LC-QDa系统监控阿达木单克隆抗体方法研究

发布时间：2019-02-21 19:27

【摘要】：随着生物药研究快速发展,到目前为止有61个抗体药物上市,但抗体药物价格昂贵,各国医保负担重;第一代重磅抗体药物专利相继到期,高性价比的生物类似药被赋予期待;另外技术上新型抗体药物层出不穷,双特异性抗体、ADC药物、抗体片段、融合蛋白技术平台等,诸多新技术推动抗体药物的快速发展。在抗体药物研发及生产中需要连续监控保证药物的安全性,以更好地快速推动研发的进展,降低研发周期。然而,当前肽图谱分析平台,例如LC-MS-Tof,LC-TQ,存在价格昂贵,操作复杂,样品处理要求高等问题。QDa检测器作为操作方便、高性价比、稳定的单四级杆检测器,在生物药研究与生产过程中可作为一种常规分析方法。利用UPLC系统,TUV光学特性与QDa质谱特性,本研究发展并验证一种新的分析生物药的肽图谱方法。并探讨本方法在实验室交叉污染溯源上应用,作为常规实验室管理方法。首先,采用LC-Q-Tof方法确定源于阿达木单抗CDR区8个肽段作为特异性肽段代表整个抗体用于分析研究。针对QDa系统对8个肽段信息进行优化,得到在QDa系统中响应值最高的质核比数据;另外评估阿达木单抗最佳酶切条件,包括蛋白酶切比例与最佳酶切时间,最后得到最佳酶切条件为37℃,1:10,3h。10个其他抗体,包括曲妥珠单抗、西妥昔单抗、德尼单抗、奥法木单抗、戈利木单抗、Evolocumab、阿仑单抗、伊匹单抗、纳武单抗、Pembrolizumab用于评估方法特异性,结果均为发现非特异性。由于UV-MS的敏感性,LC-QDa系统具有高特异性;此系统的检测限为20μg/mL;样品残留为1.69%。此外,其它的确认参数,包括稳定性等也被评估。结果表明,新的LC-UV-QDa方法作为一种简单、高性价比、稳定的肽图谱方法适用于常规质量控制分析中。最后我们采用此方法检测模拟的阿达木单抗、西妥昔单抗和曲妥珠交叉污染样品,成功检测到目标蛋白阿达木单抗及溯源检测到西妥昔、曲妥珠单抗。
[Abstract]:With the rapid development of biopharmaceutical research, 61 antibody drugs have been put on the market so far, but the price of antibody drugs is expensive and the burden of medical insurance is heavy in many countries, the patent of the first generation of heavy antibody drugs has expired one after another, and the biological similar drugs with high performance and price ratio have been given expectation. In addition, new antibody drugs emerge in endlessly, bispecific antibodies, ADC drugs, antibody fragments, fusion protein technology platform, many new technologies promote the rapid development of antibody drugs. In order to accelerate the development and reduce the development cycle, it is necessary to continuously monitor and ensure the safety of the drug in the development and production of antibody drugs. However, the current peptide map analysis platform, such as LC-MS-Tof,LC-TQ, has some problems, such as high price, complex operation, high sample processing requirements and so on. QDa detector is used as a simple, cost-effective and stable single-step bar detector. It can be used as a conventional analytical method in the research and production of biopharmaceuticals. Using the UPLC system, TUV optical properties and QDa mass spectra, a new peptide map method for the analysis of biopharmaceuticals has been developed and verified. The application of this method in the traceability of laboratory cross-contamination is discussed as a routine laboratory management method. First, eight peptides derived from the CDR region of Adamu McAb were identified by LC-Q-Tof method as specific peptides representing the whole antibody for analysis. According to the QDa system, the information of eight peptide segments was optimized to obtain the highest response value of the core ratio data in the QDa system. In addition, the optimal digestion conditions of adamumab were evaluated, including the ratio of protease digestion and the optimal digestion time. Finally, the optimal digestion conditions were obtained as follows: 37 鈩，

本文编号：2427819