INTERACTIONS BETWEEN THERAPEUTIC PROTEIN FORMULATIONS AND SURFACES by BRANDON MICHAEL TESKA B.S., University of Colorado Boulder, 2007 B.M., University of Colorado Boulder, 2007 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Pharmaceutical Sciences Program 2015 This thesis for the Doctor of Philosophy degree by Brandon Michael Teska has been approved for the Pharmaceutical Sciences Program by Thomas J. Anchordoquy, Chair John F. Carpenter, Advisor David L. Bain Theodore W. Randolph Krishna M. G. Mallela Date: 07/30/2015 ii Teska, Brandon Michael (Ph.D., Pharmaceutical Sciences) Interactions Between Therapeutic Protein Formulations and Surfaces Thesis directed by Professor John F. Carpenter ABSTRACT Therapeutic protein formulations encounter a multitude of different surfaces in every part of their production, packaging, storage and administration to patients. These in- terfaces can be very different—chemically—from the formulation’s solution chemistry and can have unintended, negative effects on the formulation. Protein molecules can adsorb to these surfaces, which can induce structural perturbations in the therapeutic and promote aggregation. Components from the formulation can be absorbed into the surfaces, altering the formulation solution conditions, which can bring about addi- tional stresses on the therapeutic. Formulations can even chemically modify surfaces they come in contact with, altering the surface properties. Understanding such inter- actions between formulations components and surfaces is critical to developing better storage and delivery devices and improved formulations. In this work, I examined the pharmaceutical compatibility of a novel syringe plungercoating—designedtobe used in silicone oil-free, pre-filled syringes—and found it to cause much less protein aggregation during agitation than was observed in a traditional siliconized syringe. Second, I found that plastics found in catheters absorbed phenolic compounds from insulin analog formulations and that this depletion had a profound impact on differ- ent insulin analogs’ assembly states and stabilities under thermal stress. Finally, I also found that zinc ions, found in insulin formulations as well, chemically damaged analytical size exclusion chromatography columns, which are used to monitor soluble aggregates of insulin in therapeutic formulations. iii The form and content of this abstract are approved. I recommend its publication. Approved: John F. Carpenter iv DEDICATION This work is dedicated to my wife, Johanne, who has been my inspiration and en- couragement throughout my graduate career. To my parents, Mike and Cindy, who encouraged and supported my aspirations to become a scientist from my early years and to my brothers and all my good friends who have been my biggest fans throughout the years. v ACKNOWLEDGEMENTS First and foremost, I would like to thank Dr. John Carpenter for his mentorship, guidance and support throughout my thesis work in his lab. John is passionate about fighting for patients, one of the many things I’ve loved about working in his lab. Because of his diligence and persistence, we’re beginning to see pharmaceutical companies, regulators and clinicians work together to identify, track and mitigate sources of immunogenicity. I feel very honored to have been a part of this—we are literally changing the face of medicine for the betterment ofthepatients. I would also like to thank the members of my committee. Dr. DaveBainfor encouraging me to work towards a deeper, mechanistic understanding of my findings, Dr. Ted Randolph for unique way of thinking about the biophysics that helped drive me to a deeper understanding of pharmaceutics, Dr. Krishna Mallela for sharing his protein characterization expertise and Dr. Tom Anchordoquy for his insightful questions and for his uncanny ability to make everyone smile. Thank you all! I am eternally grateful for your guidance and mentorship during my thesis work. Secondly, I would like to thank all of my colleagues and friends at the University during my tenure in John’s lab (in no particular order): Dr. AjayThomas,Dr.Merry Christie, Dr. Tyson Smyth, Indu Persaud, Dr. Nicole Payton, Dr. Regina Bis, Dr. Tanya Clapp, Neha Pardeshi, Dr. Pinaki Basu, Dr. Jim Barnard, Dr. Wei Qi, Dr. Pradyot Nandi, Jan Jaap Verhoef, Shyam Mehta, Chen Zhou, Dr. Aaron Krueger, Aditya Gandhi and Dr. Luke Liu. For all your stimulating conversations, advice and laughs on all things science (and non-science) over the years, thank you! Third, I would like to thank all of the students that worked for me on various parts of the work in this thesis: Brad Butler, Rahie Talukder, Isabel Randolph, Mary Whitney, Sara Brown, Brian Adams, Lindsey Ross and Samantha Egger. Thank you for all your hard work and dedication contributing to thisimportanttopicin vi pharmaceutical sciences. Fourth, I would like to that the organizations that have funded parts of my re- search. Becton Dickinson and Company, W. L. Gore and Associates, and the Phar- maceutical Research and Manufacturers of America. Lastly—but certainly not least—I would like the thank my wife, Johanne, for standing by me through thick and thin. I would also like to thank my parents, my brothers and my friends for their love and support. vii TABLE OF CONTENTS CHAPTER I INTRODUCTION 1 II INVESTIGATION OF THE PHARMACEUTICAL COMPATIBILITY OF A NOVEL SURFACE FOR USE IN THERAPEUTIC PROTEIN DELIVERY DEVICES 17 Abstract..................................... 17 Introduction . 18 Materials and Methods . 22 Materials . 22 AgitationinVials............................. 23 AgitationinSyringes ........................... 24 Micro-FlowImaging(MFI). 26 SizeExclusionChromatography(SEC) . 27 Confocal Microscopy of the Adsorption of Fluorescently Labeled IVIGtotheFluoropolymerSurface . 27 Determination of the Second Osmotic Viral Coefficient (B22)..... 29 IVIGUnfoldingwithUrea ........................ 30 Results and Discussion . 30 IncreasedAgitationStressinVials . 33 Effect of Buffer Type on Aggregation of IVIG During Agitation in Vials 34 Agitation of IVIG in Glycine Buffer . 35 Agitation of IVIG in PBS Buffer . 36 AgitationofAvastin ........................... 38 AgitationinSyringes ........................... 41 Agitation of IVIG in Glycine Buffer . 41 Agitation of IVIG in PBS Buffer . 42 viii Morphological Differences Between Protein Particles Formed From Agitation of IVIG in the Fluoropolymer Syringe and the Siliconized Syringe . 44 AgitationofAvastin ....................... 47 Mechanism(s) for Inhibition of Protein Aggregation by Polysorbate 20DuringAgitation ....................... 47 Effects of pH and Buffer on the Conformational and Colloidal StabilityofIVIG ......................... 53 The Utility of the Fluoropolymer Surface in Silicone Oil-Free Syringes 58 Acknowledgments................................ 59 III EFFECTS OF PHENOL AND META-CRESOL DEPLETION ON INSULIN ANALOG STABILITY AT PHYSIOLOGICAL TEMPERATURE 60 Abstract..................................... 60 Introduction . 61 Materials and Methods . 64 Materials . 64 Stability Study . 64 SizeExclusionChromatography(SEC) . 65 Reversed Phase Chromatography (RP) . 66 Micro-FlowImaging(MFI). 66 AtomicAbsorptionSpectrometry(AAS) . 67 AnalyticalUltracentrifugation(AUC) . 67 Results . 68 AtomicAbsorptionSpectroscopy . 68 SizeExclusionChromatography . 69 Reversed-Phase Chromatography . 71 AnalyticalUltracentrifugation . 76 Micro-FlowImaging ........................... 79 ix Discussion . 80 Acknowledgements ............................... 84 IV ANALYZING INSULIN SAMPLES BY SIZE-EXCLUSION CHROMATOGRAPHY: A COLUMN DEGRADATION STUDY 93 Abstract..................................... 93 Introduction . 93 Materials and Methods . 95 Materials . 95 Silica Resin Incubation . 95 Liquid-Liquid Extraction . 96 Size-Exclusion Chromatography . 96 Nuclear Magnetic Resonance and Gas Chromatography-Mass Spectrometry ........................... 97 Results and Discussion . 98 Conclusion . 103 Acknowledgements ............................... 103 V CONCLUSIONS AND FUTURE DIRECTIONS 107 ChapterII: NewSurfacesin Pre-filled Syringes . ... 107 ChapterIII: InsulinAnalogAssembly. 112 Chapter IV: Effects of Formulations on Surfaces . ... 119 Concluding Remarks . 120 REFERENCES 146 APPENDIX 147 A SUPPLEMENTAL FIGURES FOR CHAPTER II 147 Methods . 147 x Resonant Mass Measurement (RMM) . 147 Results and Discussion . 148 Vial Agitation: IVIG in Glycine Buffer . 148 Vial Agitation: IVIG in PBS Buffer . 150 VialAgitation:Avastin. 150 Syringe Agitation: IVIG in Glycine Buffer . 150 Syringe Agitation: IVIG in PBS Buffer . 153 SyringeAgitation:Avastin. 153 B EXTENT OF ADSORPTION OF PHENOLIC PRESERVATIVES BY DIFFERENT BRANDS OF COMMONLY USED INSULIN PUMP CATHETERS 156 Abstract..................................... 156 Introduction . 156 Materials and Methods . 157 Preparation and Incubation of the Catheter Tubing . .. 158 UVAbsorbanceSpectroscopy . 160 Results and Discussion . 160 Limitations and Future Directions . .. 160 Acknowledgments................................ 162 xi LIST OF TABLES TABLE 1 Phenolic Preservative Concentration in Various Units . ..... 85 2Mobilephasecompositionduringreversed-phasechromatography . 86 3Theoreticalsedimentationcoefficient limits for different self-assemblies of insulin glulisine. ... 117 4Cathetersetdimensions..........................159 xii LIST OF FIGURES FIGURE 1 Agitationstudyvialstoppers. 24 2Glass-onlyagitationaparatus.......................25