Huai-Hsuan Chiu (Presenter)
School of Pharmacy, National Taiwan University
Bio: My name is Huai-Hsuan Chiu, I am now a Ph.D student at the School of Pharmacy at the National Taiwan University. I got my bachelor¡¦s degree in 2011 from the National Central University, from the Department of Chemistry. In 2012, I joined a lab at the School of Pharmacy at the National Taiwan University, which focuses on tackling the issues within the fields of metabolomics and drug analysis. Our team utilizes mass spectrometery and capillary electrophoresis to develop analytical methods for both biomedical and pharmaceutical analysis. My research mainly focuses on the establishment of analytical methods for metabolomic studies and protein drug analysis. For special awards, I have received an honorable mention of poster presentation at the Annual Meeting of Chinese Chemical Society 2011, and the metabolomics society student travel award in 2014.
Authorship: Huai-Hsuan Chiu (1), I-Lin Tsai (2), Ching-Hua Kuo(1,3)
(1) School of Pharmacy, College of Medicine, National Taiwan University (2) Department of Biochemistry and Molecular Cell Biology, School of Medicine, Taipei Medical University (3) The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University
| Short Abstract Bevacizumab is a humanized immunoglobulin G (IgG) monoclonal antibody (mAb) used for metastatic colon cancer treatment. As LC-MS offers high selectivity and sensitivity, we developed a method on this platform to analyze mAb. The high complexity of human plasma is the main challenge in obtaining accurate quantification results in LC-MS methods. We used the human IgG purification method to isolate the analyte in human plasma. In-solution digestion was applied for protein digestion. Surrogate peptide was selected to quantify the bevacizumab concentration in human plasma. This proposed method was validated and used to quantify the bevacizumab concentration in patient plasma samples. |
Long Abstract
Introduction:
Bevacizumab is a humanized immunoglobulin G (IgG) monoclonal antibody (mAb) which works against human vascular endothelial growth factor (VEGF) that has been used in the treatment of many types of cancer, such as metastatic colon cancer, breast, and ovarian cancer. To understand the pharmacokinetics properties of the mAb, enzyme-linked immunosorbent assay (ELISA) is the most generally used technique. However, ELISA has its own limitations, for example, antibody product is resource- and time- consuming, there is an insufficient dynamic range, and the most serious, the selectivity can be easily affected by the presence of endogenous interference molecules in different matrices which lead to inaccurate quantification results. On the other hand, the use of liquid chromatography- mass spectrometry (LC-MS) to analyze mAb has been increasing due to its¡¦ rapid assay development, high selectivity and sensitivity. The general procedures for LC-MS based mAb quantification are divided into two parts: (1) digestion of the mAb to peptide levels, (2) selection of the surrogate peptides for quantification. However, high complexity of human plasma is one of the main challenges in obtaining accurate surrogate peptide quantification results when using LC-MS to analyze mAbs. Therefore, a good analytical method should require a suitable sample pre-treatment method and a reliable LC-MS platform for mAb quantification.
Methods: To improve sensitivity and selectivity, we used the human IgG purification method to isolate the analyte in human plasma. In-solution digestion was applied for protein digestion. Surrogate peptide was selected to quantify the bevacizumab concentration in human plasma by LC-MS. The mass spectrometry was performed in the multiple reaction monitoring mode (MRM). The LC-MS method was validated in terms of the linearity, accuracy and precision.
Results: The peptide with an m/z of 588 was selected as the surrogate peptide to provide selective quantification of bevacizumab in human plasma. Using commercial protein G magnetic beads to isolate the target analyte in human plasma greatly improved digestion efficiency and sensitivity. It also reduced the amount of interferences seen in human plasma. In-solution digestion containing reduction and alkylation of disulfide bonds provided more accessible sites for proteolysis. As a result, the concern of the digestion efficiency for the hard-to-digest regions of mAb could be eliminated. The validation results showed high precision and good accuracy. Finally, we used the established method to quantify the bevacizumab concentration in patient plasma samples.
Conclusions:
We developed a LC-MS method to quantify bevacizumab in human plasma using immunoglobulin G purification and in-solution digestion for sample pre-treatment. This sample pre-treatment method effectively trapped the target analyte (bevacizumab), and in-solution digestion displayed good digestion efficiency. The high selectivity of the surrogate peptide provided accurate quantification results which could be used for pharmacokinetic studies and therapeutic drug monitoring for bevacizumab.
References & Acknowledgements:
The authors appreciate the support from NTU Integrated Core Facility for Functional Genomics of National Research Program for Genomic Medicine of Taiwan and the Ministry of Science and Technology of Taiwan
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