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Solvent Freeze out (SFO) Technology for Protein Crystallization

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Solvent Freeze out (SFO) Technology for Protein Crystallization Solvent Freeze Out (SFO) Technology for Protein Crystallization -Optimization and Applicability- Dissertation Zur Erlangung des Doktorgrades der Ingenieurwissenschaften (Dr. -Ing.) des Zentrums für Ingenieurwissenschaften der Martin-Luther-Universität Halle-Wittenberg, vorgelegt von Frau M.Sc. Viviana Patricia Díaz Borbón geb. am 05.10.1981 in Bogotá, Kolumbien Gutachter: 1. Prof. Dr. –Ing. Dr. hc. Joachim Ulrich 2. apl. Prof. Dr. rer. nat. habil. Heike Lorenz Datum der Verteidigung: 28.03.2013 Düsseldorf, den 19.06.2013 Acknowledgments During the Christmas party of the year 2009, Professor Joachim Ulrich announced to me that I have been accepted as one of his PhD students. Since that day until today, when I am writing these sentences, I have been deeply grateful for this opportunity. I very much appreciated the support of Prof. Ulrich during the time I was his student. In our meetings and discussions, he always showed me the right way to the fulfillment of my tasks and aims. All the experiences gained through several congresses, seminars and exchange journeys are also of high value to me and I am very grateful for them. Special acknowledgments are directed to Prof. Dr. M. Pietzsch and Dr. Hertel, members of the Downstream Processing group which belongs to the Institute of Pharmacy of the MLU Halle- Wittenberg, for sharing their laboratories and valuable proteins with us. I would like to thank Dr. Spohn from Fraunhofer Institute for Mechanics of Materials IWM, for the supply of the silk proteins. Thanks to Ms. Andrea Alles, account manager of European STEM, for providing us with the STEAM Integrity 10 system used in a crucial part of my experiments. A special thank you goes to the students which were under my supervision: Steven, Annika, Steffi, Maria and Boat. We built this work together! I want to thank Tao, with whom I had some common aims and worked together in order to reach them. We had therefore, several fruitful discussions and learned a lot from each other. I would like to thank all my TVT colleagues and team members, for their support, help and especially for the moments we spent together. I wish all of them the best in their careers and personal life, for now and for the future. I thank my husband for his support, patience and love and his family, which has been my family here in my second home: Germany. The love to my parents and grandparents has been the motor of my life and their love to me has been the element which permits my life to be so precious. Thank God for letting us be. Oh! And thanks to my cat –Tiga. Because of him, I was awake and “fit” early in the morning to start a new working day. 2 Table of Contents 1. Introduction .............................................................................................................................. 5 2. Theoretical Background ........................................................................................................... 7 2.1 Basics of Crystallization ..................................................................................................... 7 2.1.1 Phase Diagrams .......................................................................................................... 8 2.1.2 Industrial Crystallization ........................................................................................... 10 2.2 Melt Crystallization .......................................................................................................... 11 2.3 Solvent Freeze Out Crystallization (SFO) Technology ..................................................... 13 2.4 Protein Crystallization ..................................................................................................... 15 2.4.1 Industrial Application of Protein Crystallization ...................................................... 18 2.5 Process Optimization ....................................................................................................... 19 3. Aim of the Work ..................................................................................................................... 22 4. Materials and Methods .......................................................................................................... 23 4.1 Chemicals ......................................................................................................................... 23 4.2 Identification of Regions for Crystallization .................................................................... 25 4.2.1 Evaluation of Solubility Curve for Lysozyme by van´t Hoff Equation ....................... 25 4.2.2 Screening Tests for Protein Crystallization .............................................................. 26 4.2.3 Turbidity Measurements for Determination of Nucleation and Solubility Curves .. 29 4.3 Solvent Freeze Out Process ............................................................................................. 30 4.3.1 Protein Crystallization from Pure Solutions ............................................................. 31 4.3.2 Protein Crystallization from Mixture........................................................................ 31 4.4 Evaluation and Characterization of the Crystals ............................................................. 32 4.5 Optimization Strategy ...................................................................................................... 32 4.5.1 Fractional Factorial Design ....................................................................................... 34 4.5.2 Central Composite Design ........................................................................................ 35 5. Results .................................................................................................................................... 37 5.1 Determination of Solubility Curves of Lysozyme ............................................................. 37 5.2 Screening Test for Lysozyme Crystallization ................................................................... 39 5.2.1 Pure Lysozyme Solutions .......................................................................................... 39 5.2.2 Lysozyme Ovalbumin Mixtures ................................................................................ 47 5.3 Pseudo-phase Diagrams: Solubility and Nucleation ........................................................ 48 5.3.1 Lysozyme in Pure Solutions ...................................................................................... 49 5.3.2 Lysozyme-Ovalbumin Mixture ................................................................................. 53 5.4 Crystallization of Lysozyme by Solvent Freeze Out ......................................................... 55 5.4.1 Crystallization of Lysozyme from Pure Solutions ..................................................... 56 5.4.2 Crystallization of Lysozyme from Lysozyme-Ovalbumin Mixtures .......................... 61 5.4.2.1 Preliminary Crystallization of Lysozyme by SFO ............................................... 61 5.4.2.2 Advanced Crystallization of Lysozyme by SFO .................................................. 64 5.5 Process Optimization ....................................................................................................... 69 5.5.1 Fractional Factorial Designs ..................................................................................... 70 5.5.2 Significant Effects ..................................................................................................... 77 5.5.3 Central Composite .................................................................................................... 78 5.5.3.1 Surface Response .............................................................................................. 82 5.5.3.2 Desirability Profile ............................................................................................. 83 6. Discussion ............................................................................................................................... 85 6.1 Determination of Pseudo-Phase Diagrams for Lysozyme ............................................... 85 6.2 Crystallization of Lysozyme from Pure Solutions and Mixtures ...................................... 88 6.2.1 Screening Tests ......................................................................................................... 88 6.2.2 Solvent Freeze Out -SFO ........................................................................................... 89 6.3 Effects of Parameters and Interactions on Process Responses ...................................... 91 6.4 Optimization of the Process Parameters in the SFO Crystallization of Lysozyme .......... 94 6.5 Considerations on the Design of Solvent Freeze Out Process ........................................ 95 7. Conclusions ........................................................................................................................... 101 8. Summary .............................................................................................................................. 102 9. Appendix............................................................................................................................... 114 Introduction 5 1. Introduction One of the greatest challenges with respect to protection and preservation of the environment is the replacement of many synthetic materials with biodegradable and environmentally friendly materials [Fro10]. Nowadays proteins, such as biopolymers, play an important
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