FUNCTIONALIZED NANOPARTICLE CROSSLINKING FOR ENHANCED AFFINITY PRECIPITATION OF MONOCLONAL ANTIBODIES by Andrew R. Swartz 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 Summer 2018 © 2018 Andrew R. Swartz All Rights Reserved FUNCTIONALIZED NANOPARTICLE CROSSLINKING FOR ENHANCED AFFINITY PRECIPITATION OF MONOCLONAL ANTIBODIES by Andrew R. Swartz Approved: __________________________________________________________ Eric Furst, Ph.D. Chair of the Department of Chemical and Biomolecular Engineering Approved: __________________________________________________________ Babatunde Ogunnaike, Ph.D. Dean of the College of Engineering Approved: __________________________________________________________ Douglas J. Doren, 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: __________________________________________________________ Wilfred Chen, 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: __________________________________________________________ Abraham Lenhoff, 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: __________________________________________________________ Christopher Roberts, 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: __________________________________________________________ Steven Traylor, Ph.D. Member of dissertation committee ACKNOWLEDGMENTS I would like to thank my advisor, Prof. Wilfred Chen, my lab group members, my dissertation committee, Prof. Abraham Lenhoff, Prof. Christopher Roberts, and Dr. Steven Traylor, and my collaborators at Bristol-Meyers Squibb (BMS) and Rensselaer Polytechnic Institute for their technical guidance, scientific expertise, and insightful discussions during my PhD research. I would like to acknowledge my funding source supported by grants from NSF (CBET1403724 and DMR1609621) and BMS via the GOALI program collaboration. I would like to thank BMS Biologics Process Development (Devens, MA) and Dr. Xuankuo Xu, Dr. Steven Traylor, and Dr. Zheng Jian Li for providing the mAb samples and for their technical and analytical support. I would also like to thank Prof. Kelvin Lee and Dr. Jongyoun Baik and for access and guidance with the Octet RED96e instrument in the NIIMBL laboratory at the University of Delaware Biotechnology Institute. I would like to acknowledge the Center of Biomedical Research Excellence Microscopy and Mechanical Testing Core (COBRE- MMT) with a grant from the National Institute of General Medical Sciences – NIGMS (1 P30 GM110758-01) at NIH for use of the Malvern Zetasizer dynamic light scattering instrument. iv TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... x LIST OF FIGURES ....................................................................................................... xi ABSTRACT ................................................................................................................. xv Chapter 1 INTRODUCTION .............................................................................................. 1 1.1 Therapeutic Monoclonal Antibody Production ......................................... 1 1.2 Protein Purification with Affinity Precipitation ........................................ 2 1.2.1 Primary Effect Affinity Ligand Crosslinking ................................ 2 1.2.2 Secondary Effect Stimuli-responsive Affinity Ligands ................ 3 1.3 Antibody Affinity Precipitation with Z-Elastin-Like Polypeptides .......... 4 1.4 Z-ELP-Nanoparticle Scaffold for Multivalent Crosslinking ..................... 5 1.5 E2 Nanocage Functionalization using Sortase A Ligation ........................ 6 1.6 Dissertation Objectives .............................................................................. 8 FIGURES ........................................................................................................... 9 REFERENCES ................................................................................................. 15 2 ANTIBODY BINDING INDUCED CROSSLINKING OF Z-DOMAIN- ELASTIN-LIKE-POLYPEPTIDE FUNCTIONALIZED E2 NANOPARTICLES ......................................................................................... 20 2.1 Introduction ............................................................................................. 21 2.2 Materials and Methods ............................................................................ 22 2.2.1 Materials ...................................................................................... 22 2.2.2 Protein Expression and Purification ............................................ 23 2.2.3 Sortase A Mediated Ligation ....................................................... 24 2.2.4 Transition Temperature Measurement ........................................ 25 2.2.5 Dynamic Light Scattering Measurement ..................................... 25 2.2.6 Antibody Affinity Precipitation ................................................... 26 2.2.7 Turbidity Measurement ............................................................... 27 v 2.3 Results and Discussion ............................................................................ 27 2.3.1 Z-ELP functionalization of E2 by sortase A ligation .................. 27 2.3.2 Effect of Z-ELP-E2 ligation density on nanoparticle properties . 28 2.3.3 Enhanced precipitation by antibody-inducible crosslinking ....... 29 2.4 Conclusion ............................................................................................... 32 FIGURES ......................................................................................................... 33 REFERENCES ................................................................................................. 39 3 ONE-STEP AFFINITY CAPTURE AND PRECIPITATION FOR IMPROVED PURIFICATION OF AN INDUSTRIAL MONOCLONAL ANTIBODY ..................................................................................................... 42 3.1 Introduction ............................................................................................. 43 3.2 Materials and Methods ............................................................................ 46 3.2.1 Materials ...................................................................................... 46 3.2.2 Experimental Methods ................................................................. 46 3.2.2.1 Protein Expression and Purification ............................. 46 3.2.2.2 High-throughput Affinity Precipitation Optimization .. 47 3.2.2.3 mAb Cell Culture Affinity Precipitation ...................... 48 3.2.3 Analytical Methods ..................................................................... 49 3.2.3.1 PLSR Model of Two Component Absorption Spectra . 49 3.2.3.2 Analytical Size Exclusion Chromatography ................ 50 3.2.3.3 Analytical Impurity Content ......................................... 50 3.2.3.4 Dynamic Light Scattering Measurement ...................... 50 3.3 Results and Discussion ............................................................................ 51 3.3.1 High-throughput optimization of mAb binding and precipitation ................................................................................. 51 3.3.2 High-throughput optimization of mAb elution and nanocage recovery ....................................................................................... 52 3.3.3 Process stability of the mAb and nanocage ................................. 54 3.3.4 Removal of process impurities .................................................... 55 3.3.5 Optimized affinity precipitation of mAb cell culture harvest...... 56 3.4 Conclusion ............................................................................................... 58 TABLES ........................................................................................................... 60 vi FIGURES ......................................................................................................... 63 REFERENCES ................................................................................................. 75 4 HIGH-EFFICIENCY AFFINITY PRECIPITATION OF INDUSTRIAL MABS AND FC-FUSION PROTEINS FROM CELL CULTURE HARVESTS ..................................................................................................... 78 4.1 Introduction ............................................................................................. 79 4.2 Materials and Methods ............................................................................ 80 4.2.1 Materials ...................................................................................... 80 4.2.2 Z-ELP-E2 Nanocage