溶解度參數計算及量測於HPLC純化胜肽程序之最佳化研究

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姓名

王唯真(Wei-Chen Wang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

溶解度參數計算及量測於HPLC純化胜肽程序之最佳化研究
(The study of solubility parameter calculations and measurements for the optimization of peptide purification by HPLC)

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摘要(中)

隨著生物技術和遺傳工程領域的迅速發展，人們對於胜肽（peptide）藥物之合成與發展，更有精確的掌握，也成為各醫藥公司於先導化合物（lead compound）及新藥研發的重要方向之一，更是原料藥廠積極投入生產之項目。胜肽藥物在製藥業中是一個逐年成長的領域，預計至2013年可達到115億歐元的價值[1]。
固相胜肽合成法（solid-phase peptide synthesis，SPPS）成功，更加推動了胜肽藥物的發展。然而，在SPPS的化學合成過程中，可能由於消旋反應（racemization）、合成中的胺基酸缺少（deletion）或插入（insertion），而產生不純物，及此不純物與欲合成之產品在物理及化學性質上的相近，也增加後續純化的困難。這也是整個利用SPPS生產之過程中最困難解決及整個生產程序中固定投資成本，操作成本最多的程序。
因此，考慮了現行純化過程中，仍以管柱層析之操作為主，及為了可以達到生產操作成本之降低及最佳化之純化，本研究根據溶解度參數（solubility parameter，δ）理論與管柱層析之應用，針對胜肽藥物之純化做探討，並藉由實驗與理論計算確立之溶解度參數值對於胜肽於管柱中的滯留行為做有效地描述與控制。此理論原則之建立將可於面對不同胜肽藥物之合成與純化需求時，能更有原則性的建立生產程序，減低嘗試錯誤之需求等多重目的。
論文主要分為三個部份：第一部分為層析系統中分析物（胜肽）、移動相（沖提液）及固定相（管柱）之溶解度參數值的決定。並藉由層析實驗對於管柱及胜肽溶解度參數值做相互的驗證；第二部份則著重於層析移動相的選擇，當原本做為移動相的雙成分溶劑，無法達到分離標準時，本研究進一步根據相同溶解度參數即有相同沖提強度的觀念，對於移動相雙成份或三成份組成以及比例之選擇做較好的推測，進而達到最佳化的分離。第三部份，本研究也針對胜肽鏡像異構物的分離，以分子間溶解度參數差值（△δ）越小，即相溶性越好的觀念，及配合了分子模擬計算，有效地分離產物與其鏡像異構物。

摘要(英)

With the rapid development of biotechnology and genetic engineering over this decade, peptide drug synthesis has precisely controlled to become an important direction for searching and developing of lead compounds in pharmaceutical companies. The market capitalization of peptide drugs was estimated about ?11.5 billion until 2013. The solid-phase peptide synthesis (SPPS) gave a strong impetus to the therapeutic peptide field. However, the peptide purification process usually dominated the overall cost. Also, the synthetic peptides often contained impurities due to amino acid deletion or insertions; in particular, enantiomeric impurities arising from racemization during peptide synthesis. The almost similar physical and chemical properties between impurity and desired peptides were the major problems for peptide separation. Therefore, the optimization of peptide purification process was to be an important course for minimizing the cost. In this study, the solubility parameter (δ) theory was utilized to build up an optimization strategy for peptide purification by HPLC. Under the experimental and theoretical methods, we hope to predict and descript the retention behaviors of peptide in column. This thesis is divided into three parts: First, to determine the solubility parameter of analyte (peptide), mobile phase (solvent) and stationary phase (column) in HPLC system; then, to find the proper ranging of mobile phase and composition; finally, to combine the solubility parameter calculation and molecular dynamics simulation for isomer separation. The results revealed that the isomers could be separated by transferring from one binary mobile phase to an iso-eluotropic (the same eluotropic strength) binary mixture arisen from different solubility parameter, and owned a good enantiomer separation.

關鍵字(中)

★ 胜肽純化
★ 溶解度參數
★ 管柱層析

關鍵字(英)

★ isomer separation
★ chromatography
★ peptide purification
★ solubility parameter

論文目次

中文摘要 i
Abstract iii
誌謝 v
目錄 vi
圖目錄 x
表目錄 xvii
符號說明 xix
第一章緒論 1
第二章文獻回顧 3
2.1 胜肽之合成 3
2.1-1 液相胜肽合成法（solution-phase peptide synthesis） 4
2.1-2 固相胜肽合成法（solid-phase peptide synthesis） 4
2.1-3 保護基（protecting group）的使用 6
2.2 不純物的生成 9
2.3 管柱層析（chromatography）簡介 10
2.3-1 分子大小排斥層析 11
2.3-2 疏水交互作用層析 12
2.3-3 逆相層析 14
2.3-4 正相層析 15
2.4 層析參數計算 16
2.5 胜肽於高效能液相層析中的分離與純化 19
2.6 影響胜肽於層析滯留性之因素 24
2.6 -1 溫度 24
2.6 -2 不同的有機修飾劑（organic modifier） 27
2.6 -3 不同的移動相添加劑（mobile phase additives） 29
2.6 -4 沖提程序（elution program）改變 33
2.7 溶解度參數（solubility parameter）理論 35
2.7-1 分散力（dispersion force） 40
2.7-2 偶極力（dipolar intermolecular force） 41
2.7-3 氫鍵作用力（hydrogen bonding） 42
2.7-4 溶解度參數的假設及限制 43
2.8 管柱溶解度參數（solubility parameter）計算 44
2.8-1等溫吸附曲線之數學模式 44
2.8-2 平衡常數與溶解度參數之關係 46
2.9 溶解度參數應用於層析移動相之選擇 49
2.9-1 The Regular Mixing Rule 49
2.9-2 容積因子與溶解度參數之關係式 50
2.9-3 層析移動相之選擇 51
2.9-4 層析移動相（binary，ternary）之最佳化選擇 54
第三章實驗藥品、儀器設備與實驗方法 63
3.1 實驗藥品 63
3.1-1 層析用溶劑及藥品 63
3.1-2 胜肽鏈 64
3.2 儀器設備 65
3.2-1 一般設備 65
3.2-2 高效能液相層析儀 66
3.3 實驗步驟與分析 67
3.3-1 管柱基材之溶解度參數測定 67
3.3-2 胜肽之溶解度參數測定 75
第四章結果與討論 81
4.1 以層析法求得管柱填充基材之溶解度參數 81
4.2 以層析法與Hoy software求得胜肽之溶解度參數 87
4.3 溶解度參數﹙δd、δp、δh﹚對胜肽純化分離之影響 100
4.4 胜肽鏡像異構物之純化分離 108
第五章結論 119
第六章參考文獻 120
附錄 130

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2011-7-8

推文