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Chromatographic Separation and Stability Analysis of Small Interfering RNA And Chromatographic Separation and Stability Analysis of Small Interfering RNA and Lipid Vehicles Using Ion-Pair Reversed Phase Liquid Chromatography A Thesis Submitted to the Faculty of Drexel University by Li Li In partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2017 © Copyright 2017 Li Li. All Rights Reserved. Dedications This thesis is dedicated to my loving parents, Lanyin Cao and Yisong Li, for their unconditional love, support and encouragement. Acknowledgements I would like to thank my advisor Prof. Joe Foley for an incredible support of my study and research at Drexel. I am grateful for his patience, motivation, and immense knowledge in separation science. His encouragement and guidance helped me greatly throughout the research and the writing of this thesis. I would also like to thank my research co-advisor, Dr. Roy Helmy, for his scientific guidance and for creating a supportive environment at Merck so I can focus and complete my Ph.D. study. I am very grateful that Dr. Helmy has taken time from his busy schedule to be my co-advisor, and without his help and encouragement, this would not have been possible. I would like to thank my research committee members: Dr. Frank Ji (chair), Dr. Lynn Penn, Dr. Dan King, Dr. Peter Wade, and Dr. Ezra Wood. Special thanks to Dr. Mark Schure from the University of Delaware for serving in my research committee. Thank you all for your time and effort reviewing my work and thesis and for providing valuable comments. I also want to express my sincere appreciation to the faculty and staff of Drexel University Chemistry Department and my fellow graduate students for their support during my time at Drexel. Your friendship made this journey a truly memorable chapter of my life. I want to express my most profound gratitude to my employer Merck for the financial support and lab resources during my study. I also want to thank my current and past mentors: Dr. Chris Welch, Dr. Tony Leone, Dr. Yun Mao, Dr. Mark Mowery and Dr. Peter Wuelfing, who have supported me to pursue my dream. Finally, I want to thank my parents and siblings for always being there for me when I need the help to navigate through a challenging schedule. Special thanks to my husband Xiaoping and daughter Yuyan. Words cannot express how grateful I am to you all for the sacrifices that you've made on my behalf. Your unconditional love and support have been the constant driving force during this incredible journey. i Table of Contents List of Tables ..................................................................................................................... vi List of Schemes ................................................................................................................. vii List of Figures .................................................................................................................. viii List of Symbols ..................................................................................................................xv List of Abbreviations ...................................................................................................... xvii Abstract ..............................................................................................................................xx Chapter 1 Introduction to HPLC .........................................................................................1 1.1 A brief history of HPLC ..............................................................................................1 1.2 HPLC theory and principles ........................................................................................3 1.2.1 Key factors impacting chromatographic resolution ....................................................3 1.2.2 Fundamental principles of band broadening and zone separation ..............................5 1.2.2.1 Column contributions to band dispersions and Van Deemter equation ...................5 1.2.2.2 Fundamental principles of solute-zone separation ...................................................7 1.3 HPLC methodology .......................................................................................................8 1.3.1 Normal-phase liquid chromatography ........................................................................9 1.3.2 Reversed-phase liquid chromatography ......................................................................9 1.3.3 Ion-pair chromatography ..........................................................................................11 1.3.4 Ion-exchange chromatography..................................................................................12 1.3.5 Size exclusion chromatography ................................................................................14 List of references................................................................................................................16 ii Chapter 2 Overview of small interfering RNA-based therapy, delivery technology, and analytical characterization of lipid nanoparticle formulations ...........................................19 2.1 RNAi: mechanism of action and biologic significance ...............................................19 2.2 siRNA delivery platforms ............................................................................................23 2.2.1 Lipid-based delivery systems ....................................................................................25 2.2.1.1 Cationic lipids as carriers for siRNA delivery .......................................................26 2.2.1.2 Anionic lipids as carriers for siRNA delivery........................................................28 2.2.2 Polymer-based delivery systems ...............................................................................29 2.2.2.1 Nanoparticle-based polymer delivery systems ......................................................29 2.2.2.1.1 Natural polymers as carriers for siRNA delivery ...............................................30 2.2.2.1.2 Synthetic polymers as carriers for siRNA delivery ............................................31 2.2.2.2 Covalent bond based delivery system: dynamic polyconjugate ............................36 2.2.2.2.1 Polyvinylether-based polyconjugate ...................................................................36 2.2.2.2.2 PolyLycine-based polyconjugate .......................................................................38 2.2.3 Summary of siRNA delivery platforms ....................................................................39 2.3 Overview of analytical separation and stability characterization of LNP ...................40 2.3.1 Chemical degradation of RNA and lipids in LNP ....................................................40 2.3.2 Overview of separation techniques for oligonucleotides ..........................................44 2.3.2.1 Ion-pair reversed phase liquid chromatography ....................................................46 2.3.2.2 Ion-exchange chromatography...............................................................................49 2.3.2.3 Capillary electrophoresis ......................................................................................52 2.3.3 Overview of lipid separation and analysis ................................................................55 2.4 Research objectives and rationales .............................................................................59 iii List of References ..............................................................................................................64 Chapter 3 Separation of siRNA stereoisomers using ion-pair reversed phase liquid chromatography .................................................................................................................71 3.1 Introduction ..................................................................................................................71 3.2 Material and methods ...................................................................................................73 3.2.1 Chemicals ..................................................................................................................73 3.2.2 Instrument .................................................................................................................73 3.2.3 IP-RP Chromatographic conditions ..........................................................................74 3.2.4 Differential scanning calorimetry method condition ................................................74 3.2.5 Desulfurization of siRNA duplex using Iodine ........................................................75 3.3 Results and Discussion ................................................................................................75 3.3.1 The impact of column stationary phase on the separation of siRNA stereoisomers .... ............................................................................................................................................77 3.3.2 The impact of ion-pair reagents on the separation of siRNA diastereomers ............79 3.3.3 The impact of organic modifier on the separation of siRNA diastereomers ............83 3.3.4 Method optimization for the separation of siRNA stereoisomers ............................85 3.3.5 Separation of desulfurization products of siRNA stressed with Iodine ....................87 3.4 Conclusions ..................................................................................................................88 List of References
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