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Investigation of Lyophilized Antibody Formulations to Enable Short Freeze-Drying Cycles and Storage at Room Temperature

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Investigation of Lyophilized Antibody Formulations to Enable Short Freeze-Drying Cycles and Storage at Room Temperature Investigation of lyophilized antibody formulations to enable short freeze-drying cycles and storage at room temperature Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Christina Häuser aus Deutschland Basel, 2020 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von: Erstbetreuer: Dr. P. Goldbach Zusätzliche Erstbetreuerin: Dr. A. Allmendinger Zweitbetreuer: Prof. J. Huwyler Externer Experte: Prof. G. Winter Basel, den 19. November 2019 ................................................................... Prof. Dr. Martin Spiess Dekan FÜR MEINE ELTERN f The important thing is not to stop questioning. ~Albert Einstein~ ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS First, I would like to generally thank all my supervisors for the excellent support I enjoyed throughout this PhD project. Foremost, I would like thank my doctoral supervisor Prof. Dr. Jörg Huwyler for giving me the opportunity to write my dissertation within his research group. Thank you for your enthusiasm for my project and for always having an open door. Additional thanks go to the entire Pharmaceutical Technology group of the University of Basel for creating a warm and welcoming atmosphere and the fun times during our sledging trip and sailing course and particularly Dr. Maxim Puchkov, Dr. Klara Kiene, Dr. Dominik Witzigmann, and Viktoria Schreiner, for the time we were having together during the supervision of the practical courses. Maxim thank you also for introducing me into the image processing features of ImageJ. Dr. Leonie Wagner, thank you for the energizing lunch breaks we are having together. I would like to particularly express my deepest gratitude to both my Roche supervisors, Dr. Andrea Allmendinger and Dr. Pierre Goldbach. Thank you for your scientific guidance, for teaching me to view a problem in its entirety, and to critically question my data. Through your continuous encouragement and support throughout the past years I grew from a professional point of view but also personally and feel affirmed in having chosen the right path for my professional life. Special thanks go to Prof. Dr. Wolfgang Friess for supporting my PhD project. I highly appreciated the encouraging discussions and valuable scientific advice. Thank you for also integrating me into the Pharmaceutical Technology group of the LMU Munich to extend my network and gather even broader scientific input. I am grateful for giving me the opportunity to perform some last freeze-drying experiments in the laboratories of the LMU. Many thanks also to Ivonne Seifert in this context, for your great ad-hoc organizational support during my visit. I would like to acknowledge Prof. Dr. Gerhard Winter for taking over the co-referee. Thank you for taking the time to read and evaluate my dissertation. I am grateful to Dr. Michael Adler and Dr. Silke Mohl who made it possible that I could conduct my PhD research project within Late-Stage Pharmaceutical and Processing Development. In addition, I would like to thank Carmen Lema Martinez und Dr. Jörg Lümkemann for giving me the opportunity to continue my further professional career within the department of Pharmaceutical Development and Supplies. I would like to thank the entire Late-Stage Pharmaceutical Development and Processing group for the warm and supportive atmosphere. Thank you Jasmin John, Severine Ughetti, Monika Gisin, and especially Sonja Omlin for your help with the analysis of stability samples and preparation of formulations for freeze-drying. Martin Worgull, Thomas Steffen, and particularly my Roche PhD fellow Daniel Kullmann – many thanks for your hands-on support with technical issues of the freeze-dryers and troubleshooting. Additional thanks go to my Roche PhD fellow Tim Dreckmann and my lab colleagues from the research lab, Dr. Christoph Grapentin, Inas El-Bialy, and particularly Dr. Monica de Bardi, Dr. Dominique Ditter, and Dr. Hannah Kientz. Thank you for the pleasant working atmosphere, the scientific exchange, and the several coffee and lunch breaks. Especially Hannah, we were a great team during the last year and you really grew to my heart. i ACKNOWLEDGEMENTS Furthermore, I would like to thank my colleague Dr. Michael Göllner from solid state analytics for the collaboration and support with micro-computed tomography measurements and the helpful scientific discussions on data processing. Additionally, Joerg Voelkle and Thomas Zumstein from the Robotics group within Roche are acknowledged for their help with designing and manufacturing the vial cutting device and the sealable plastic cups and Hendrik Rother from Roche Diagnostics Mannheim for the 3D laser scanning during the lyophilisate imaging studies. Moreover, I would like to thank Anna-Katharina Heidenreich from analytics in Roche Penzberg and particularly Anja Bathke from analytics in Basel for the mass spectrometry measurements of dextran-based stability samples. Anja, thank you for the many intense discussions on interpretation of the data. Above all, I would like to thank my parents, Gabi and Heinz. Without them I would not be where I am today. Thank you for your continuous support and for always believing in me. Thank you also to my brother Stefan. It is great knowing that I can always count on you. Last but not least, my greatest thanks go to Johannes. I am exceedingly thankful for your endless support and your patience. Thank you for always motivating me, for the relaxing moments together, and additionally for proofreading my thesis. I am really happy to have you by my side. ii SUMMARY SUMMARY Antibodies are dominating the biopharmaceutical market and are expected to grow further. Aiming to improve existing treatments, new antibody derivatives of improved efficacy and safety are being developed in a competitive market. Antibody derivatives include but are not limited to antibody-drug conjugates and Fc-fusion proteins. Due to their higher complexity, they are often less stable as liquids, increasing the demand for lyophilized formulations to ensure protein storage stability over the desired shelf life. In many cases, these antibody formats require lower doses, posing challenges to formulation and freeze-drying process development. Commercialized lyophilized antibodies typically contain disaccharides, most frequently sucrose, as a stabilizer and bulking agent. The low glass transition temperature of sucrose requires time- and energy-intensive, thus expensive freeze-drying cycles. At lower protein concentrations, this becomes even more relevant, raising the risk of product collapse during freeze-drying. Collapse occurs when primary drying is performed above the glass transition temperature or collapse temperature of the formulation. It is current dogma to design freeze-drying cycles that provide pharmaceutically elegant lyophilisates as collapse leads to batch inhomogeneity causing rejects, higher complaint rates, and most importantly may potentially be detrimental to protein storage stability. Thus, there is a need to look into alternative excipients for future freeze-dried antibody formulations. The presented work investigated amorphous excipients to be used as alternative excipients to sucrose for freeze-dried antibody formulations, increasing the formulation’s glass transition temperature. The main objectives were to investigate their ability to render pharmaceutically elegant lyophilisates upon short freeze-drying cycles, and to stabilize antibodies during freeze-drying and subsequent storage. Special focus was given to storage stability at elevated temperatures with the aim to study the potential for room temperature stable formulations. At first, an imaging technique was established to evaluate the impact of excipients and freeze-drying cycles on cake appearance and structure. Different imaging techniques were compared regarding qualitative and quantitative characterization of the entire lyophilisate, and their potential for non- invasive evaluation of structure and morphology in the glass vial (Chapter 1). The comparative analysis revealed limitations of scanning electron microscopy, the current state of the art technique to characterize cake morphology. Micro-computed tomography was introduced as a technique allowing for comprehensive and reproducible imaging of cake structure and morphology. Having established a method for evaluation of cake appearance, the next step of this work was focused on formulation development. Dextrans of different molecular weight from 1 to 500 kDa (Chapter 2) followed by HPBCD-based formulations in combination with other amorphous compounds (Chapter 3) were investigated. Their impact on thermal properties, cake appearance, other physico-chemical product quality attributes, and protein stability of two model antibodies was characterized. In particular, HPBCD was found to be a promising excipient, while dextran showed several limitations. Large dextrans of 40 kDa or higher were shown to increase the viscosity of the formulations leading to long reconstitution times, and did not sufficiently stabilize the antibodies during freeze-drying compared to smaller dextrans and HPBCD. The work highlighted limitations of dextrans with regards to protein stability, due to antibody glycation during storage at elevated temperatures. HPBCD iii SUMMARY rendered lyophilisates with good product quality attributes and ensured antibody
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