Analytical Development, Johnson and Johnson Pharmaceutical Research and Development, Malvern, PA, USA.
J Pharm Biomed Anal. 2010 Dec 15;53(5):1236-43. doi: 10.1016/j.jpba.2010.07.029. Epub 2010 Jul 30.
A capillary sodium dodecyl sulfate gel electrophoresis (cSDS) method has been developed and qualified for purity and impurity analysis of monoclonal antibodies. This method was optimized and qualified for the analysis of monoclonal antibody (mAb1) under reduced and non-reduced conditions. Some of the sample preparation parameters including sample buffer pH, incubation temperature and duration, alkylation conditions with iodoacetamide (IAM), and reduction conditions with 2-mercaptoethanol (2-ME) were optimized. It was observed that under slightly acidic conditions (pH 5.5-6.5) the thermally induced fragmentation of non-reduced mAb1 was greatly decreased. As such, a citrate-phosphate buffer at pH 6.5 was used for sample preparation to replace the original Beckman sample buffer (pH 9.0). The optimal sample preparation conditions were found to be as follows: (1) incubation temperature and duration (reduced and non-reduced), 65 degrees C for 5 min; (2) alkylation condition, 10 microL of 0.25 M IAM; (3) reduction condition, 10 microL of 5-fold diluted 2-ME. The method was qualified by evaluating specificity, accuracy, precision, limit of quantitation (LOQ), and linearity. The method exhibited no interference from sample buffer matrix. The method was found to be linear, accurate, and precise in the range of 0.25-3.0mg/mL protein concentration. The LOQ of the method was determined to be 0.02 mg/mL for reduced and non-reduced mAb1. In addition, some aspects of sample stability were examined during qualification.
已开发并验证了一种毛细管十二烷基硫酸钠凝胶电泳（cSDS）方法，用于分析单克隆抗体的纯度和杂质。该方法经过优化和验证，可用于分析还原和非还原条件下的单克隆抗体（mAb1）。对一些样品制备参数进行了优化，包括样品缓冲液 pH 值、孵育温度和时间、碘乙酰胺（IAM）的烷基化条件以及 2-巯基乙醇（2-ME）的还原条件。结果表明，在略酸性条件（pH 5.5-6.5）下，非还原 mAb1 的热诱导片段化大大减少。因此，使用柠檬酸盐-磷酸盐缓冲液（pH 6.5）替代原始 Beckman 样品缓冲液（pH 9.0）进行样品制备。优化后的样品制备条件如下：（1）孵育温度和时间（还原和非还原），65°C 5 分钟；（2）烷基化条件，10μL 0.25M IAM；（3）还原条件，10μL 5 倍稀释的 2-ME。通过评估特异性、准确性、精密度、定量限（LOQ）和线性来验证方法。该方法未显示出样品缓冲液基质的干扰。该方法在 0.25-3.0mg/mL 蛋白浓度范围内表现出线性、准确和精密度。该方法的 LOQ 分别为 0.02mg/mL 还原和非还原 mAb1。此外，在验证过程中还检查了样品稳定性的一些方面。
