Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
J Am Soc Mass Spectrom. 2017 May;28(5):811-817. doi: 10.1007/s13361-016-1566-y. Epub 2016 Dec 21.
As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure. Graphical Abstract ᅟ.
随着治疗性单克隆抗体（mAbs）变得更加人性化，用于测量血清中生物制剂的传统胰蛋白酶肽方法变得更加具有挑战性，因为用于定量 mAb 的独特克隆型肽也可能存在于正常血清多克隆背景中。另一种方法是使用液相色谱-质谱（LC-MS）监测 mAb 完整轻链部分的独特分子量。当确定血清中的定量下限（LOQ）时，区分治疗性 mAb 与患者正常的多克隆免疫球蛋白（Ig）谱是主要的限制因素。从血清中选择性提取亚类特异性 Ig 的能力可降低样品中的多克隆背景。我们在此介绍了一种用于定量血清中依库珠单抗的 LC-MS 方法。依库珠单抗是一种补体成分 5（C5）结合 mAb，它完全人源化，包含 IgG2 和 IgG4 亚类的部分。我们的团队开发了一种使用 Life Technologies CaptureSelect IgG4（Hu）亲和基质的方法。我们在这里展示了通过从多克隆背景中去除更高丰度的 IgG1、IgG2 和 IgG3 来定量依库珠单抗的能力，LOQ 为 5 mcg/mL，使这种方法成为一种简单有效的方法。
