探討聚乙二醇對溶菌酶與管柱層析樹脂間交互作用之影響

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姓名

徐民晏(Min-Yen Hsu) 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

探討聚乙二醇對溶菌酶與管柱層析樹脂間交互作用之影響
(Effects of Polyethylene Glycol on the Interactions Lysozyme with Chromatography Resins)

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摘要(中)

製藥產業由早期從自然界中取得或合成的小分子藥物，到二十世紀末拜基因技術工程之賜，蛋白質藥物和胜肽藥物等生物製劑開始蓬勃發展。生物製劑之進一步於藥效提昇、副作用降低、具標靶治療等所謂”biobetters藥物”的相關探討為現今之研究重點。
目前已有相當多不同的改良方式能使生物製劑的藥效提昇，其中，由於聚乙二醇（polyethylene glycol, PEG）具有良好的生物相容性，所以也被廣泛應用在biobetters藥物的研究發展上。然而，改良後需要將有效藥物成份分離純化會遭遇許多困難，目前也尚未有研究可以完整說明聚乙二醇改質至生物製劑上後，對生物製劑之物理化學及生物性質的影響。
本研究以針對生物製劑純化時，聚乙二醇分子對生物製劑於層析分離行為之影響為研究重點。我們藉由等溫吸附曲線和恆溫滴定卡計來量測以及探討溶菌酶與管柱層析樹脂間的交互作用中，調控環境之鹽離子種類和濃度，以及額外加入不同分子量、不同濃度聚乙二醇分子看吸附平衡常數以及吸附焓之改變，希望可以藉此得知聚乙二醇於溶液中的行為及其在溶菌酶與管柱層析樹脂間交互作用中所扮演的角色，並執行及觀察聚乙二醇分子改質至溶菌酶上的相關化學反應。
由實驗結果可得知，添加的鹽種類不同，會對溶菌酶與管柱層析樹脂間吸附行為有不同影響，且行為符合Hofmerister series之論述。而加入聚乙二醇時，聚乙二醇的水合會導致類似kosmotropes的鹽析效應（salting-out effect）產生，且影響程度依分子量大小而有差異。另外，聚乙二醇分子在不同種鹽類溶液中的行為亦會受到鹽類之鹽析或鹽溶效應（salting-in effect）影響，故當溶液中存在有不同種鹽類時，聚乙二醇之影響亦隨之改變。本研究也初步合成聚乙二醇化溶菌酶，並初步探討PEGylation之化學合成與聚乙二醇化溶菌酶於管柱層析之行為。

摘要(英)

Since the development of genetic engineering, protein and peptide drugs now play an important role in pharmaceutical industry. Unfortunately, a short circulating half-life time of protein/peptide drugs limits their pharmaceutical applications. A new product through grafting polyethylene glycol (PEG), a well-known biocompatible polymer, with protein/peptide drugs is called PEGylated product. The PEGylayed drug is able to avoid clearance of kidney or attack of immune system, thus prolong the circulating half-life time. This product is known as “Biobetters” which could improve the efficacy of biologics over the originals. However, the chromatographic purification process of PEGylation protein/peptide drugs is lacking of a general guidence. In particular, for the effects of PEG on protein/peptide drugs and PEG in solution on the chromatographic behavior are required for the guidence. In this study, we investigated the effects of buffer solution contains of the PEG molecules on the interaction between lysozyme and anionic resin (SP Sepharose) via thermodynamic analysis. By combining the analysis of isothermal titration calorimeter and adsorption isotherms, the results revealed that the bindings of lysozyme and resin both in saline buffer and in PEG-contained solution are dominated by enthalpy, indicating the binding adopts electrostatic interactions driven manner. Furthermore, we considered that the solution behavior of PEG on lysozyme-resin binding may be described as a “kosmotrope-like” polymer, which means the existence of PEG will facilitate the salting-out effect in the solution phase. In kosmotropic type saline buffer, such as ammonium sulfate, the addition of PEG molecules would facilitate the increase of binding affinity between lysozyme and resin which resulting in the decrease of binding entropy. Apparently, the hydration of PEG molecules reduces the tightly bounded water molecules around lysozyme surface to reduce the binding entropy gain, i.e. the hydration of PEG molecules lead to the less water molecules being repelled from the protein surface. On the contrary, the addition of PEG molecules would also facilitate the increase of binding affinity between lysozyme and resin which resulting in the increase of binding entropy in chaotropic salt, such as sodium chloride. We suggested that the PEG molecules may have a higher solubility in salting in solution; therefore, the tightly bounded water molecules still preserve around the lysozyme surface resulting in high entropic gain. Consequently, this study provided an important implication on the hydration role of PEG molecules to mediate the binding of protein on resin.

關鍵字(中)

★ 聚乙二醇化
★ 聚乙二醇
★ 等溫吸附曲線
★ 恆溫滴定微卡計
★ 管柱層析樹脂

關鍵字(英)

★ polyethylene glycol
★ PEGylation
★ chromatography resins
★ isothermal titration microcalorimetry
★ adsorption isotherms

論文目次

摘　要 i
ABSTRACT iii
致　謝 v
目　錄 vii
圖　目　錄 x
表　目　錄 xiv
一、　緒論 1
二、　文獻回顧 4
2-1　層析法之簡介 4
2-1-1　層析用樹脂之簡介與分類 5
2-2　溶菌酶之簡介與應用 6
2-3　聚乙二醇化之簡介與應用 7
2-3-1　聚乙二醇化之化學合成 10
2-3-2　聚乙二醇化分子之分離純化 14
2-3-3　聚乙二醇化藥物 18
2-5　Langmuir adsorption isotherm model 23
2-6　恆溫滴定微卡計 24
2-6-1　裝置及運作原理之簡介 25
2-6-2　熱量量測與吸附焓之換算 27
三、　實驗藥品、儀器及方法 28
3-1　實驗藥品 28
3-2　儀器設備 30
3-2-1　一般設備 30
3-2-1　恆溫滴定微卡計 31
3-2-2　高效能液相層析儀 31
3-3 實驗步驟 32
3-3-1溶液之配置 32
3-3-2 等溫吸附曲線實驗 33
3-3-3 恆溫滴定微卡計實驗 34
3-3-3-1 實驗步驟 34
3-3-3-2 清洗步驟 36
3-3-4 圓二色光譜儀實驗 37
3-3-5 螢光光譜儀實驗 37
3-3-6 聚乙二醇化溶菌酶製備 38
3-3-6-1 以PEG-aldehyde製備 38
3-3-6-2 以PEG-SPA製備 38
3-3-7 管柱層析實驗 39
四、　結果與討論 40
4-1　批次等溫吸附曲線實驗 40
4-1-1　疏水交互作用層析樹脂 44
4-1-1-1　鹽類效應 45
4-1-1-2　聚乙二醇之影響 47
4-1-2　強陽離子交換樹脂 50
4-1-2-1　鹽類效應 50
4-1-2-2　聚乙二醇之影響 55
4-2　恆溫滴定卡計量測吸附熱力學 62
4-2-1　鹽類效應 64
4-2-2　聚乙二醇之影響 66
4-2-2-1 於鹽類中添加或不添加聚乙二醇之影響 66
4-2-2-2 於鹽類中添加不同分子量之聚乙二醇的影響 69
4-3　聚乙二醇化溶菌酶 75
4-3-1　聚乙二醇化溶菌酶之製備 76
4-3-2　聚乙二醇化溶菌酶之分離純化 78
五、　結　論 80
六、　參考文獻 82

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2012-7-6

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