Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations January 2015 Thiol-Disulfide Exchange in Human Growth Hormone Saradha Chandrasekhar Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Recommended Citation Chandrasekhar, Saradha, "Thiol-Disulfide Exchange in Human Growth Hormone" (2015). Open Access Dissertations. 1449. https://docs.lib.purdue.edu/open_access_dissertations/1449 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. i THIOL-DISULFIDE EXCHANGE IN HUMAN GROWTH HORMONE A Dissertation Submitted to the Faculty of Purdue University by Saradha Chandrasekhar In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2015 Purdue University West Lafayette, Indiana ii To my parents Chandrasekhar and Visalakshi & To my fiancé Niranjan iii ACKNOWLEDGEMENTS I would like to thank Dr. Elizabeth M. Topp for her tremendous support, guidance and valuable suggestions throughout. The successful completion of my PhD program would not have been possible without her constant encouragement and enthusiasm. Through the last five years, I’ve learned so much as a graduate student in her lab. My thesis committee members: Dr. Stephen R. Byrn, Dr. Gregory T. Knipp and Dr. Weiguo A. Tao, thank you for your time and for all your valuable comments during my oral preliminary exam. I would also like to thank Dr. Fred Regnier for his suggestions with the work on human growth hormone. I am grateful to all my lab members and friends for their assistance and support. I would like to especially thank Dr. Andreas S. Sophocleous, Dr. Jun Zhang and Dr. Shenbaga Moorthy Balakrishnan for helpful discussions, answering questions and for training on me on different instruments in the lab. Special thanks and gratitude go to Daniel E. Epling for his help with the temperature studies, Ruichao Xie for performing peptide studies and to John O. Grady for training me on the Perfinity Flash digest protocol. iv I am very grateful to my parents, who have encouraged and believed in me from the beginning. Thank you for being so patient and for giving me the opportunity to pursue my dreams. I am also thankful to my fiancé, Niranjan, for supporting me through the good and bad times of my graduate student life. I will be forever indebted to you all. v TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ........................................................................................................... xi LIST OF ABBREVIATIONS .......................................................................................... xix ABSTRACT ............................................................................................................ xxi CHAPTER 1. OVERVIEW OF THERAPEUTIC PROTEINS ................................... 1 1.1 Introduction .....................................................................................................1 1.2 Recombinant DNA technology .......................................................................3 1.3 Stability of protein drugs ................................................................................4 1.3.1 Physical degradation ............................................................................. 5 1.3.2 Chemical degradation ........................................................................... 7 1.4 Thiols and disulfide bonds ..............................................................................9 1.5 Disulfide bond formation in proteins ............................................................10 1.5.1 Disulfide bond disruption and rearrangement ..................................... 14 1.6 Factors that influence disulfide bond reactivity ............................................18 1.6.1 Intrinsic factors ................................................................................... 18 1.6.2 Extrinsic factors .................................................................................. 20 1.7 Impact on protein physical stability and biological activity .........................23 1.8 Formulation strategies for Cys-containing proteins .....................................25 1.8.1 Formulating proteins with a reducing agent ....................................... 25 1.8.2 Chemical modification of thiol groups ............................................... 25 1.8.3 Protein engineering ............................................................................. 27 1.9 Lyophilization of peptide and protein drugs .................................................29 1.9.1 Thiol-disulfide exchange in the solid-state ......................................... 30 vi Page 1.10 Disulfide bond mapping ...............................................................................32 1.11 Research objectives and specific aims ..........................................................36 1.12 Research Significance ...................................................................................38 CHAPTER 2. ELUCIDATING THE MECHANISM AND KINETICS OF THIOL- DISULFIDE EXCHANGE IN PEPTIDES DERIVED FROM HUMAN GROWTH HORMONE IN AQUEOUS SOLUTION ........................................................................ 41 2.1 Abstract .........................................................................................................41 2.2 Keywords ......................................................................................................42 2.3 Introduction ...................................................................................................42 2.4 Materials .......................................................................................................46 2.5 Methods ........................................................................................................47 2.5.1 Preparation of peptide stock solutions and disulfide linked peptides for kinetic studies........................................................................................................ 47 2.5.2 Homodimer formation ........................................................................ 47 2.5.3 Buffer preparation ............................................................................... 48 2.5.4 Quantification of reactants and products by HPLC ............................ 49 2.5.5 Determination of reaction order .......................................................... 52 2.5.6 Thiol-disulfide exchange reactions ..................................................... 52 2.5.7 Determining activation parameters for the reaction of T20 with T20-T21 ............................................................................................................. 53 2.6 Data analysis .................................................................................................54 2.7 Results ...........................................................................................................57 2.7.1 Addition of oxidation suppressants to favor the thiol-disulfide exchange reaction ............................................................................................................. 57 2.7.2 Apparent order of reaction; reaction of T20 and T20-T21 ................. 60 2.7.3 Effect of oxidation suppressants on thiol-disulfide exchange ............ 62 2.7.4 Effect of pH on the reaction of T20 with T20-T21 ............................. 64 2.7.5 Activation parameters for the reaction of T20 with T20-T21 ............. 71 2.7.6 Effect of peptide cyclization on thiol-disulfide exchange .................. 74 vii Page 2.8 Discussion .....................................................................................................77 2.9 Conclusion ....................................................................................................81 CHAPTER 3. INVESTIGATING THE EFFECT OF LYOPHILIZATION AND SUBSEQUENT STORAGE IN THE SOLID STATE ON THIOL-DISULFIDE EXCHANGE IN PEPTIDES DERIVED FROM HUMAN GROWTH HORMONE .... 83 3.1 Abstract .........................................................................................................83 3.2 Keywords ......................................................................................................84 3.3 Introduction ...................................................................................................84 3.4 Materials .......................................................................................................88 3.4.1 Quantification of reactants and products by HPLC ............................ 89 3.4.2 Thiol-disulfide exchange reactions ..................................................... 90 3.4.3 Lyophilization of peptide samples ...................................................... 93 3.4.4 Peptide adsorption to ice ..................................................................... 94 3.4.5 Method for detecting reaction intermediates ...................................... 95 3.4.6 Data analysis ....................................................................................... 96 3.5 Results ...........................................................................................................98 3.5.1 Effect of lyophilization on thiol-disulfide exchange .......................... 98 3.5.2 Disulfide bond stability during lyophilization .................................