Peters Björn-Hendrik, Bautista James, Slaney Thomas R, Guo Hongyue, Huang Richard Y-C, Krause Mary E, Zeng Ming, Cheng Julie, Chen Zhi
Drug Product Development, Bristol Myers Squibb, New Brunswick, New Jersey, USA.
Biologics Development, Bristol Myers Squibb, New Brunswick, New Jersey, USA.
Electrophoresis. 2022 May;43(9-10):1059-1067. doi: 10.1002/elps.202100259. Epub 2022 Apr 27.
Antibody-based therapeutic proteins have highly complex molecular structures. The final therapeutic protein product may contain a wide range of charge variants. Accurate analysis of this charge variant composition is critical to determine manufacturing process consistency and protein stability and ultimately helps to ensure that patients receive a safe and efficacious product. Here, a highly sialylated bispecific antibody (bsAb-1) challenged the ability to monitor stability by imaged capillary isoelectric focusing (iCIEF). This challenge was overcome by optimization of the iCIEF master mix buffer (adjustment of urea concentration, addition of l-arginine) and enzymatic removal of sialic acid. The method was qualified by assessing linearity, precision, LOD, LOQ, accuracy, and robustness in accordance with ICH guidance. Main species loss detectability increased up to approximately fivefold compared to the iCIEF method without desialylation when monitoring changes in stressed samples. Importantly, the results of the iCIEF method with desialylation correlated with results obtained through LC-MS tryptic peptide mapping and enabled analysis of formulation development stability samples. Finally, this analytical method shows the potential to assess low-concentration formulation development samples down to a sample concentration of 0.1 mg/ml.
基于抗体的治疗性蛋白质具有高度复杂的分子结构。最终的治疗性蛋白质产品可能包含多种电荷变体。准确分析这种电荷变体组成对于确定生产过程的一致性和蛋白质稳定性至关重要，最终有助于确保患者获得安全有效的产品。在此，一种高度唾液酸化的双特异性抗体（bsAb-1）对通过成像毛细管等电聚焦（iCIEF）监测稳定性的能力提出了挑战。通过优化iCIEF主混合缓冲液（调整尿素浓度、添加L-精氨酸）和酶促去除唾液酸克服了这一挑战。该方法根据ICH指南通过评估线性、精密度、检测限、定量限、准确度和稳健性进行了验证。在监测应激样品的变化时，与未去唾液酸化的iCIEF方法相比，主要物种损失的可检测性提高了约五倍。重要的是，去唾液酸化的iCIEF方法的结果与通过LC-MS胰蛋白酶肽图谱获得的结果相关，并能够分析制剂开发稳定性样品。最后，这种分析方法显示了评估低浓度制剂开发样品至0.1 mg/ml样品浓度的潜力。
