LC-MS/MS approach therapeutic proteins for quantification of in plasma using a protein internal standard and 2D-solid-phase extraction cleanup
LC-MS/MS approach therapeutic proteins for quantification of in plasma using a protein internal standard and 2D-solid-phase extraction cleanup
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DOI：
10.1021/ac0712502
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发表时间：
2007-12-15
影响因子：
7.4
通讯作者：
Tse, Francis L. S.
中科院分区：
文献类型：
--
作者：
Yang, Ziping;Hayes, Michael;Tse, Francis L. S.
对体内治疗蛋白研究产生的血浆样本的生物分析在生物制药行业中越来越受关注。传统的酶联免疫吸附试验方法检测时间较长，限制了蛋白质类药物的早期发现和开发。在这项研究中，建立了一种LC-MS/MS生物测定方法，用于测定生长激素和一种治疗性的人类单抗。该方法以牛胎蛋白为内标，采用二维固相萃取法净化血浆消化液。样品萃取物在分析尺寸的色谱柱上使用6分钟的LC梯度进行解析，并使用三重四重质谱仪进行分析。生长激素的线性范围为1~1000µg/m L，准确度和精密度均在15%以内。该LC-MS方法还用于治疗性单抗的大鼠药代动力学研究，其定量下限为0.5微克/毫升。该方法具有更高的准确度和精密度，对体内研究样品的分析结果与用ELISA法获得的数据具有良好的一致性。本研究的结果表明，LC-MS生物分析是一种简单可行的方法，可用于治疗蛋白的生物分析，以支持早期药物发现和开发中的体内研究。
The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC-MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 mu g/mL with accuracy and precision within 15%. This LC-MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 mu g/mL. The LCMS assay had improved accuracy and precision, and the results from analysis of in vivo study samples showed good agreement with the data obtained with an ELISA. The results from this study indicate that the LC-MS bioassay is a simple and feasible approach for the bioanalysis of therapeutic proteins to support in vivo studies during early drug discovery and development.