MECHANISTIC INSIGHTS INTO THE ROLE OF PROTEIN INTERACTIONS ON THE AGGREGATION BEHAVIOR OF ANTI-STREPTAVIDIN IMMUNOGLOBULIN GAMMA-1 by Gregory V. Barnett A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Engineering Fall 2015 c 2015 Gregory V. Barnett All Rights Reserved ProQuest Number: 10014766 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest 10014766 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 MECHANISTIC INSIGHTS INTO THE ROLE OF PROTEIN INTERACTIONS ON THE AGGREGATION BEHAVIOR OF ANTI-STREPTAVIDIN IMMUNOGLOBULIN GAMMA-1 by Gregory V. Barnett Approved: Abraham M. Lenhoff, Ph.D. Chair of the Department of Chemical Engineering Approved: Babatunde Ogunnaike, Ph.D Dean of the College of Engineering Approved: Ann L. Ardis, Ph.D. Interim Vice Provost for Graduate and Professional Education I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: Christopher J. Roberts, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: David W. Colby, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: Eric M. Furst, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: Vladimir I. Razinkov, Ph.D Member of dissertation committee ACKNOWLEDGEMENTS I would like to thank many people for their support during my time at the University of Delaware. First and foremost, I want to thank my advisor, Chris Roberts, for his guidance and support over the years. Chris encouraged many collaborative opportunities that has enriched this work and my experience here. He challenges students to think deeper about protein interactions and behavior. I will always value the rigorous approach Chris brings to chemical engineering and protein biophysics, and I will strive towards that standard in my career. I want to thanks the many collaborators and colleagues who invited me to work in their labs. A special thanks to Amgen for supporting this work and Vladimir Razinkov who meet biweekly for almost four years. I thank you for your patience, helpfulness and practical industrial perspective. Thank you to Tom Laue for guid- ance on electrophoresis measurements, collaborators at Malvern Instruments, Wei Qi, Samiul Amin, and Neil Lewis, who allowed me to visit their lab and work with the DLS-Raman prototype. I enjoyed many scientific discussions with Wei and Samiul. Thanks to Yun Liu and Paul Butler at the NIST Center for Neutron Research who where always friendly and helpful discussing SANS experiments or data analysis. I want to thank the current and past members of the Roberts group, especially Nayoung Kim, Marco Blanco, Chris O'Brien, and Ranendu Ghosh for their support, patience, and help with many long hours fixing and troubleshooting experiments and instruments. I could always count on open doors and thorough scientific discussions. Lastly I want to thanks my friends and family. Jon, Ian and friends at Delaware have made the last four years fun and exciting. Most importantly, I owe a special thanks to my Mom and Dad for encouragement and education throughout the years and always having a welcoming home nearby. iv TABLE OF CONTENTS LIST OF TABLES :::::::::::::::::::::::::::::::: x LIST OF FIGURES ::::::::::::::::::::::::::::::: xi ABSTRACT ::::::::::::::::::::::::::::::::::: xviii Chapter 1 INTRODUCTION :::::::::::::::::::::::::::::: 1 1.1 Motivation ::::::::::::::::::::::::::::::::: 1 1.2 Objectives ::::::::::::::::::::::::::::::::: 5 1.3 Non-native aggregation :::::::::::::::::::::::::: 6 1.3.1 Rate determining steps for non-native aggregation ::::::: 7 1.4 Aggregation mechanisms ::::::::::::::::::::::::: 8 1.5 Modeling aggregation kinetics :::::::::::::::::::::: 8 1.5.1 Colloidal aggregation ::::::::::::::::::::::: 9 1.5.2 Lumry-eyring nucleation polymerization (LENP) model :::: 10 1.6 Factors affecting protein aggregation :::::::::::::::::: 10 1.6.1 Solution pH :::::::::::::::::::::::::::: 11 1.6.2 Salts :::::::::::::::::::::::::::::::: 12 1.6.3 Neutral osmolytes ::::::::::::::::::::::::: 13 1.6.4 Other osmolytes :::::::::::::::::::::::::: 15 1.7 Predicting aggregation :::::::::::::::::::::::::: 16 1.7.1 Non-arrhenius behavior :::::::::::::::::::::: 17 1.8 Anti-streptavidin IgG1 :::::::::::::::::::::::::: 18 1.8.1 IgG1 structure :::::::::::::::::::::::::: 18 v 1.8.2 Prior work with AS-IgG1 ::::::::::::::::::::: 19 1.9 Organization of the dissertation ::::::::::::::::::::: 20 2 SPECIFIC-ION EFFECTS ON IGG1 AGGREGATION BEHAVIOR :::::::::::::::::::::::::::::::::: 22 2.1 Introduction :::::::::::::::::::::::::::::::: 22 2.2 Materials and methods :::::::::::::::::::::::::: 23 2.2.1 Sample preparation :::::::::::::::::::::::: 23 2.2.2 Size exclusion chromatography with inline light scattering (SEC-MALS) ::::::::::::::::::::::::::: 24 2.2.3 Protein-protein interactions via laser light scattering ::::: 25 2.2.4 Determination of radius of gyration for small oligomers with SAXS ::::::::::::::::::::::::::::::: 26 2.2.5 IgG1 aggregate hydrodynamic radius from dynamic light scattering ::::::::::::::::::::::::::::: 26 2.2.6 IgG1 net charge (valence) via electrophoretic light scattering : 27 2.2.7 Aggregate morphology using small angle neutron scattering (SANS) :::::::::::::::::::::::::::::: 28 2.3 Aggregation mechanism(s) from SEC-MALS :::::::::::::: 29 2.4 Aggregate mass to size scaling :::::::::::::::::::::: 34 2.5 Aggregate morphology using SANS and SAXS. ::::::::::::: 36 2.6 Protein-protein interactions based on static light scattering :::::: 40 2.7 Protein net echarge determined by electrophoretic light scattering (ELS) 42 2.8 Role of electrostatic protein-protein interactions on IgG1 aggregation mechanims :::::::::::::::::::::::::::::::: 46 2.9 Aggregate morphology from scattering ::::::::::::::::: 49 ∗ 2.10 G22: semi-quantitative tool to predict aggregation mechanism :::: 51 2.11 Summary and Conclusions :::::::::::::::::::::::: 53 3 PARALLEL TEMPERATURE INITIAL RATES: PH, AND COUNTERION EFFECTS ON IGG1 AGGREGATION RATES 55 3.1 Introduction :::::::::::::::::::::::::::::::: 55 3.2 Materials and Methods :::::::::::::::::::::::::: 57 3.2.1 Differential scanning calorimetry (DSC) :::::::::::: 57 3.2.2 Quantifying Aggregation Rates ::::::::::::::::: 57 vi 3.2.3 Parallel Temperatures Initial Rates (PTIR) :::::::::: 58 3.3 AS-IgG1 thermal unfolding using differential scanning calorimetry (DSC) ::::::::::::::::::::::::::::::::::: 60 3.4 Aggregation Rates from PTIR and Standard Isothermal Approaches : 62 3.5 Effects of pH, buffer, and NaCl on Temperature-Dependent Rates :: 69 3.6 Summary and Conclusions :::::::::::::::::::::::: 73 4 OSMOLYTE EFFECTS ON MONOCLONAL ANTIBODY STABILITY AND CONCENTRATION-DEPENDENT PROTEIN INTERACTIONS WITH WATER AND COMMON OSMOLYTES 74 4.1 Introduction :::::::::::::::::::::::::::::::: 74 4.2 Materials and methods :::::::::::::::::::::::::: 77 4.2.1 Sample preparation :::::::::::::::::::::::: 77 4.2.2 Partial specific volume via densimetry ::::::::::::: 78 4.2.3 Model prediction of preferential interaction via solvent accessible surface area (ASA) :::::::::::::::::::::::: 79 4.2.4 AS-IgG1 unfolding via differential scanning calorimetry :::: 81 4.3 Partial specific volume via density measurements ::::::::::: 81 4.4 Partial specific volume of AS-IgG1 in neutral osmolytes :::::::: 83 4.5 Kirkwood-Buff integrals for protein-water and protein-osmolyte interactions :::::::::::::::::::::::::::::::: 86 4.6 AS-IgG1 native state chemical potential :::::::::::::::: 89 4.7 AS-IgG1 unfolding via DSC ::::::::::::::::::::::: 92 4.8 Preferential interactions and their effect on protein unfolding and stability :::::::::::::::::::::::::::::::::: 95 4.9 Summary and conclusions :::::::::::::::::::::::: 100 5 AGGREGATE STRUCTURAL CHANGES AND MECHANISMS AT ELEVATED CONCENTRATION ::::::::::::::::: 102 5.1 Introduction :::::::::::::::::::::::::::::::: 102 5.2 Materials and Methods :::::::::::::::::::::::::: 103 5.2.1 Sample Preparation :::::::::::::::::::::::: 103 5.2.2 Ex-situ monomer loss kinetics and light scattering using SEC-MALS :::::::::::::::::::::::::::: 104 5.2.3 Circular Dichroism :::::::::::::::::::::::: 104 5.2.4 Second derivative UV absorption :::::::::::::::: 105 vii 5.2.5 In-situ dynamic light scattering with Raman spectroscopy (DLS-Raman) ::::::::::::::::::::::::::: 106 5.2.6 Small angle neutron