Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan.
J Biosci Bioeng. 2013 Jan;115(1):104-10. doi: 10.1016/j.jbiosc.2012.07.021. Epub 2012 Aug 24.
In the pharmaceutical industry, analysis of soluble aggregates in pharmaceutical formulations is most commonly performed using size-exclusion chromatography (SEC). However, owing to concerns that aggregates can be overlooked by SEC analysis, it has been suggested that its results should be confirmed with orthogonal methods. One of the main alternative methods for SEC is analytical ultracentrifugation sedimentation velocity (AUC-SV), which has been indicated as an important tool for the measurement of protein aggregation. The present study aimed to show that AUC-SV can be effectively applied for the characterization of marketed immunoglobulin pharmaceutical preparations to support the results obtained by SEC. In addition, the present research aimed to assess the appropriateness of two integration approaches for the quantitative analysis of the SEC results. Thus, the aggregates were measured in seven different preparations of human immunoglobulins by AUC-SV and SEC, and the acquired chromatographic data were processed by using either the vertical drop method or the Gaussian skim approach, implemented in the Empower II chromatography data software (Waters, Tokyo, Japan). The results of aggregation measurements performed using AUC-SV were in good agreement with those obtained using SEC. As expected, the Gaussian skim integration approach inherently provided lower estimates of aggregation content than the results of the vertical drop method. The finding of this study confirmed the complementary nature of AUC-SV to SEC for aggregate composition analysis and underscored the important role that the different integration methods can play in the quantitative interpretation of chromatographic results.
在制药行业中，药物制剂中可溶性聚集体的分析最常使用尺寸排阻色谱法（SEC）进行。然而，由于担心 SEC 分析可能会忽略聚集体，因此有人建议应使用正交方法对其结果进行确认。SEC 的主要替代方法之一是分析超速离心沉降速度（AUC-SV），它已被证明是测量蛋白质聚集的重要工具。本研究旨在表明 AUC-SV 可有效地应用于市售免疫球蛋白药物制剂的特性描述，以支持 SEC 获得的结果。此外，本研究旨在评估两种定量分析 SEC 结果的积分方法的适用性。因此，通过 AUC-SV 和 SEC 测量了七种不同的人免疫球蛋白制剂中的聚集体，并用垂直下降法或高斯削峰法对获得的色谱数据进行处理，高斯削峰法在 Empower II 色谱数据软件（Waters，东京，日本）中实现。使用 AUC-SV 进行的聚集测量结果与使用 SEC 获得的结果非常吻合。正如预期的那样，高斯削峰积分方法固有地提供了比垂直下降法更低的聚集含量估计值。这项研究的结果证实了 AUC-SV 与 SEC 对聚集体组成分析的互补性，并强调了不同积分方法在色谱结果定量解释中所起的重要作用。
