MRC Centre for Drug Safety Science, Department of Pharmacology & Therapeutics, University of Liverpool, Liverpool, United Kingdom.
Mol Pharm. 2012 May 7;9(5):1291-301. doi: 10.1021/mp200587m. Epub 2012 Apr 18.
PEGylation of therapeutic proteins is commonly used to extend half-lives and to reduce immunogenicity. However, reports of antibodies toward PEGylated proteins and of poly(ethylene glycol) (PEG) accumulation suggest that efficacy and safety concerns may arise. To understand the relationship among the pharmacology, immunogenicity, and toxicology of PEGylated proteins, we require knowledge of the disposition and metabolic fate of both the drug and the polymer moieties. The analysis of PEG by standard spectrophotometric or mass spectrometric techniques is problematic. Consequently, we have examined and compared two independent analytical approaches, based on gel electrophoresis and nuclear magnetic resonance (NMR) spectroscopy, to determine the biological fate of a model PEGylated protein, (40K)PEG-insulin, within a rat model. Both immunoblotting with an antibody to PEG and NMR analyses (LOD 0.5 μg/mL for both assays) indicated that the PEG moiety remained detectable for several weeks in both serum and urine following intravenous administration of (40K)PEG-insulin (4 mg/kg). In contrast, Western blotting with anti-insulin IgG indicated that the terminal half-life of the insulin moiety was far shorter than that of the PEG, providing clear evidence of conjugate cleavage. The application of combined analytical techniques in this way thus allows simultaneous independent monitoring of both protein and polymer elements of a PEGylated molecule. These methodologies also provide direct evidence for cleavage and definition of the chemical species present in biological fluids which may have toxicological consequences due to unconjugated PEG accumulation or immunogenic recognition of the uncoupled protein.
聚乙二醇（PEG）化治疗性蛋白通常用于延长半衰期和降低免疫原性。然而，关于针对聚乙二醇化蛋白的抗体和聚乙二醇（PEG）蓄积的报道表明，可能会出现疗效和安全性问题。为了了解聚乙二醇化蛋白的药理学、免疫原性和毒理学之间的关系，我们需要了解药物和聚合物部分的处置和代谢命运。使用标准分光光度法或质谱法分析 PEG 存在问题。因此，我们检查并比较了两种独立的分析方法，基于凝胶电泳和核磁共振（NMR）光谱，以确定模型聚乙二醇化蛋白（40K）PEG-胰岛素在大鼠模型中的生物学命运。用针对 PEG 的抗体进行免疫印迹和 NMR 分析（两种测定的检测限均为 0.5 μg/mL）均表明，在静脉内给予（40K）PEG-胰岛素（4 mg/kg）后，PEG 部分在血清和尿液中可检测数周。相比之下，用抗胰岛素 IgG 的 Western blot 分析表明，胰岛素部分的半衰期远短于 PEG，这提供了明显的缀合物断裂的证据。以这种方式应用组合分析技术可以同时独立监测聚乙二醇化分子的蛋白质和聚合物部分。这些方法还为生物流体中存在的化学物质的断裂和定义提供了直接证据，由于未结合的 PEG 蓄积或未偶联的蛋白质的免疫识别，这些化学物质可能具有毒理学后果。
