Abstract

Pharmaceutical application of therapeutic proteins has been continuously expanded for the treatment of various diseases. Efficient and reliable bioanalytical methods are essential to expedite the identification and successful clinical development of therapeutic proteins. In particular, selective quantitative assays in a high-throughput format are critical for the pharmacokinetic and pharmacodynamic evaluation of protein drugs and to meet the regulatory requirements for new drug approval. However, the inherent complexity of proteins and many interfering substances presented in biological matrices have a great impact on the specificity, sensitivity, accuracy, and robustness of analytical assays, thereby hindering the quantification of proteins. To overcome these issues, various protein assays and sample preparation methods are currently available in a medium- or high-throughput format. While there is no standard or universal approach suitable for all circumstances, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay often becomes a method of choice for the identification and quantitative analysis of therapeutic proteins in complex biological samples, owing to its high sensitivity, specificity, and throughput. Accordingly, its application as an essential analytical tool is continuously expanded in pharmaceutical R&D processes. Proper sample preparation is also important since clean samples can minimize the interference from co-existing substances and improve the specificity and sensitivity of LC-MS/MS assays. A combination of different methods can be utilized to improve bioanalytical performance and ensure more accurate quantification. This review provides an overview of various protein assays and sample preparation methods, with particular emphasis on quantitative protein analysis by LC-MS/MS.

摘要

治疗性蛋白质的药物应用不断扩大，用于治疗各种疾病。高效可靠的生物分析方法对于加快治疗性蛋白质的鉴定和成功临床开发至关重要。特别是，高通量格式的选择性定量分析对于蛋白质药物的药代动力学和药效学评估以及满足新药批准的监管要求至关重要。然而，蛋白质固有的复杂性和生物基质中存在的许多干扰物质对分析检测的特异性、灵敏度、准确性和稳健性有很大影响，从而阻碍了蛋白质的定量。为了克服这些问题，各种蛋白质测定和样品制备方法目前以中等或高通量形式提供。虽然没有适用于所有情况的标准或通用方法，但液相色谱-串联质谱 (LC-MS/MS) 测定通常成为复杂生物样品中治疗性蛋白质鉴定和定量分析的首选方法，因为其高灵敏度、特异性和通量。因此，其作为基本分析工具的应用在药物研发过程中不断扩展。正确的样品制备也很重要，因为干净的样品可以最大限度地减少共存物质的干扰，并提高 LC-MS/MS 分析的特异性和灵敏度。可以利用不同方法的组合来提高生物分析性能并确保更准确的量化。本综述概述了各种蛋白质测定和样品制备方法，特别强调了通过 LC-MS/MS 进行的定量蛋白质分析。