發展恆溫滴定微卡計成為探討生物分子於溶液系統中作用行為之新技術平台

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姓名

吳松桓(Sung-huan Wu) 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

發展恆溫滴定微卡計成為探討生物分子於溶液系統中作用行為之新技術平台
(The development of Isothermal Titration Calorimetry as an emerging tool for the intermolecular interactions behavior studies of bio-molecules in solution phase)

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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 第二維里係數
★ 恆溫滴定微卡計
★ 自我反應管柱層析

關鍵字(英)

★ Isothermal Titration Calorimetry
★ Self-interaction Chromatography
★ second virial coefficient

論文目次

中文摘要 I
Abstract III
誌謝 V
目錄 VII
圖目錄 X
表目錄 XIV
第一章緒論 1
第二章文獻回顧 4
2.1 第二維里係數（Second Virial Coefficient） 4
2.1.1 第二維里係數之介紹 4
2.1.2 第二維里係數之應用 6
2.2 第二維里係數之量測方法 9
2.2.1 自我反應管柱層析（Self-interaction Chromatography，SIC） 9
2.2.2 恆溫滴定微卡計（Isothermal Titration Calorimetry，ITC） 22
2.2.3 其他量測方法 28
2.3 溶液添加物對蛋白質分子間作用力之影響（Effects of solution additives） 37
2.3.1 鹽效應（Hofmeister effect） 40
2.3.2 有機溶劑效應（Organic solvent effect） 50
2.3.3 胺基酸效應（Amino acid effect） 53
2.4 蛋白質聚集之量測方法 56
2.5 恆溫滴定微卡計（ITC） 62
2.5.1 卡計之基本介紹 62
2.5.2 VP-ITC之介紹 63
第三章實驗藥品、儀器與方法 66
3.1 實驗藥品 66
3.2 儀器設備 67
3.3 實驗方法 68
3.3.1 VP-ITC操作步驟 68
3.3.2 稀釋焓之量測 70
第四章結果與討論 71
4.1 ITC系統穩定度及b2值可靠度之測試 71
4.2 鹽效應（Salt effect） 75
4.3 有機溶劑效應（Organic solvent effect） 84
4.4 胺基酸效應（Amino acid effect） 92
第五章結論 98
第六章參考文獻 100
Appendix 106

參考文獻

1. Chayen, N. E., Tackling the bottleneck of protein crystallization in the post-genomic era. Trends Biotechnol 2002, 20 (3), 98-98.
2. Haynes, C. A., Tamura, K., Korfer, H. R., Blanch, H. W., and Prausnitz, J. M., Thermodynamic properties of aqueous a-chymotrypsin solutions from membrane osmometry mesurements. J. Chem. Phys. 1992, 96;
3. Wu, J. Z.; Prausnitz, J. M., Osmotic pressures of aqueous bovine serum albumin solutions at high ionic strength. Fluid Phase Equilibr. 1999, 155 (1), 139-154.
4. Curtis, R. A.; Montaser, A.; Prausnitz, J. M.; Blanch, H. W., Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes. Biotechnol. Bioeng. 1998, 58 (4), 451-451;
5. Velev, O. D.; Kaler, E. W.; Lenhoff, A. M., Protein interactions in solution characterized by light and neutron scattering: Comparison of lysozyme and chymotrypsinogen. Biophys. J. 1998, 75 (6), 2682-2697;
6. Piazza, R.; Pierno, M., Protein interactions near crystallization: A microscopic approach to the Hofmeister series. J. Phys-Condens Mat. 2000, 12 (8A), 443-449.
7. Behlke, J.; Ristau, O., Analysis of the thermodynamic nonideality of proteins by sedimentation equilibrium experiments. Biophys. Chem. 1999, 76, 13-23.
8. Bonnete, F.; Finet, S.; Tardieu, A., Second virial coefficient: variations with lysozyme crystallization conditions. J. Cryst. Growth 1999, 196 (2-4), 403-414;
9. Vivares, D.; Bonnete, F., X-ray scattering studies of Aspergillus flavus urate oxidase: towards a better understanding of PEG effects on the crystallization of large proteins. Acta Crystallogr. D 2002, 58, 472-479.
10. Gripon, C.; Legrand, L.; Rosenman, I.; Vidal, O.; Robert, M. C.; Boue, F., Study of protein-protein interactions in undersaturated and supersaturated lysozyme solutions in heavy water as a function of temperature. Cr Acad Sci Ii B 1996, 322 (7), 565-571;
11. Gripon, C.; Legrand, L.; Rosenman, I.; Vidal, O.; Robert, M. C.; Boue, F., Lysozyme-lysozyme interactions in under- and super-saturated solutions: a simple relation between the second virial coefficients in H2O and D2O. J. Cryst. Growth 1997, 178 (4), 575-584.
12. Bloustine, J.; Berejnov, V.; Fraden, S., Measurements of protein-protein interactions by size exclusion chromatography. Biophys. J. 2003, 85 (4), 2619-2623.
13. Henry et al., Screening for physical stability of a Pseudomonas amylase using self-interaction chromatography. Analytical Biochemistry 2006, 357 (1), 35-42.
14. Tessier, P. M.; Lenhoff, A. M.; Sandler, S. I., Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography. Biophys. J. 2002, 82 (3), 1620-1631.
15. Patro S. Y.; Przybycien T. M., Self-Interaction chromatography: A tool for the study of protein-protein interactions in bioprocessing environments, Biotechnol. Bioeng. 1996, 52. 193-203
16. Chen, W. Y.; Kuo, C. S.; Liu, D. Z., Determination of the second virial coefficient of the interaction between microemulsion droplets by microcalorimetry. Langmuir 2000, 16 (2), 300-302.
17. Neal, B. L.; Asthagiri, D.; Lenhoff, A. M., Molecular origins of osmotic second virial coefficients of proteins, Biophys. J. 1998, 75, 2469-2477.
18. Neal, B. L.; Asthagiri, D.; Lenhoff, A. M.; Velev, O. D., and Kaler E. W., Why is the osmotic second virial coefficient related to protein
crystallization? J. Cryst. Growth 1999, 196, 377-387.
19. Ho, J. G. S.; Middelberg, A. P. J.; Ramage, P.; Kocher, H. P., The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient. Protein Science 2003, 12 (4), 708-716.
20. Watanabe K., Segawa T., Nakamura K., Kodaka M., Konakahara T., Okuno H., Identification of the molecular interaction site of amyloid β peptide by using a fluorescence assay, J. Peptide Res. 2001, 58, 342-346.
21. Tomlinson, I. M., Next-generation protein drugs. Nat Biotechnol 2004, 22 (5), 521-522.
22. Cheney, M. L.; Shan, N.; Healey, E. R.; Hanna, M.; Wojtas, L.; Zaworotko, M. J.; Sava, V.; Song, S. J.; Sanchez-Ramos, J. R., Effects of crystal form on solubility and pharmacokinetics: A crystal engineering case study of Lamotrigine. Cryst. Growth Des. 2010, 10 (1), 394-405.
23. Deshpande, K.; Ahamed, T.; van der Wielen, L. A. M.; ter Horst, J. H.; Jansens, P. J.; Ottens, M., Protein self-interaction chromatography on a microchip. Lab Chip 2009, 9 (4), 600-605.
24. McQuarrie, D. A., Statistical Mechanics. Harper Collins: New York, 1976.
25. DePhillips P.; Lenhoff A. M., Pore size distribution of cation-exchange adsorbents determined by inverse size-exclusion chromatography. J. Chromatogr. A 2000, 883, 39-54.
26. Ahamed, T.; Ottens, M.; Dedem, G. W. K.; Van Der Wielen, L. A. M., Design of self-interaction chromatography as an analytical tool for predicting protein phase behavior. J. Chromatogr. A 2006, 1115 (1-2), 272-272;
27. Ahamed, T.; Ottens, M.; van Dedem, G. W. K.; van der Wielen, L. A. M., Design of self-interaction chromatography as an analytical tool for predicting protein phase behavior. J. Chromatogr. A 2005, 1089 (1-2), 111-124.
28. Teske C. A., Blanch H. W., Praustinz J. M., Chromatographic measurement of interactions between unlike proteins, Fluid Phase Equilib. 219 (2004), p. 139
29. Huang, S. L.; Lin, F. Y.; Yang, C. P., Microcalorimetric studies of the effects on the interactions of human recombinant interferon-alpha 2a. European J. Pharm. Science 2005, 24 (5), 545-552.
30. Wang S. C.; Chang F. M.; Tsao H. K., Second virial coefficients of Poly(ethylene glycol) in aqueous solutions at freezing point. Macromolecules 2002, 35, 9551-9555.
31. Kratochvil, P., In classical light scattering from polymer solutions, Elsevier: Amsterdam and New York, 1987;
32. Wyatt, P. J., Light-scattering and the absolute characterization of macromolecules, Analytica Chimica Acta 1993, 272. 1-40
33. Zukoski, C. F.; Rosenbaum, D. F.; Zamora, P. C., Aggregation in precipitation reactions: Stability of primary particles. Chemical Engineering Research & Design 1996, 74; 723-731
34. Muschol, M.; Rosenberger, F., Interactions in undersaturated and supersaturated lysozyme solutions: Static and dynamic light scattering results. J. Chem. Phys. 1995, 103; 10424-10432
35. Wilson, W. W., Light scattering as a diagnostic for protein crystal growth - A practical approach. Journal of Structural Biology 2003, 142. 56-65
36. Neal, B. L.; Asthagiri, D.; Velev, O. D.; Lenhoff, A. M.; Kaler, E. W.; Why is the osmotic second virial coefficient related to protein crystallization? J. Cryst. Growth 1999, 196; 377-387
37. Velev, O. D.; Kaler, E. W.; Lenhoff, A. M.; Protein interactions in solution characterized by light and neutron scattering: Comparison of lysozyme and chymotrypsinogen. Biophys. J. 1998, 75. 2682-2697
38. Wang, W., Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm 2005, 289 (1-2), 1-30.
39. Hardy, J.; Selkoe, D. J., Medicine - The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics. Science 2002, 297 (5580), 353-356.
40. Soto, C.; Kindy, M. S.; Baumann, M.; Frangione, B., Inhibition of Alzheimer's amyloidosis by peptides that prevent beta-sheet conformation. Biochem Bioph Res Co 1996, 226 (3), 672-680.
41. Shiraki, K., Hamada, H., and Arakawa, T., Effect of additives on protein aggregation. Current Pharmaceutical Biotechnology 2009, 10 (4), 400-407.
42. EJ., C., The solubility of protein. In Protein, amino acids, and peptides, JT, C. E. E., Ed. Reinhold: New York, 1943; pp 586-622.
43. Valente, J. J.; Verma K. S.; Manning M. C.; Wilson W. W.; Henry C. S.; Second virial coefficient studies of cosolvent-induced protein self-interaction. Biophys. J. 2005, 89, 4211-4218.
44. Melander, W.; Horvath, C., Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. Arch. Biochem. Biophys. 1977, 183, 200-215.
45. Casassa, E. F.; Eisenberg, H.; Thermodynamic analysis of multi-component solutions. Adv Protein Chem. 1964, 19, 287-393.
46. Lee, J. C.; Gekko, K.; Timasheff, S. N.; Measurements of preferential solvent interactions by denisimetric techniques. Meth Enzymol 1979, 61, 26-49.
47. Scopes, R. K., Protein purification. Three ed.; Springer Science: New York, 1994.
48. von Hippel, P. H.; Schleich, T.; Ion effects on the solution structure of biologival macromolecules. Acc Chem Res 1969, 2, 257-265;
49. Robinson, D. R.; Jencks. W. P.; The effect of concentrated salt solutions on the activity coefficient of acetyltetraglycine ethyl ester. J. Am. Chem. Soc. 1965, 87, 2470-2479;
50. Curtis, R. A.; Ulrich, J.; Montaser, A.; Prausnitz, J. M.; Blanch, H. W.; Protein-protein interactions in concentrated electrolyte solutions: Hofmeister-series effects. Biotechnol. and Bioeng. 2002, 79.
51. Nandi, P. K.; Robinson, D. R.; The effects of salts on the free energy of the peptide group. J. Am. Chem. Soc. 1972, 94, 1299-1307.
52. Hofmeister, F.; Zur Lehre von der Wirkung der Salze. Arch Exp Pathol Pharmakol 1888, 24, 247-260.
53. Collins, K. D.; Washabaugh, M. W.; The Hofmeister effect and the behavior of water at interfaces. Q Rev Biophys. 1985, 18, 323-421.
54. Tessier, P. M.; Johnson, H. R.; Pazhianur, R.; Berger, B. W.; Prentice, J. L.; Bahnson, B. J.; Sandler, S. I.; Lenhoff, A. M., Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients. Proteins 2003, 50 (2), 303-311.
55. Tanford, C.; Hauenstein, J. D.; Hydrogen ion equilibria of ribonuclease. J. Am. Chem. Soc. 1956, 78, 5287-5291.
56. Kuntz, I. D.; Hydration of macromolecules. 3. Hydration of polypeptides. J. Am. Chem. Soc. 1971, 93, 514-516.
57. Rao, D. G., Introduction to Biochemical Engineering. 2nd ed.; McGraw Hill: 2010.
58. Shah, D.; Shaikh, A. R.; Peng, X. X.; Rajagopalan, R., Effects of arginine on heat-induced aggregation of concentrated protein solutions. Biotechnol Progr 2011, 27 (2), 513-520.
59. Burke et al., The adsorption of proteins to pharmaceutical container surfaces. International Journal of Pharmaceutics 1992, 86 (1), 89-93.
60. Li, Y., Lubchenko, V., and Vekilov, P. G., The use of dynamic light scattering and Brownian microscopy to characterize protein aggregation. Review of Scientific Instruments 2011, 82 (5).
61. Roufik, S., Paquin, P., and Britten, M., Use of high-performance size exclusion chromatography to characterize protein aggregation in commercial whey protein concentrates. International Dairy Journal 2005, 15 (3), 231-241.
62. Ortega-Vinuesa, J. L., Tengvall, P., and Lundstrom, I., Aggregation of HSA, IgG, and fibrinogen on methylated silicon surfaces. Journal of Colloid and Interface Science 1998, 207 (2), 228-239.
63. Santos et al., Whey protein adsorption onto steel surfaces - effect of temperature, flow rate, residence time and aggregation. Journal of Food Engineering 2006, 74 (4), 468-483.
64. Pihlasalo et al., High sensitivity luminescence nanoparticle assay for the detection of protein aggregation. Analytical Chemistry 2011, 83 (4), 1163-1166.
65. Harma et al., Sensitive quantitative protein concentration method using luminescent resonance energy transfer on a Layer-by-Layer Europium(III) chelate particle sensor. Analytical Chemistry 2008, 80 (24), 9781-9786.
66. Eisenberg, D., McLachlan, A. D. , Solvation energy in protein folding and binding. Nature 1986, 319 (6050), 199-203.
67. Curtis, R. A.; Steinbrecher, C.; Heinemann, A.; Blanch, H. W.; Prausnitz, J. M., Hydrophobic forces between protein molecules in aqueous solutions of concentrated electrolyte. Biophys Chem 2002, 98 (3), 249-265.
68. Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C. V.; Sarma, R.; Structure of hen egg-white lysozyme. Nature 1965, 22, 757-761.
69. Hamaguchi, K., Conformation and enzymatic activity of lysozyme. Tampakushitsu. Kakusan Kosa 1968, 13.
70. Valente, J. J.; Verma K. S.; Manning M. C.; Wilson W. W.; Henry C. S.; Collodial behavior of proteins: Effect of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution. Currently Pharm. Biotechnology 2005, 6, 427-436.

指導教授

陳文逸(Wen-yih Chen)

審核日期

2011-7-26

推文