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研究生： 高偉紘
Wei-hung Kao
論文名稱： 利用逆相管柱層析並輔以溶解度參數計算進行聚乙烯二醇化人類副甲狀腺素位置異構物之分離
Separation of Positional Mono-PEGylated Teriparatide Isomers Using Reverse-Phased Chromatography Assisted by Solubility Parameter Calculation
指導教授： 陳文逸
Wen-yih Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
論文出版年： 2013
畢業學年度： 101
語文別： 中文
論文頁數： 78
中文關鍵詞： 聚乙烯二醇化 、胜肽位置異構物 、人類副甲狀腺素 、逆相管柱曾析 、溶解度參數
外文關鍵詞： PEGylation, Peptide positional isomers, Human parathyroid hormone, Reversed-phase chromatography, Solubility parameter
相關次數： 點閱：11  下載：0 
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    • Teriparatide為人類副甲狀腺素(human parathyroid hormone, PTH)中1到34胺基酸片段。於2002年，美國食品藥物管理局核可Teriparatide作為治療療骨質疏鬆症(osteoporosis)的胜肽藥物。Teriparatide主要是藉由皮下注射方式進行給藥，但藥物進入血液中的穩定性常因蛋白脢分解或是肝腎排除，而造成藥物穩定性下降或是藥效減弱。藥物聚乙烯二醇化(PEGylation)可降低肝腎排除的機率，並同時延長藥物在人體作用的循環時間。因此本研究以Teriparatide為目標分子，分別從聚乙烯二醇化的反應條件、初步分離、接枝位置的鑑定、二級結構分析，以及耐酵素降解測試，進一步的以逆相管柱層析分離聚乙烯二醇化胜肽之位置異構物，並透過移動相pH值調控以及溶解度參數(solubility parameters)計算的方式，了解影響異構物分離之間的作用力，提出分離純化聚乙烯二醇化胜肽的優化程序。

    在合成 PEGylated Teriparatide方面，本研究藉由調控反應溶劑之pH值可得不同單一PEG5K接枝位置之Teriparatide位置異構物，經由Lysine C切割證明當反應溶劑在pH 6時，PEG5K大多接枝於Teriparatide 的N端；而反應溶劑在pH 8時，PEG5K多接於Teriparatide 的Lys13。此外也發現Teriparatide的結構螺旋性(Helicity)會因PEG5K的接枝而稍微降低，但此二位置異構物抵抗胰蛋白脢水解之能力也因PEG接枝而大幅提升。在逆相層析純化分離N端與Lys13接枝PEG5K的Teriparatide方面，結果發現隨著沖堤溶劑之pH 值提高，由於一級胺質子化程度之差異，導致於異構物間的極性不同，使得此二位置異構物有較佳之分離效果。進一步的以溶解度參數計算發現，可藉由調整溶劑之組成，使二位置異構物與溶劑間之極性項(delta P; polarity term of solubility parameter)的差異增加，以利於基線分離，此結果與調整移動相溶劑pH值之結果相符。由本研究得知，溶劑組成與pH值之調整均可改變分子的極性差異，以利於N端與Lys13接枝PEG的胜肽異構物達基線分離。

    Teriparatide, a peptide drug for treating to osteoporosis by once-daily injection, is the 1-34 segment of recombinant human parathyroid hormone. However, Teriparatide is proteolytically instable in human serum resulting in short circulation half-life (less than 1 hour). Therefore, conjugation with polyethylene glycol (PEGylation) to Teriparatide may shield it from proteolysis to prolong the circulation half-life. For the PEGylated Teriparatide, the positional isomers are usually formed with random PEGylation. We obtained the Nter-PEGylated and K13-PEGylated Teriparatide while the synthesis conditions using pH 6.0 and pH8.0 phosphate buffer, respectively. In this investigation, we intended to directly separate the isomers by tuning mobile phase pH in reversed-phase chromatography (RPC) operation. The results showed that the baseline separation of two isomers can be achieved by tuning the pH value of mobile phase from 7.0 to 9.0, and Nter-PEGylated Teriparatide is much retained in RPC. Form the solubility parameter measurement, we examined that the key factor for the separation of these two isomers is the polarity difference rather than hydrogen bonding or dispersion force. From the circular dichroism measurement, the K13- PEGylated Teriparatide with higher helixity shows less retained in RPC. The results are coherent with our previously proposed structure-retention relationships for peptide isomer retention prediction in RPC.

    摘要......................................................I Abstract................................................II 圖目錄..................................................III 表目錄.................................................VIII 第一章 緒論................................................1 1.1 研究背景與動機..........................................1 1.2 研究目的...............................................2第二章 文獻回顧.............................................3 2.1 人類副甲狀腺素荷爾蒙(1-34)...............................3 2.2 聚乙烯二醇化胜肽藥物.....................................4 2.3 聚乙烯二醇化藥物之反應化學................................5 2.4 聚乙烯二醇化藥物純化分離.................................10 2.5 利用溶解度參數分析胜肽與管柱間作用力.......................11 2.5.1 溶解度參數...........................................11 2.5.2 管柱基材溶解度參數之求得...............................13 2.5.3 胜肽溶解度參數之求得...................................14 2.5.3.1 Hoy Software軟體計算法.............................14 2.5.3.2 以層析法求得胜肽溶解度參數............................14 2.5.3.3 以管柱層析輔以溶解度參數計算分離胜肽非鏡相異構物..........16 第三章 材料與實驗方法........................................18 3.1 藥品與材料.............................................18 3.2 儀器設備...............................................19 3.2.1 一般設備.............................................19 3.2.2 高效能液相層析儀......................................19 3.3 實驗方法...............................................20 3.3.1 PEGylated Teriparatide後接枝化學.....................20 3.3.2 Mono-PEGylated Teriparatide 之初步分離...............20 3.3.3 以基質輔助脫附離子化質譜儀對接枝程度檢測..................21 3.3.4 Mono-PEGylated Teriparaide純度鑑定..................21 3.3.5 藉Lys-C酵素反應判定Mono-PEGylated Teriparaide接枝位置.22 3.3.6 以圓二色光譜分析位置異構物二級結構......................22 3.3.7 耐酵素降解反應性測試...................................23 3.3.8 移動相pH值調控以分離Mono-PEGylated Teriparaide位置異構物........................................................233.3.9 溶解度參數分析用於分離Mono-PEGylated Teriparaide位置異構物........................................................24 第四章 結果與討論...........................................25 4.1 利用後接枝法合成聚乙烯二醇化胜肽...........................25 4.1.1 反應溶劑的pH值之影響...................................26 4.1.2 PEG對胜肽莫耳比之影響..................................30 4.2 PEGylation胜肽位置異構物的初步分離與物性鑑定...............33 4.2.1 以逆相管柱層析對PEGylation胜肽做初步分離.................33 4.2.2 以酵素Lys-C判定PEG接枝位置.............................34 4.2.3 以圓二色光譜分析位置異構物二級結構.......................39 4.2.4 耐酵素穩定性測試......................................41 4.3 分離Mono-PEGylated Teriparatide位置異構物...............45 4.3.1 逆相管柱層析移動相pH值調控..............................45 4.3.2 逆相管柱層析移動相組成調控..............................51 4.3.3 溶解度參數分析Mono-PEGylated Teriparatide位置異構物之分離機制........................................................53 4.3.4 胜肽異構物之結構穩定性與滯留行為關聯......................56 第五章 結論................................................60 第六章 參考文獻.............................................62

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