FACULTY OF PHARMACEUTICAL SCIENCES DRUG Quality & Registration (D RU QUA R) Lab TOWARDS A PEPTIDE IMPLANT : ANALYTICS , DRY HEAT BEHAVIOR AND FUNCTIONAL CHARACTERIZATION Thesis submitted to obtain the degree of Doctor in Pharmaceutical Sciences Matthias D’Hondt Promotor Prof. Dr. Bart DE SPIEGELEER FACULTY OF PHARMACEUTICAL SCIENCES Drug Quality & Registration (DruQuaR) Lab TOWARDS A PEPTIDE IMPLANT: ANALYTICS, DRY HEAT BEHAVIOR AND FUNCTIONAL CHARACTERIZATION Matthias D’HONDT Master of Science in Drug Development Promoter Prof. Dr. Apr. Bart DE SPIEGELEER 2015 Thesis submitted to obtain the degree of Doctor in Pharmaceutical Sciences COPYRIGHT COPYRIGHT The author and the promotor give the authorisation to consult and to copy parts of this thesis for personal use only. Any other use is limited by the Laws of Copyright, especially the obligation to refer to the source whenever results from this thesis are cited. Ghent, 16 January 2015 The promoto r The author Prof. Dr. Bart De Spiegeleer Matthias D’Hondt . 3 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Writing this doctoral thesis would have been impossible without the help and support of many people. First of all, I would like to thank my promoter, prof. Dr. Bart De Spiegeleer , for passing along or rather infecting me with his interests in peptide medicines and analytical technology. From our first meeting, his enthusiastic, stimulating and science-driven state-of-mind was immediately clear. Our countless discussions throughout the past few years have thought me invaluable lessons in numerous pharmaceutical (and other) aspects and were crucial for this work. It was a real honour completing this thesis under his guidance and hope that we may continue working together in the future, one way or the other. I also like to express my sincere gratitude to the members of the reading and examination committee (i.e. prof. Dr. Jean Paul Remon as chairman, prof. Dr. Deirdre Cabooter, prof. Dr. Catherine Delesalle, prof. Dr. Ralf Hoffmann, prof. Dr. Chris Vervaet, prof. Dr. Bruno De Geest and prof. Dr. Marleen Temmerman) for taking the time to read this work with a critical view; the fruitful, dynamic and open-minded discussions revealed aspects which were suited for improvement and truly pushed the quality of this thesis to a higher level. I would also like to acknowledge the Ghent University and IWT (Instituut voor Innovatie door Wetenschap en Technologie Vlaanderen; mandate 101529), who provided me with the necessary funding. Without their financial help, it would have been impossible for me to pursue and successfully complete this Ph.D. project. Next, I am grateful to my former DruQuaR colleagues Dr. Valentijn Vergote and Dr. Sylvia Van Dorpe, for familiarizing me with peptide mass spectrometry. Dr. Jente Boonen, Elien Vangheluwe and the previous lab technicians for guiding me during my first few months, teaching me the ropes of chromatography, and Dr. Bram Baert for introducing me into the quality assurance aspect of the DruQuaR lab. My gratitude also goes out to my present colle agues for their support during experiments and writing, scientific discussions, fun intermezzo’s and the general DruQuaR atmosphere: Dr. Evelien Wynendaele, Mathieu Verbeken, Nathalie Bracke, Sultan Suleman, Sofie Stalmans, Kirsten Vandercruyssen, Lieselotte Veryser, Lien Taevernier, Bert Gevaert, Frederick Verbeke, Han Hao, Xiaolong Xu and Marianne Lauwers. 5 ACKNOWLEDGEMENTS Furthermore, I would like to thank the people I worked with during our collaborations. I’m grateful to Bart De Spiegeleer Jr. for his help in the relative response factor calculations, Dr. Herwig Jansen (Dafra Pharma International), Dr. Ilse Koijen and Dr. Jacques van Gompel (both Janssen Pharmaceutica N.V) for providing necessary drug substances and their help in the AMES test experiments. I’m also indebted to Dr. Maria Fedorova (University of Leipzig, Faculty of Chemistry) for her help in identifying the peptide degradants and prof. Dr. Chien-Yu Peng (Academia Sinica, Institute of Statistical Science) for his contribution to the kinetic degradation models. I’m very thankful to Pieter Wuytens and prof. Dr. Andre Skirtach (UGhent, Department of Molecular Biotechnology) for the confocal Raman images of the implant and Dr. Steven Mullens (Flemish Institute for Technological Research (VITO)) for the R a determination of the mortar surfaces. I wish to thank my friends for providing timely distractions and sometimes showing me there’s more to life than work, be it at my tennis club or whilst attending a football game. Last but foremost, I wish to thank my family , especially my parents and sister, for their encouragement, help and support during this long “school” career and their understanding nature. Matthias 6 LIST OF ABBREVIATIONS AND SYMBOLS LIST OF ABBREVIATIONS AND SYMBOLS 2-MBT 2-mercaptobenzothiazole 2D-COSY 2D-correlated spectroscopy 2D-TOCSY 2D-total correlation spectroscopy A Frequency factor AA Amino acid Acm Acetamidomethyl ACN Acetonitrile ACTH Adrenocorticotropic hormone API Active Pharmaceutical Ingredient As Asymmetry factor BEH Bridged ethylene hybrid Bpa p-benzoylphenylalanine BSE Bovine spongiform encephalopathy BZL Benzyl CAGR Compound annual growth rate CAD Charged aerosol detection CCR5 C-C chemokine receptor type 5 CD Circular dichroism CD4 Cluster of differentiation 4 CE Capillary electrophoresis CLND Chemiluminescent nitrogen detection CPP Cell-penetrating peptide CRF Chromatographic response factor CSH Charged surface hybrid CXCR4 C-X-C chemokine receptor type 4 DIC Diisopropylcarbodiimide DMSO Dimethylsulfoxide Ea Activation energy EDQM European Directorate for the Quality of Medicines and HealthCare EDT Ethanedithiol ELSD Evaporative light scattering detection EMA European Medicines Agency 7 LIST OF ABBREVIATIONS AND SYMBOLS ENN Enniatin ESI Electrospray ionization EU European Union FA Formic acid FDA Food and Drug Administration FDP Finished drug product Fmoc Fluorenylmethyloxycarbonyl Fmoc-OSu Fluorenylmethoxycarbonyloxy succinimide FSH Follicle-stimulating hormone FT-ICT Fourier Transform Ion Cyclotron Resonance FTIR Fourier transform infrared FTN Flow through needle GMP Good manufacturing practice GnRH Gonadotropin-releasing hormone GP Glycoprotein HBTU O-Benzotriazole-N,N,N’,N’-tetramethyluroniumhexafluorophosphate HCA Hierarchical Cluster Analysis HCMV Human cytomegalovirus HETP Height Equivalent to a Theoretical Plate HF Hydrofluoric acid HIV Human immunodeficiency virus HLB Hydrophilic-Lipophilic balance HME Hot-melt extrusion HPLC High Performance Liquid Chromatography HR Heptad repeat IBS Irritable bowel syndrome ICH International conference on harmonization ID Internal diameter IEC Ion-exchange chromatography IRDS Infant respiratory distress syndrome IS Internal standard IUPAC International Union of Pure and Applied Chemistry KPL Kinetic performance limit LC Liquid chromatography LH Luteinizing hormone 8 LIST OF ABBREVIATIONS AND SYMBOLS LoD Limit of detection LogD Distribution coefficient LoQ Limit of quantification MeOH Methanol MRM Multiple reaction monitoring MS Mass spectrometry MW Molecular weight NMR Nuclear magnetic resonance NOD2 Nucleotide-binding oligomerization domain-containing 2 Np Peak capacity OBt Benzotriazole ester ORD Optical rotation dispersion PC Peak capacity PC Principal Component PCA Principal Component Analysis PDA Photodiode array PEG Polyethylene glycol pEVA Poly(ethylene-vinylacetate) PG Protecting group Ph. Eur. European Pharmacopoeia Phg Phenylglycine pI Isoelectric point QC Quality control QbD Quality by design QSPR Quantitative structure-property relationship R&D Research and development RID Refractive index detector RMS Root mean square ROS Reactive oxygen species RP Reversed-phase RRT Relative retention times Rs corr Time-corrected resolution product S Separation factor S/N Signal to noise SBS Short bowel syndrome 9 LIST OF ABBREVIATIONS AND SYMBOLS SCX Strong cation exchange SEC Size-exclusion chromatography SEM Standard error on mean SER Surface-Enhanced Raman SFC Supercritical fluid chromatography SIM Selected ion monitoring SM Starting material SMILES Simplified Molecular Input Line Entry Specification SPC Summary of product characteristics SPE Solid-phase extraction SPP Superficially porous particle SPPS Solid-Phase Peptide Synthesis SST System suitability test p/v Peak to valley ratio PAT Process analytical technology PGA Polyglycolic acid PLA Polylactic acid PLGA Poly(lactide-co-glycolide) tBoc Tert-butyloxycarbonyl tBu Tertiary butyl TCS Tetracosactide TFA Trifluoroacetic acid TIC Total ion chromatogram TIS Triisopropylsilane TLC Thin-layer chromatography TSE Transmissible spongiform encephalopathy TQ-S Triple quadrupole mass spectrometer UPLC Ultra Performance Liquid Chromatography USA United States of America USP United States Pharmacopeia UV Ultraviolet VCD Vibrational circular dichroism WHO World Health Organisation WCX Weak cation exchange 10 TABLE OF CONTENTS TABLE OF CONTENTS Chapter I: Introduction 15 1. Peptides 17 2. Peptide stability 25 3. Peptide administration 26 4. Peptides with extended release potential 26 5. Peptide processing 29 6. Study objectives 30 7. Thesis outline 32 8. Referenc es 35 Chapter II: LC column selection: Derringer desirability and kinetic plots 39 1. Introduction 41 2. Materials and methods 43 3. Results and discussion 47 4. Conclusions 56 5. References 57 Chapter III: QC analysis of peptide APIs 59 1. Introduction 61 2. Materials and methods 62 3. Results 66 4. Discussion 72 5. Conclusions 75 6. References 77 Chapter IV: Peak