蛋白質混合溶液膜過濾之界面現象研究

Interfacial Phenomena Analysis on Crossflow Ultrafiltration for Protein Mixtures

李佳玲(Chia-Ling Li)

指導教授 ： 童國倫

中原大學/工學院/化學工程研究所/碩士(2004年)

https://doi.org/10.6840/cycu200400313

摘要

在生化分離技術中，如何有效純化蛋白質混合液，為目前主要研究方向。其中薄膜過濾是一種簡易且可大規模連續操作的分離方式。但超過濾膜純化蛋白質，受限於蛋白質-蛋白質與蛋白質-膜之間的作用、濾速衰減及濃度極化現象，降低膜分離蛋白質的效率，故本研究以改變各種操作變數的方式，探討蛋白質混合液膜過濾之界面現象，藉此改善超過濾膜分離蛋白質的選擇性。
本研究以膜過濾之界面現象的實驗探討為主，在此使用聚?膜，以改變pH值、離子濃度及薄膜親疏水性，針對單成份蛋白質(BSA、OV及LY)，探討靜電作用及膜面親疏水性對過濾行為之影響，之後針對雙成份蛋白質(BSA+LY及BSA+OV)，以改變pH值、離子濃度、掃流速度、透膜壓差、溶質濃度比及薄膜親疏水性，探討分離機制，最後根據以上結果，尋找適當的分離條件，純化三成份蛋白質混合液(BSA+OV+LY)，並與真實情況下所面對的蛋白質混合液(CON+OV+LY)進行比較。
研究結果指出，過濾程序中，蛋白質粒子的沉積與通過膜孔的行為，主要是依據靜電作用的強弱，而與膜面的吸附行為、蛋白質之間的作用與穩定濾速，則是取決於粒子帶電性質。若兩蛋白質帶相反電荷(BSA/LY)，操作在兩者吸引力最強的情況，LY有較高的穿透率，且隨離子濃度的增加，可增加濾速及LY的穿透率。在操作條件方面，藉由透膜壓差的調整可降低膜面結垢，減少濃度極化現象產生；亦可以掃流速度改變濾餅組成，調整濾餅之靜電作用，增加蛋白質之穿透率。若對於粒徑差異小的蛋白質(BSA/OV)，發現使用電漿改質之親水化薄膜有助於疏水性的蛋白質(OV)通過薄膜，增加膜分離的選擇性；且研究結果發現靜電作用力大於疏水性作用力，使用疏水膜時，操作在等電點時會有最大蛋白質吸附量，造成蛋白質穿透率減小。以上述結果所得之最適操作條件分離三成份蛋白質混合液(BSA+OV+LY)，發現電漿改質之親水化薄膜較未改質之薄膜，可增加膜分離三成份蛋白質混合液之選擇性。最後並以氧氣電漿改質之親水化薄膜純化真實系統中所面對的蛋白質混合液(CON+OV+LY)，可在濾液端收集到純度90%以上的OV。
本研究藉由蛋白質混合液膜過濾之界面現象的實驗探討為主，提出了蛋白質混合液超過濾之最佳操作準則，可有效大量純化所需之蛋白質。雖然膜過濾所得之純度不如層析分離高，但大規模處理的優勢，可進行初步的純化，有助於減低層析操作的負擔，降低純化成本。
在未來可以程序設計之方式，分別針對斥回液及濾液，以分段調整溶液環境之方式，逐步純化回收目標蛋白質，亦可以親和性膜分離的方式，即外加親和性膠體，吸附目標蛋白質，提升膜分離之純化效果。

關鍵字

掃流過濾 ； 蛋白質 ； 超過濾 ； 電漿改質 ； 聚醚?膜

並列摘要

Purification of the desired protein from the isolated protein mixture plays a vital role in the downstream processing of biotechnology industry. However, among the bioseparation steps, purification is aimed at highly selective for the product and removes impurities of similar physical properties and chemical functionality. How to purify desired protein from isolated mixture containing tremendous diversity of proteins effectively and continuously is an anathema for many engineers. In the past decade, membrane filtration is reported to be an essential mode of filtration for recovering valuable biological materials from manufacturing processes. Although ultrafiltration is frequently regarded to be effective for protein purification, the concentration polarization and membrane fouling remain the main obstacles to the application of UF for protein purification.
Purpose of this study is to reveal the membrane fouling mechanism by interfacial analyses. These include the role of protein-protein and protein-membrane interactions, the effects of operating conditions and the membrane surface hydrophobicility. Firstly, UF of single protein of bovine serum albumin (BSA), ovalbumin (OV) and lysozyme (LY), respectively, under various operating conditions of pH, ionic concentration and polyethersulfone (PES) membrane surface hydrophobicility were investigated to analyze the protein-membrane interfacial phenomena. Then, binary protein mixtures of BSA/LY and BSA/OV were conducted by changing the pH, ionic concentration, crossflow velocity, transmembrane pressure, solute composition and the PES membrane surface hydrophobicility were depicted to obtain the optimum operating conditions for the purpose of high flux and selectivity. Finally, the obtained criteria were applied to multiple protein mixtures of BSA+OV+LY and CON+OV+LY in order to examine the validity of the optimum operating criteria for protein UF.
The preliminary results indicate that the electrostatic strength plays a vital role in the deposition and transmission of protein, while the steady state flux, protein adsorption and interaction among proteins depend mainly on the net charge of protein. For binary protein mixture with opposite electric charge of BSA+LY, there is a highest transmission for LY under the circumstance of strongest attraction between BSA and LY. The transmission of LY as well as steady-state flux is also increase as the increase of ionic concentration. Experimental analysis on the effect of operation conditions on the protein UF indicate that increase of transmembrane pressure will reduce the concentration polarization problem and result in a higher steady-state flux. Adequate control of crossflow velocity can also effectively reduce membrane fouling and improve the concentration polarization problem due to the change of protein composition in the of cake layer and regulating the electrostatic distribution on the cake surface.
On the effect of hydrophibility of membrane on the performance of protein UF, the hydrophobic membrane would cause serious protein adsorption on the membrane and result in a lowest amount of transmission of protein at its isoelectric pH value. For binary protein mixture with similar molecular weight in size, say BSA+OV, the hydrophilic treated PES membrane facilitated the transmission of hydrophobic protein OV through the membrane and thus improves the selectivity of membrane separation. However, experimental results show that the electrostatic interaction plays a more important role than hydrophobic interaction on the performance of protein UF.
Finally, an optimal operating criterion has been proposed and validated by UF of multiple protein mixtures of BSA+OV+LY and chicken egg white of CON+OV+LY. The proposed criteria would be helpful for engineering design of continuously operating equipment for protein purification by UF.

並列關鍵字

ultrafiltration ； crossflow ； plasma modification ； protein ； PES

參考文獻

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