【摘要】：隨著生物藥研究快速發(fā)展,到目前為止有61個抗體藥物上市,但抗體藥物價格昂貴,各國醫(yī)保負(fù)擔(dān)重;第一代重磅抗體藥物專利相繼到期,高性價比的生物類似藥被賦予期待;另外技術(shù)上新型抗體藥物層出不窮,雙特異性抗體、ADC藥物、抗體片段、融合蛋白技術(shù)平臺等,諸多新技術(shù)推動抗體藥物的快速發(fā)展。在抗體藥物研發(fā)及生產(chǎn)中需要連續(xù)監(jiān)控保證藥物的安全性,以更好地快速推動研發(fā)的進(jìn)展,降低研發(fā)周期。然而,當(dāng)前肽圖譜分析平臺,例如LC-MS-Tof,LC-TQ,存在價格昂貴,操作復(fù)雜,樣品處理要求高等問題。QDa檢測器作為操作方便、高性價比、穩(wěn)定的單四級桿檢測器,在生物藥研究與生產(chǎn)過程中可作為一種常規(guī)分析方法。利用UPLC系統(tǒng),TUV光學(xué)特性與QDa質(zhì)譜特性,本研究發(fā)展并驗證一種新的分析生物藥的肽圖譜方法。并探討本方法在實(shí)驗室交叉污染溯源上應(yīng)用,作為常規(guī)實(shí)驗室管理方法。首先,采用LC-Q-Tof方法確定源于阿達(dá)木單抗CDR區(qū)8個肽段作為特異性肽段代表整個抗體用于分析研究。針對QDa系統(tǒng)對8個肽段信息進(jìn)行優(yōu)化,得到在QDa系統(tǒng)中響應(yīng)值最高的質(zhì)核比數(shù)據(jù);另外評估阿達(dá)木單抗最佳酶切條件,包括蛋白酶切比例與最佳酶切時間,最后得到最佳酶切條件為37℃,1:10,3h。10個其他抗體,包括曲妥珠單抗、西妥昔單抗、德尼單抗、奧法木單抗、戈利木單抗、Evolocumab、阿侖單抗、伊匹單抗、納武單抗、Pembrolizumab用于評估方法特異性,結(jié)果均為發(fā)現(xiàn)非特異性。由于UV-MS的敏感性,LC-QDa系統(tǒng)具有高特異性;此系統(tǒng)的檢測限為20μg/mL;樣品殘留為1.69%。此外,其它的確認(rèn)參數(shù),包括穩(wěn)定性等也被評估。結(jié)果表明,新的LC-UV-QDa方法作為一種簡單、高性價比、穩(wěn)定的肽圖譜方法適用于常規(guī)質(zhì)量控制分析中。最后我們采用此方法檢測模擬的阿達(dá)木單抗、西妥昔單抗和曲妥珠交叉污染樣品,成功檢測到目標(biāo)蛋白阿達(dá)木單抗及溯源檢測到西妥昔、曲妥珠單抗。
[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，

本文編號：2427819