Statistical Equivalence Testing of Higher-Order Protein Structures with Differential Hydrogen Exchange-Mass Spectrometry (HX-MS)
Statistical Equivalence Testing of Higher-Order Protein Structures with Differential Hydrogen Exchange-Mass Spectrometry (HX-MS)
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DOI：
10.1021/acs.analchem.0c05279
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发表时间：
2021-04-29
影响因子：
7.4
通讯作者：
Weis, David D.
中科院分区：
文献类型：
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作者：
Hageman, Tyler S.;Wrigley, Michael S.;Weis, David D.
氢交换质谱法（HX-MS）因其在建立蛋白质高阶结构的等效性方面的潜在效用而被广泛认可，特别是在可比性和相似性方面。然而，最近在HX- ms数据统计分析方面的进展将重点放在了显著性检验上，以确定两种或多种蛋白质状态之间HX有显著差异的蛋白质区域。在涉及评估不同蛋白质样品（如生物类似药）的高阶结构的相似性或等效性的情况下，HX-MS数据的显著性检验是不合适的。为了满足这一需求，我们对HX-MS数据采用了单变量双单侧检验（TOST）等效检验方法。等效接受标准是通过随机重新采样真实等效样本的最大偏差来确定的，以定义混合等效标准（真实等效的最大偏差，MDTE）。将TOST-MDTE测试应用于野生型和突变型麦芽糖结合蛋白的HX-MS差异测量表明，等效测试方法是合适的。基于差异HX-MS测量，发现三种英夫利昔单抗生物类似药（Remsima， Renflexis和Inflectra）与其Remicade参考产品等效，而5%去糖基化的NIST单抗与未修饰的NIST单抗参考产品在统计学上不等效。
Hydrogen exchange-mass spectrometry (HX-MS) is widely recognized for its potential utility for establishing the equivalence of the higher-order structures of proteins, particularly in comparability and similarity contexts. However, recent progress in the statistical analysis of HX-MS data has instead placed an emphasis on significance testing to identify regions of proteins where there are significant differences in HX between two or more protein states. In the cases involving assessment of similarity or equivalence of the higher-order structure of different protein samples (e.g., biosimilars), significance testing of HX-MS data is unsuitable. To meet this need, we have adapted the univariate two one-sided test (TOST) equivalence testing method for HX-MS data. Equivalence acceptance criteria were determined using maximum deviations from randomized resampling of truly equivalent samples to define hybrid equivalence criteria (maximum deviation of true equivalents, MDTE). Application of the TOST-MDTE test on differential HX-MS measurements of wild-type and mutated maltose-binding proteins demonstrates that the equivalence testing method was fit-for-purpose. Three infliximab biosimilars (Remsima, Renflexis, and Inflectra) were found to be equivalent to their Remicade reference product based on differential HX-MS measurements, while 5% deglycosylated NIST mAb was not statistically equivalent to the unmodified NIST mAb reference.