Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography-mass spectrometry
Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography-mass spectrometry
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DOI：
10.1016/j.ab.2011.03.004
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发表时间：
2011-07-01
影响因子：
2.9
通讯作者：
Tarcsa, Edit
中科院分区：
文献类型：
--
作者：
Liu, Hongcheng;Manuilov, Anton V.;Tarcsa, Edit
在研究中使用包括蛋白A纯化、有限Lys-C消化和质谱分析的方法来定量食蟹猴血清中的重组单克隆抗体。使用同位素标记的相同抗体作为内标。首先通过蛋白A纯化，然后通过蛋白A结合IgG（1）（包括猴和重组IgG）的有限Lys-C消化，显著降低血清蛋白的干扰。通过LC-MS直接分析重组人IgG的Fab片段，而猴IgG和重组人IgG的Fc片段仍与蛋白A树脂结合。通过测量来自重组人IgG的Fab的峰强度并将其与来自稳定同位素标记的内标物的Fab的峰强度进行比较来实现定量。结果与ELISA法测定结果一致。因此，LC-MS可用作ELISA的补充方法，以定量血清中的重组单克隆抗体，用于药代动力学研究，并且还可用于特异性试剂（如抗原）不易用于ELISA的情况。(c)2011 Elsevier Inc. All rights reserved.
A method including protein A purification, limited Lys-C digestion, and mass spectrometry analysis was used in the study to quantify a recombinant monoclonal antibody in cynomolgus monkey serum. The same antibody that was isotopically labeled was used as an internal standard. Interferences from serum proteins were first significantly reduced by protein A purification and then by limited Lys-C digestion of protein A bound IgG(1) including both monkey and the recombinant IgG. Fab fragment of the recombinant human IgG was analyzed directly by LC-MS, while monkey IgG and the Fc fragment of the recombinant human IgG remained bound to protein A resin. Quantitation was achieved by measuring the peak intensity of the Fab from the recombinant human IgG and comparing it to that of the Fab from the stable isotope-labeled internal standard. The results were in good agreement with the values from ELISA. LC-MS can therefore be used as a complementary approach to ELISA to quantify recombinant monoclonal antibodies in serum for pharmacokinetics studies and it can also be used where specific reagents such as antigens are not readily available for ELISA. (c) 2011 Elsevier Inc. All rights reserved.