| 摘要: | 生物科技具有低污染、天然資源需求少且偏重腦力、知識密集等特質，而二十一世紀正是一個重視環境保護及知識經濟的時代；因此，生物科技將可成為本世紀最有潛力的新興產業，也是我國經濟邁向另一個高峰的原動力。近年來，生技產業的蓬勃發展，已帶動我國經濟重要之成長；而生技產業又以蛋白質藥物生產之成就，為產業發展之指標。蛋白質藥物之生產包括：蛋白質藥物於生產程序（process）、配方（formulation）與藥導（delivery），而其中，扮演最關鍵之基礎與應用知識即為分子間交互作用力。 本研究即整合現今量測蛋白質分子於溶液中之交互作用行為，亦即所謂第二維里係數（second virial coefficient，B22），進一步配合本實驗室具多年經驗之恆溫滴定微卡計（Isothermal Titration Calorimetry，ITC）之理論與實驗設計，發展出更簡易、省時、省樣本及能多方探討B2值之熱力學方法。實驗上，以核醣核酸酶（Ribonuclease A）及溶菌酶（Lysozyme）當作研究之模型蛋白，於不同常用添加物（NaCl、n-propanol、Arginine）之條件下，利用ITC來獲得分子間交互作用力之資訊。ITC研究結果顯示，鹽類之鹽析效應符合Hofmeister Series；有機溶劑效應之探討，在低濃度範圍，蛋白質溶解度隨n-propanol增加而增加；但在高濃度範圍時，其結果卻相反；胺基酸效應之研究結果顯示：在溶液含有低濃度鹽類時，分子間作用力與胺基酸濃度無關；而含有高濃度鹽類時，蛋白質溶解度隨著胺基酸濃度增加而增加。 此外，ITC研究結果與現今常用之SIC（self-interaction chromatography）比較顯示，當一蛋白質表面電荷分布不均勻且環境pH值較接近蛋白質之pI值之條件下，蛋白質於SIC系統中之固定化的方向性（orientation）將導致固定相上的蛋白質與移動相中的蛋白質分子之間的作用力之量測無法完全表示溶液中蛋白質分子間的作用行為；反之，ITC獲得B22值之環境為溶液系統，蛋白質分子不需要被固定化，因此沒有方向性的問題，其所獲得之B22值與SIC相較之下，較可確切描述蛋白質分子於該溶液條件下之交互作用行為。 Understanding and overcoming analytic, formulation, manufacturing, and regulatory challenges of protein drugs or biosimilars covers the latest trends and challenges of both academic and R&D of pharmaceutical companies. The focus are on: understanding and controlling protein aggregation, improving detection and quantitation of aggregates, analyzing subvisible and visible particles with various techniques, understanding aggregates as an inducing factor in immungenicity, and improving structural analysis and modeling to predict protein aggregation. In a word to describe all the above attentive focus is “second virial coefficient B22” of protein in solution. Here we cope with the theoretical developments of the current methods for B22 measurements, especially focus is on Self-interaction Chromatography (SIC). Moreover, based on our extensive experience on Isothermal Titration Calorimetry (ITC), we derive a statistic thermodynamics model to obtain B22 of protein to describe protein behaviors in solution by means of the dilution enthalpy measurement of protein solution. And, the B22 value can be applied for the protein purifcation, protein conformational disease (PCD) and protein crystallization. In this work, we focus on the studies of solution additive effects on B22, including NaCl, n-propanol, and Arginine. It shows that protein-protein interactions change from repulsive(electrostatic dominant) to attractive(hydrophobic dominant) with increasing NaCl conc. from the ITC experimental result. From the study of organic solvent effect, Protein-protein repulsive interactions increased with increasing np. conc. at the lower conc. range; however, protein-protein attractive interactions increased with increasing np. conc. was shown at the range of higher np. conc. Amino acid effect on B22 of protein in solution exhibits that protein-protein interactions aren’t function of Arg. conc. as the present of lower NaCl conc.(2% w/v). But, protein-protein repulsive interactions increased with increasing Arg. conc. at the solution condition of NaCl conc.5% (w/v). Comparison ITC experimental results with SIC, we can discover that unexpected results occur at SIC system as the surface charge distribution of protein isn’t uniform and the pH of solution is closer to the pI of protein due to immobilization of protein molecules on the solid support. The orientation concern of the immobilized protein molecules is the most drawback for SIC methodology to determine B22. On the contrary, we can describe precisely protein behaviors in solution by ITC because of the fully degree of rational freedom of protein at ITC solution system. Besides, the B22 values measured by ITC has the advantage on costs including the materials and time, especially for the studies of those pharmaceutical proteins. |
