Abstract：
The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry (LC-MS) peptide mapping technique that has been proposed as a replacement for several conventional quality control (QC) methods for therapeutic proteins. In addition to quantification of multiple product quality attributes (PQAs), MAM can also monitor impurities using a new peak detection (NPD) feature. Here, results are provided from method validation and NPD studies of an MAM approach applied to rituximab as a model monoclonal antibody (mAb). Twenty-one rituximab PQAs were monitored, including oxidation, pyroglutamination, deamidation, lysine clipping, and glycosylation. The PQA monitoring aspect of the method was validated according to ICH Guidance. Accuracy, precision, specificity, detection and quantitation limits, linearity, range, and robustness were demonstrated for this MAM approach with minimal issues. All PQAs were successfully validated except for several oxidation sites, which did not pass intermediate precision criteria. The variability found in oxidation measurements was attributed to artificial oxidation during sample preparation and could likely be alleviated through several approaches. The NPD aspect of the method was also evaluated. A spike-in approach was used to assess the limits of detection and quantitation (LOD/LOQ) of the NPD feature of MAM. For NPD, the peak intensity threshold was found to be the most critical parameter for accurate detection of impurities since a low threshold can result in false positives while a high threshold can obscure the detection of true peaks. Overall, the MAM approach presented and validated here has been demonstrated to be suitable for both targeted monitoring of rituximab PQAs and non-targeted detection of new peaks that represent impurities.
摘要：
多属性方法（MAM）是一种液相色谱-质谱（LC-MS）肽作图技术，已被提议替代治疗性蛋白质的几种常规质量控制（QC）方法。除了量化多个产品质量属性(PQA)之外，MAM还可以使用新的峰值检测(NPD)功能监测杂质。这里,结果来自方法验证和应用于利妥昔单抗作为模型单克隆抗体(mAb)的MAM方法的NPD研究.监测21名利妥昔单抗PQA，包括氧化，焦谷氨酰胺，脱酰胺,赖氨酸剪切，和糖基化。该方法的PQA监测方面根据ICH指南进行了验证。准确性,精度,特异性，检测和定量限，线性度范围,并证明了这种MAM方法的鲁棒性，问题最少。除几个氧化位点外，所有PQA均已成功验证，没有通过中间精度标准。氧化测量中发现的可变性归因于样品制备过程中的人工氧化，并且可能通过几种方法来缓解。还评估了该方法的NPD方面。使用加标方法来评估MAM的NPD特征的检测和定量极限（LOD/LOQ）。对于NPD来说,发现峰强度阈值是准确检测杂质的最关键参数,因为低阈值可导致假阳性,而高阈值可模糊真实峰的检测。总的来说,本文提出并验证的MAM方法已被证明既适用于利妥昔单抗PQA的靶向监测,也适用于代表杂质的新峰的非靶向检测.
