Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Inc. , West Point, Pennsylvania, United States.
Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Inc. , Palo Alto, California, United States.
Anal Chem. 2018 Jan 2;90(1):866-871. doi: 10.1021/acs.analchem.7b03716. Epub 2017 Dec 20.
Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents. The reagents were selective for the heavy and light chain of a monoclonal antibody, which when used consecutively, ensures that only the intact form of the antibody is represented by the surrogate peptide. The approach provided comparable results to a traditional sandwich IA assay indicating similar capture populations. The use of an initial ISAC assessment of affinity capture purification, should add a degree of confidence in the use of a single IA-LC-MS/MS quantitation assay.
LC-MS/MS 通常通过自上而下或所谓的替代肽测量来定量大分子，以推断整个分子的浓度。这可能会导致对测定中实际测量的内容（完整药物和/或其他相关药物物质）产生疑问。本研究开发了一种完整的顺序亲和捕获（ISAC）测定法，使用了两种不同的免疫亲和（IA）试剂。这些试剂对单克隆抗体的重链和轻链具有选择性，连续使用时，可确保仅由替代肽代表完整形式的抗体。该方法与传统的夹心 IA 测定法相比提供了可比的结果，表明具有相似的捕获群体。在使用单一 IA-LC-MS/MS 定量测定法时，使用初始 ISAC 评估亲和捕获纯化，应该可以增加对其的信心。
