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Specific Enzymatic Digestion and Fragment Isolation for the Critical Quality Attribute Assessment of Igg1

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Specific Enzymatic Digestion and Fragment Isolation for the Critical Quality Attribute Assessment of Igg1 ZURICH UNIVERSITY OF APPLIED SCIENCES DEPARTMENT OF LIFE SCIENCES AND FACILITY MANAGEMENT INSTITUTE OF BIOTECHNOLOGY Specific enzymatic digestion and fragment isolation for the critical quality attribute assessment of IgG1 Master thesis by Rappo, Martin Alexander Master of Life Sciences 20.10.2017 Pharmaceutical Biotechnology Academic supervisor: Prof. Dr. Jack Rohrer Head of the Cell Physiology and Cellular Engineering Group ZHAW Life Sciences und Facility Management, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland Industrial supervisor: Dr. Christoph Rösli Senior Fellow in the Physicochemical Analytics Group Novartis Pharma AG, Werk Klybeck, Postfach, 4002 Basel, Switzerland ii Imprint Keywords Critical Quality Attributes, CQA, Monoclonal Antibody, mab, Fab, Fc, F(ab’)2, Large hinge IgG fragment, LHF, IdeS, IgdE, Lysine Gingipain, Kgp, EndoS2 Please cite this report as followed: M. A. Rappo, Specific enzymatic digestion and fragment isolation for the critical quality attribute assessment of IgG1, ZHAW LSFM (2017) Contact information Author Martin Alexander Rappo Speiserstrasse 12 4600 Olten Switzerland Tel. +41 79 537 64 76 E-Mail: [email protected] Academic Supervisor Prof. Dr. Jack Rohrer ZHAW Life Sciences und Facility Management Einsiedlerstrasse 31 8820 Wädenswil Switzerland Tel. +41 58 934 57 17 E-mail: [email protected] Industrial Supervisor Dr. Christoph Rösli Novartis Pharma AG Werk Klybeck Postfach 4002 Basel Switzerland Tel. +41 61 696 14 39 E-Mail: [email protected] Basel 20.10.2017 iii Acknowledgement I would like to express my very great appreciation to Christoph Rösli. I would like to thank him for giving me the chance to work on this interesting project, the stimulating scientific discussions and useful inputs given during the time I spend at Novartis. I am particular grateful for the support and training given by Olivia Guerre. Also the interesting exchange helped to approach problems with a different perspective. I would like to offer my special thanks to Philippe Simeoni, who always offered this time whenever a question or issue regarding CE came up. Also Francois Griaud I would like to thank for giving me an introduction on antibody digestion. A thanks also goes to Wolfram Kern for being so kind to let me use his work bench and all his equipment. The refreshing exchange with Manuel Diez inside and outside the office has been a great help to keep motivated and stay open-minded. I would like to thank Genovis for kindly providing me a free test sample of FabALACTICA™. A special thanks also goes to Raphael Dreier for all the coffee and lunch breaks we had together, the support during stressful times, extended brain-storming sessions and all the laughs we shared. iv Abstract Recent ICH quality guideline updates introduced the enhanced drug development which is based on Quality by Design approach. For this a Quality Risk Management has to be conducted based on scientific knowledge. Information gaps are closed through analysis of molecules with domain-specific modifications. The aim of this thesis was to evaluate methods to prepare a model IgG1 for such studies. The antibody fragments F(ab’)2, LHF, Fab and Fc and the Fc-glycosylation cleaved IgG with residual N-acetylglucosamine were selected. A representative IgG1 was digested with the recently discovered enzymes IdeS, Kgp, IgdE and EndoS2 and the digestion products were purified with common biotechnological down-stream methods. The F(ab’)2 was successfully isolated with a yield of 55% and a purity of 96%. After an initial digestion parameter optimization the LHF, Fab and Fc could be generated and an isolation method was established. A following scale-up was unsuccessful, due to reproducibility issues. A promising alternative was identified. The enzymatic glycan cleavage and product isolation was successful with a yield of 80% and a purity of 99%. Zusammenfassung Die kürzlich von der ICH aktualisierten Qualitätsrichtlinien führten den erweiterten Wirkstoffentwicklungsvorgangs ein, welcher auf dem Quality-by-Design Prinzip basiert. Dazu wird ein Risikomanagement, basierend wissenschaftlichen Fakten durchgeführt. Um Informationslücken zu schliessen müssen proteindomänenspezifisch veränderte Moleküle untersucht werden. Das Ziel dieser Arbeit war die Evaluation von Methoden zur Vorbereitung eines Modell IgG1 für solche Studien mit minimaler unspezifischer Veränderungen. Ausgewählt wurden die IgG-Fragmente F(ab‘)2, LHF, Fab und Fc und das Fc-Polysaccharid gespaltene IgG mit restlichem N-Acetylglucosamin. Ein repräsentatives IgG1 wurde mit den kürzlich entdeckten Enzymen IdeS, Kgp, IgdE und EndoS2 verdaut und mittels üblichen biotechnologisch Reinigungsmethoden aufgereinigt. Das F(ab‘)2 Fragment konnte mit einer Ausbeute von 55% und Reinheit von 96% isoliert werden. Nach einer initialen Optimierung der Verdauungsparameter konnte das LHF, Fab und Fc erfolgreich hergestellt und eine Methode zur Fragmentisolation gefunden werden. Die Herstellung einer grösseren Menge war erfolglos, jedoch konnte eine alternative Lösung identifiziert werden. Die enzymatische Spaltung des Polysaccharids und Isolation war erfolgreich mit einer Ausbeute von 80% und einer Reinheit von 99%. v Table of content 1 INTRODUCTION ............................................................................................................ 1 2 THEORETICAL BACKGROUND.................................................................................... 5 2.1 ANTIBODIES ............................................................................................................... 5 2.1.1 Structure ............................................................................................................ 5 2.1.2 Fragment nomenclature ..................................................................................... 7 2.1.3 Glycosylation ...................................................................................................... 8 2.1.4 Mechanism of action .......................................................................................... 8 2.2 ENZYMES ................................................................................................................... 8 2.2.1 Immunoglobulin-degrading enzyme of Streptococcus pyogenes ........................ 9 2.2.2 Lysine gingipain of Porphyromonas gingivalis .................................................. 11 2.2.3 Immunoglobulin G degrading enzyme of Streptococcus agalactiae .................. 13 2.2.4 Endo-beta-N-acetylglucosaminidase of Streptococcus pyogenes .................... 13 2.3 KINETICS MODEL FOR THE KGP DIGESTION ................................................................. 15 3 METHODS AND MATERIALS ...................................................................................... 17 3.1 INSTRUMENTS, CONSUMABLES, REAGENTS AND SOFTWARE. ....................................... 17 3.2 MODEL ANTIBODY ..................................................................................................... 17 3.3 BUFFER PREPARATION .............................................................................................. 17 3.4 PHYSICOCHEMICAL METHODS ................................................................................... 18 3.4.1 Cation exchange chromatography .................................................................... 18 3.4.2 Size exclusion chromatography ........................................................................ 19 3.4.3 Capillary electrophoresis – sodium dodecyl sulfate .......................................... 20 3.5 DIGESTION AND ISOLATION ........................................................................................ 22 3.5.1 Fab, Fc and LHF .............................................................................................. 22 3.5.2 F(ab’)2 .............................................................................................................. 27 3.5.3 Chitobiose cleavage ......................................................................................... 29 3.6 ECOLOGY, SAFETY AND DISPOSAL ............................................................................. 30 4 RESULTS AND DISCUSSION ..................................................................................... 31 4.1.1 Fab, Fc and LHF .............................................................................................. 31 4.1.2 F(ab’)2 .............................................................................................................. 44 4.1.3 Chitobiose cleavage ......................................................................................... 46 5 CONCLUSION .............................................................................................................. 48 6 OUTLOOK .................................................................................................................... 49 7 REFERENCES ............................................................................................................. 50 7.1 LIST OF ILLUSTRATIONS ............................................................................................ 55 7.2 LIST OF TABLES ........................................................................................................ 56 7.3 LIST OF EQUATIONS .................................................................................................. 57 ANNEX .................................................................................................................................. I vi Abbreviations
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