探討聚乙烯二醇化對於聚乙烯二醇化人類副甲狀腺素 (1-34) 與疏水介面間交互作用之影響

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中川小夜子(Hsiao-Yeh-Tzu Chung-Chuan) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

探討聚乙烯二醇化對於聚乙烯二醇化人類副甲狀腺素 (1-34) 與疏水介面間交互作用之影響
(Effect of PEGylation on the Interactions between PEGylated Teriparatide and Hydrophobic Surface)

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摘要(中)

近年來蛋白質藥物和胜肽藥物等生物製劑開始蓬勃發展，此類生物製劑之進一步於藥效提升、副作用降低與具標靶治療等之相關研究非常重要。目前解決生物製劑之血液穩定性之方式為聚乙烯二醇化，聚乙烯二醇化可增加生物製劑之溶解度、降低肝腎排除的機率與避免血液中酵素的分解，因而延長藥物在人體內之循環時間。由於聚乙烯二醇具有良好的生物相容性，所以被廣泛應用在改良生物製劑之用途。在聚乙烯二醇化的過程中，時常伴隨著未接枝、不同接枝程度以及不同接枝位置之產物的生成，然而不同接枝情形皆會影響生物製劑之特性，因此聚乙烯二醇化後之生物製劑的純化是非常重要的。
本研究以Teriparatide作為目標分子，探討胜肽以及聚乙烯二醇化胜肽於逆相管柱層析中之吸附機制。Teriparatide為人類副甲狀腺素中1到34胺基酸片段，於2002年經美國食品藥物管理局核可作為治療骨質疏鬆症之胜肽藥物，主要是藉由皮下注射方式進行給藥。
我們利用圓二色光譜量測Teriparatide與聚乙烯二醇化Teriparatide異構物之二級結構，發現經過聚乙烯二醇化後，其二級結構會發生改變，且其結構穩定性較未接枝聚乙烯二醇時來的高。另外，我們量測Teriparatide與聚乙烯二醇化Teriparatide異構物於水相以及有機相中之溶解度，發現所有樣品在水相皆有良好的溶解度；但在有機相中，Teriparatide的溶解度低，而聚乙烯二醇化會使Teriparatide在有機相中之溶解度增加。最後，我們以逆相管柱層析量測並計算樣品於管柱中作用之熱力學參數，由結果發現N端或C端聚乙烯二醇化Teriparatide吸附到逆相管柱層析中之樹脂上時，為一焓主導之吸附，即吸附過程伴隨熱量的釋放並且亂度減少；反觀Lys13聚乙烯二醇化Teriparatide吸附到管柱層析中之樹脂上時，為一熵主導之吸附，即吸附過程亂度增加，但須吸收熱量。由熱力學參數發現N端或C端接枝聚乙烯二醇化Teriparatide與Lys13接枝聚乙烯二醇化 Teriparatide兩者之吸附機制截然不同，因此在本研究最後嘗試以調控溫度之方式，分離上述之位置異構物，雖未成功地基線分離上述之位置異構物，但發現原本同時被沖堤出來之異構物，經由調控溫度仍然可以明顯地看出N端或C端聚乙烯二醇化Teriparatide與Lys13聚乙烯二醇化Teriparatide有不同的滯留時間。

摘要(英)

In recent years, protein and peptide drugs play an important role in pharmaceutical industry. Unfortunately, these protein and peptide drugs are proteolytically instable in human serum resulting in short circulation half-life. A new product through grafting the polyethylene glycol (PEG), a well-known biocompatible polymer, with protein and peptide drugs is able to avoid clearance of kidney or attack of immune system, thus prolong the circulating half-life time. However, for the PEGylated protein and peptide drugs, the positional isomers are usually formed with random PEGylation. The chromatographic purification process of PEGylated protein and peptide drugs is still without a general guideline to carrier out, in particular for the role of PEG in protein and peptide drugs. In this study, we use Teriparatide as a model peptide drug, investigating the effect of PEGylation on the interactions between PEGylated peptide and hydrophobic surface. Teriparatide, a peptide drug for treating to osteoporosis by once-daily injection, is the 1-34 segment of recombinant human parathyroid hormone. After PEGylation, we found that the retention time increase and we determine the thermodynamic parameters between peptides and hydrophobic surface by reversed-phase chromatography. From the results, we concluded that the PEG chain will expose to the solution phase when the N- or C-PEG-Teriparatide adsorbed to the hydrophobic surface, resulting in numerous hydration reactions. In the contrary, when the Lys13-PEG-Teriparatide adsorbed to the hydrophobic surface, PEG chain will attach to the hydrophobic surface, release the water molecules, leading to increase the entropy of system. In addition, from the results of circular dichroism, the secondary structure of PEGylated Teriparatides are stronger than native Teriparatide in 30% ACN. Therefore, Teriparatide is more soluble in this condition, lead to the weaker binding strength between peptide and hydrophobic surface. Furthermore, the structure of Teriparatide in different solution conditions will change more than PEGylated Teriparatides. We are able to consider that PEGylated Teriparatides are more rigid.

關鍵字(中)

★ 聚乙烯二醇
★ 疏水介面
★ 逆相管柱層析
★ 熱力學參數

關鍵字(英)

論文目次

摘要 I
ABSTRACT III
目錄 V
圖目錄 VII
表目錄 IX
第一章緒論 1
第二章文獻回顧 4
2-1　層析法之簡介 4
2-1-1　逆相管柱層析（Reversed-phase chromatography） 5
2-2 人類副甲狀腺素荷爾蒙(1-34) 6
2-3 聚乙烯二醇化之簡介與應用 7
2-3-1 聚乙烯二醇化胜肽藥物 8
2-3-2 聚乙烯二醇化藥物之化學合成 9
2-3-3 聚乙烯二醇化藥物之分離純化 11
2-4 以熱力學研究生化分子與界面間之交互作用 12
2-4-1 鍵結機制之探討 13
2-4-2 以van’t Hoff方程式研究吸附行為 15
第三章材料與實驗方法 17
3-1 藥品與材料 17
3-2 儀器設備 17
3-2-1 高效能液相層析儀 17
3-2-2 圓二色光譜儀 18
3-2-3 其它設備 18
3-3 實驗方法 18
3-3-1 聚乙烯二醇化胜肽化學合成 18
3-3-2 聚乙烯二醇化胜肽之分離純化 19
3-3-3 以圓二色光譜分析聚乙烯二醇化胜肽之二級結構 20
3-3-4 溶解度測試 20
3-3-5 以管柱層析計算熱力學參數 21
3-3-6 調控溫度分離聚乙烯二醇化Teriparatide之異構物 21
第四章結果與討論 23
4-1 聚乙烯二醇化Teriparatide之合成、純化與鑑定 23
4-1-1 聚乙烯二醇化Teriparatide之合成 24
4-1-2 聚乙烯二醇化Teriparatide之純化 24
4-1-3 聚乙烯二醇化Teriparatide之接枝位置判定 26
4-2 聚乙烯二醇化Teriparatide之性質測定 29
4-2-1 以圓二色光譜分析聚乙烯二醇化Teriparatide之二級結構 29
4-2-2 聚乙烯二醇化Teriparatide之溶解度測試 32
4-3 以van’t Hoff方程式探討聚乙烯二醇化Teriparatide之鍵結機制 34
4-3-1 Teriparateide之鍵結機制 34
4-3-2 N端或C端聚乙烯二醇化Teriparatide之鍵結機制 35
4-3-3 Lys13聚乙烯二醇化Teriparatide之鍵結機制 36
第五章結論 40
第六章參考文獻 42

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指導教授

陳文逸

審核日期

2014-8-29

推文