蛋白 A 色谱，用于估计效价，第二维为阳离子交换色谱 (CEX)，用于分析电荷变异体。该方法在两个维度上都使用挥发性盐，并且易于与 MS 耦合。所提出的 2DLC 方法显示出与传统方法相似的电荷变异体谱，并且每个变体的质谱鉴定仅需 15 分钟。在效价估计后，通过 CEX 分析分离出六种电荷变体，包括从 5 μg 到 160 μg 注射 mAb 样品的线性评估。该方法成功地对 mAb 创新药及其 4 种生物类似药的电荷变异体进行了估计，展示了其在生物类似药研究中的适用性。因此，2D ProA CEX MS 方法允许在单个工作流程中直接估计 mAb 的效价和电荷变异体。
