A Novel and Cost Effective Method of Removing Excess Albumin from Plasma/Serum Samples and Its Impacts on LC-MS/MS Bioanalysis of Therapeutic Proteins
A Novel and Cost Effective Method of Removing Excess Albumin from Plasma/Serum Samples and Its Impacts on LC-MS/MS Bioanalysis of Therapeutic Proteins
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DOI：
10.1021/ac501837t
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发表时间：
2014-08-19
影响因子：
7.4
通讯作者：
Shen, Jim X.
中科院分区：
文献类型：
--
作者：
Liu, Guowen;Zhao, Yue;Shen, Jim X.
我们开发了一种从血浆/血清样品中去除白蛋白的创新方法，用于治疗性蛋白质的 LC-MS/MS 定量。筛选了有机溶剂和酸的不同组合从血浆和血清样品中去除白蛋白的能力。通过白蛋白中的两种特征肽（QTALVELVK 和 LVNEVTEFAK）监测去除效率。发现异丙醇与 1.0% 三氯乙酸是去除白蛋白同时保留目标蛋白的最有效组合。我们的方法与商业白蛋白去除试剂盒在白蛋白去除效率和目标蛋白回收率方面进行了比较。我们已经证明，我们的方法可以去除人血浆样本中 95% 的总白蛋白，同时对测试的三种治疗蛋白中的两种蛋白保留接近 100%，第三种蛋白的保留率为 60-80%。与我们的方法相比，商业试剂盒去除了 98% 的白蛋白，但所有治疗性蛋白质的回收率损失了至少 50%。与单独的全血浆蛋白消化方法相比，使用 BMS-C 作为探针化合物，结合白蛋白去除方法提高了测定灵敏度和耐用性。基于这种新方法开发并验证了 LC-MS/MS 方法，用于分析猴血清中的 BMS-C。该测定已成功应用于毒理学研究。当白蛋白去除方法用于另一种临床 LC-MS/MS 方法时，与典型的沉淀消化方法相比，灵敏度提高了 10 倍，达到 50 ng/mL LLOQ。
We have developed an innovative method to remove albumin from plasma/serum samples for the LC-MS/MS quantitation of therapeutic proteins. Different combinations of organic solvents and acids were screened for their ability to remove albumin from plasma and serum samples. Removal efficiency was monitored by two signature peptides (QTALVELVK and LVNEVTEFAK) from albumin Isopropanol with 1.0% tridiloroacetic acid was found to be the most effective combination to remove albumin while retaining the protein of interest. Our approach was compared with a commercial albumin depletion kit on both efficiency of albumin removal and recovery of target proteins. We have demonstrated that our approach can remove 95% of the total albumin in human plasma samples while retaining close to 100% for two of three therapeutic proteins tested, with the third one at 60-80%. The commercial kit removed 98% of albumin but suffered at least 50% recovery loss for all therapeutic proteins when compared to our approach. Using BMS-C as a probe compound, the incorporation of the albumin removal approach has improved both assay sensitivity and ruggedness, compared to the whole plasma protein digestion approach alone. An LC-MS/MS method was developed and validated based on this new approach for the analysis of BMS-C in monkey serum. This assay was successfully applied to a toxicological study. When the albumin removal method was used in another clinical LC-MS/MS method, the sensitivity improved 10-fold to 50 ng/mL LLOQ comparing to a typical pellet digestion method.