Thesis by Publication Development of Aggregation- Resistant Monoclonal Antibodies Through Antibody Engineering and Formulation Approaches Mouhamad Reslan This thesis is submitted in full satisfaction of the requirements for the degree of Doctor of Philosophy at the University of Sydney. School of Pharmacy Faculty of Medicine and Health 2018 i Statement of Authentication This thesis is submitted to the University of Sydney in fulfilment of the requirement for the degree of Doctor of Philosophy. The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. Signature: Date: 13th of August 2018 ii Acknowledgements I start by acknowledging the traditional owners of this land - the Gadigal people of the Eora nation - on which I have spent the last three to four years conducting research and developing new friendships. To my supervisor, Associate Professor Veysel Kayser, I thank you for your patience, support and guidance during this time. I thank you for embarking on this journey with me, while giving me the freedom to decide my own path. To my co-supervisor Associate Professor Serdar Kuyucak and the Biophysics Team, thank you for the time we worked together, the words of support as we hit roadblocks along the way, and the celebrations as we broke through and graduated. To my colleagues, Vicki, Esteban, Yusuf and the many students I met on the way, I could not have asked for a better network of friends to work alongside and to enjoy this experience with. Thank you for all the laughter and fun we had together (and all those games of ping pong!). To the amazing people I met along the way including Meryem, Zehra and David – thank you for being mentors, your time and effort has been invaluable to myself and others and has contributed significantly to the success of the work within this thesis. Thank you to everyone in the office, and the wonderful staff and academics at the School of Pharmacy, for your continued support and words of encouragement. Thank you to Sheng and Donna for your time training me and assisting with my research at MBF. Thank you to Mario, for sharing your expertise with us when we first began and supporting us throughout. Finally, thank you to my family for your constant support during the challenges and moments of joy; I can now close this chapter of my life, having gained new insights, experiences, and ideas. I look forward to writing the next chapter. iii Authorship attribution statement Chapter 1 of this thesis (with omitted sections) is published as: Elgundi, Z., Reslan, M., Cruz, E., Sifniotis, V., Kayser, V. (2017). The state-of-play and future of antibody therapeutics. Advanced Drug Delivery Reviews, 122:2-19. M. Reslan wrote several sections of the review including: parts of the section ‘Biobetters’, all ‘Physical and chemical degradation of antibodies’ including ‘Computational design tools’, made minor edits to ‘optimisation of antibody bioavailability and delivery’ and contributed to writing the abstract, introduction, and conclusion. He also critically reviewed and edited the entire manuscript to prepare it for submission. In the thesis introduction, he updated the information in each included section with current literature. Chapter 2 of this thesis is published as: Reslan, M., Demir, Y., Trout, B., Chan, H., Kayser, V. (2017). Lack of a synergistic effect of arginine-glutamic acid on the physical stability of spray-dried bovine serum albumin. Pharmaceutical Development and Technology, 22:785-791. M. Reslan co-designed the study, prepared the formulations and performed most of the formulation characterisation. He analysed most of the data and wrote the manuscript. Chapter 3 of this thesis is published as: Reslan, M., Kayser, V. (2016). The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin. Pharmaceutical Development and Technology, 1-7. M. Reslan co-designed the study, performed all experiments and data analysis, and wrote the manuscript. Chapter 4 of this thesis is published as: Reslan, M., Kayser, V. (2018). Ionic liquids as biocompatible stabilizers of proteins. Biophysical Reviews. doi:10.1007/s12551-018-0407-6 M. Reslan performed the literature searches and co-wrote the review. iv Chapter 5 of this thesis is prepared for publication as: Reslan, M., Kayser, V. (2018). Analysis of the aggregation kinetics of Herceptin® (trastuzumab). European Journal of Pharmaceutics and Biopharmaceutics. M. Reslan co-designed the study, performed all experiments and data analysis, and wrote the manuscript. Chapter 6 of this thesis published as: Reslan, M., Vijayaraghavan R., Macfarlane D. R., Kayser, V. (2018). Choline ionic liquid enhances the stability of Herceptin® (trastuzumab). Chemical Communications, 54:10622-10625. M. Reslan co-designed the study, prepared the formulations, performed all stability characterisation experiments and data analysis, and wrote the manuscript. Chapter 7 of this thesis is published as: Elgundi, Z., Sifniotis, V., Reslan, M., Cruz, E., Kayser, V. (2017). Laboratory Scale Production and Purification of a Therapeutic Antibody. Journal of Visualized Experiments, 119: e55153. M. Reslan assisted with the writing of the manuscript. He designed and wrote all sections relating to the purification and characterisation of the antibody following expression, performed those experiments and prepared the figures. Chapter 8 of this thesis is prepared for publication as: Reslan, M., Sifniotis, V., Cruz, E., Sumer-Bayraktar, Z., Kayser, V. (2018). Enhancing the stability of adalimumab by engineering additional glycosylation motifs. European Journal of Pharmaceutics and Biopharmaceutics. M. Reslan co-designed the experiments and conducted most of the experimental work including mutagenesis, sequencing analysis, expression of the antibody variants, purification and characterisation. He also wrote most sections of the manuscript and compiled it together for publication. In addition to the statements above, in cases where I am not the corresponding author of a published item, permission to include the published material has been granted by the corresponding author. v Mouhamad Reslan, Signature: 13th of August 2018 As supervisor for the candidature upon which this thesis is based, and corresponding author for the publications, I can confirm that the authorship attribution statements above are correct. Veysel Kayser, Signature: 13th of August 2018 vi Table of Contents Statement of authentication .................................................................................... ii Acknowledgements ................................................................................................. iii Authorship attribution statement ........................................................................... iv Thesis abstract ........................................................................................................ xi Thesis introduction ................................................................................................ xii Conferences and publications .............................................................................. xv Chapter 1: The state-of-play and future of antibody therapeutics ...................... 1 Graphical abstract ................................................................................................... 1 Abstract ................................................................................................................... 2 1. Introduction ........................................................................................................ 2 2. Antibody discovery strategies ............................................................................ 2 3. Manufacture of antibodies ................................................................................. 4 4. Biobetter antibodies ........................................................................................... 6 4.1. Fc engineered antibodies with enhanced effector function ......................... 12 5. Physical and chemical degradation of antibodies ............................................ 13 5.1. Aggregation ................................................................................................ 14 5.2. Denaturation ............................................................................................... 14 5.3. Fragmentation ............................................................................................ 15 5.4. Deamidation ............................................................................................... 15 5.5. Oxidation .................................................................................................... 16 6. Computational design tools ............................................................................. 17 7. Optimisation of antibody bioavailability and delivery ........................................ 19 8. Conclusion ....................................................................................................... 21 Acknowledgements ............................................................................................... 22 Disclosures ........................................................................................................... 22 References ...........................................................................................................