Validation of LC-MS/MS bioanalytical methods for protein therapeutics.
Validation of LC-MS/MS bioanalytical methods for protein therapeutics.
复制标题
蛋白质治疗的 LC-MS/MS 生物分析方法的验证。
DOI：
10.4155/bio.14.125
复制
发表时间：
2014
期刊：
影响因子：
1.8
通讯作者：
Jeffrey X. Duggan
中科院分区：
文献类型：
--
作者：
F. Vazvaei;Jeffrey X. Duggan
背景目前有超过100种蛋白质和肽类药物获得美国FDA批准，目前全球制药公司正在积极开发超过100种药物[1]。蛋白质治疗剂的定量传统上依赖于配体结合测定（LBA）。如果使用适当的试剂适当地开发，这样的测定可以是非常灵敏和选择性的，但是由于循环配体、抗药抗体（ADA）、交叉反应和各种形式的非特异性结合而对干扰敏感。LC-MS/MS分析已成为蛋白质生物分析的有效定量工具。特别令人感兴趣的是通过定量酶消化产生的独特替代肽对治疗性蛋白质进行LC-MS/MS分析，这是一种广泛使用的技术[1-4]。为简单起见，我们将这种类型的分析称为蛋白质消化LC-MS/MS（PrD-LC-MS）。PrD-LC- MS可用作LBA的替代和补充方法，近年来已广泛讨论[1-4]。此类LC-MS/MS方法（其中蛋白质生物酶在LC-MS/MS之前直接在生物基质中消化）可提供ADA和其他循环配体干扰较低的生物分析数据[5]。这些方法可以产生更有代表性的总药物定量，但它们可能具有有限的灵敏度。在需要更高灵敏度的情况下，可以在[6]或[7]蛋白水解消化之前或之后添加分析物富集或亲和捕获浓缩步骤。这些富集方法可以通过消除来自生物基质本身或来自肽LC-MS/MS之前的消化物的背景信号来提高灵敏度。随着PrD-LC-MS方法的成熟，将越来越需要在受监管的环境中使用这些测定法，用于药物非临床研究质量管理规范（GLP）毒理学研究和临床试验中的受监管样品生物分析。最近的EMA指南[8]和FDA生物分析方法验证（BMV）指南草案[9]均未专门讨论PrD-LC-MS的验证或其在受监管生物分析中的应用。对于任何受监管的PrD-LC-MS分析验证，需要解决几个问题：
Background There are more than 100 protein and peptide drugs currently approved by the US FDA and now over 100 more are currently in active development by pharmaceutical companies worldwide [1]. The quantification of protein therapeutics has traditionally relied upon ligand-binding assays (LBAs). Such assays can be very sensitive and selective if properly developed using appropriate reagents, but are nevertheless susceptible to interferences due to circulating ligands, antidrug antibodies (ADAs), cross reactions and various forms of non-specific binding. LC– MS/MS analysis has emerged as an effective quantitative tool for protein bioanalysis. Of particular interest is LC–MS/MS analysis of therapeutic proteins via quantification of unique surrogate peptides that are produced by enzymatic digestion, a technique that is being widely used [1–4]. For simplicity, we will call this type of analysis protein digestion LC–MS/MS (PrD-LC–MS). PrD-LC– MS can be used as an alternative and supplemental approach to LBAs, and this has been extensively discussed in recent years [1–4]. Such LC–MS/MS methods, where the protein biotherapeutic is digested directly in the biomatrix prior to LC–MS/MS, can provide bioanalytical data with low interference from ADAs and other circulating ligands [5]. These methods can yield more representative quantification of total drug, but they may have limited sensitivity. Where higher sensitivity has been required, analyte enrichment or affinity capture concentration steps can be added either before [6] or after [7] proteolytic digestion. These enrichment methods can improve sensitivity by eliminating background signals either from the biomatrix itself or from the digest prior to peptide LC–MS/MS. As the methodology for PrD-LC–MS matures, there will be an increased need to use these assays in a regulated environment, both for good laboratory practice (GLP) toxicology studies and for regulated sample bioanalysis in clinical trials. Neither the recent EMA guidance [8], nor the FDA draft Bioanalytical Method Validation (BMV) guidance [9] has specifically discussed the validation of PrD-LC–MS or its application to regulated bioanalysis. Several questions need to be addressed for any regulated PrD-LC–MS assay validation: