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首頁   >>   公司動態   >>   電泳技術與高分辨質譜聯用,助力復雜蛋白治療產品深入表征

電泳技術與高分辨質譜聯用,助力復雜蛋白治療產品深入表征

閱讀:627      發布時間:2022-6-30
  近年來,隨著人們對醫療健康行業需求的不斷增長,生物制藥行業也在隨之蓬勃發展。近兩年的疫情全球大流行,在改變人們日常生活的同時更是催生了生物制藥行業對于先進分析技術的需求。
 
  蛋白質分離、純化和分析是生物zhi療藥物開發中的關鍵組成部分,但該過程可能復雜且具挑戰性。而全柱成像等電毛細管電泳(whole column imaged capillary isoelectric focusing, WC-iCIEF)技術,可以根據蛋白質的等電點(isoelectric point, pI)差異將其分離,在此基礎上,將iCIEF與高分辨質譜聯用,可以借助質譜的高靈敏度、高分辨率和高質量精度使各種蛋白質變異體的鑒定更容易、更準確。從2021年6月開始,我們與蛋白質成像技術專家 Advanced Electrophoresis Solutions Ltd (AES)宣布達成協議,將蛋白質分離技術與質譜相結合,通過簡化表征來推進zhi療性蛋白質藥物的開發。
 
  到目前為止,通過將iCIEF技術與高分辨質譜聯合使用,我們已經對單抗、ADC和融合蛋白等多種產品進行了各種層面的表征。下圖1~3展示了iCIEF技術對單抗\ADC\融合蛋白的分離結果,可見對于不同種類的重組生物zhi療性產品,均可根據pI差異將其電荷變異體進行分離,且系統具有優異的穩定性與重現性。
 
圖1 iCIEF-UV分析帕博利珠單抗電荷變異體,8針平行進樣(點擊查看大圖)
 
圖2 iCIEF-UV分析恩美曲妥珠電荷變異體,3針平行進樣
 
圖3 iCIEF-UV分析依那西普電荷變異體,3針平行進樣
 
  我們使用的CEInfinite iCIEF平臺(AES)除了高質量的iCIEF-UV功能外,還可以與高分辨質譜直接在線串聯,直接測定電荷變異體的分子量,無需額外轉換接口。下圖4展示了我們使用iCIEF-MS直連技術分析帕博利珠單抗的結果,可見即使是pI僅差0.02的堿峰B1和主峰,也可以在iCIEF上得到基線分離,隨后的高分辨質譜分子量測定結果顯示該堿峰與主峰相比,主要差異是其中一條重鏈的N端未發生焦谷氨酸環化。另外觀察原始質譜譜圖不難發現,得益于Orbitrap高分辨質譜的靈敏度,即使是強度比主峰低2~3個數量級的堿峰B3,仍可得到糖型分布清晰的譜圖。
 
  圖4 iCIEF-MS在線直聯分析帕博利珠電荷變異體。上,iCIEF-UV分離圖譜。中,堿峰B1與主峰解卷積結果鏡像圖對此。下,主峰與所有堿峰原始質譜圖對比。(點擊查看大圖)
 
  與市面上其他供應商相比,AES的CEInfinite iCIEF平臺具有一個*優勢:可以實現全自動的餾分收集。我們選擇了帕博利珠單抗,對其電荷變異體的每個峰離線收集后進行酶解,隨后上樣至高分辨液質聯用平臺進行肽圖分析。圖5展示了酸/堿峰中各種CQA含量的變化,可見輕重鏈末端、重鏈糖型和側鏈常見PTM的變化趨勢。另外通過表1中分離之前/之后特定CQA含量對比可以很明顯的發現,經iCIEF分離后,酸/堿峰中特定修飾的比率有明顯zhen高,可見基于pI差異,將生物zhi療性產品的電荷變異體進行分離后,接下來采用肽圖進行深入表征的分析策略,能夠幫助研究人員將導致電荷異質性的修飾精確定位到氨基酸位點的層面。
 
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