Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Sugar-Based Surfactants for Pharmaceutical Protein Formulations Lars Kurt Johann Schiefelbein aus Stade 2011 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von Herrn Prof. Dr. Wolfgang Frieß betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, den 11. Mai 2011 ............................................................................. (Lars Schiefelbein) Dissertation eingereicht am 18.05.2011 1. Gutacher Prof. Dr. Wolfgang Frieß 2. Gutachter Prof. Dr. Gerhard Winter Mündliche Prüfung am 10.06.2011 Acknowledgments The presented thesis was written at the Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics at the Ludwig-Maximilians-University in Munich under supervision of Prof. Dr. Wolfgang Frieß. First of all I would like to take the chance and thank my supervisor Prof. Dr. Wolfgang Frieß for giving me the opportunity to work in his research group. I always felt greatly supported by you, even when the experimental results were demanding. On a personal and on a professional level interaction with you was always a pleasure. Thank you for the possibilities you opened me to increase my scientific knowledge even with research stays and congress participations abroad. I would also like to thank Prof. Dr. Gerhard Winter for being co-referee of this thesis. Furthermore, I had a wonderful time at your chair, with a lot of interesting discussions, social events and enjoyed the working conditions. I deeply appreciate Prof. Dr. Peter Westh at Roskilde University for letting me work in his lab and giving me numerous tips and help with my calorimetric experiments. Mange Tak! Also thanks to my colleagues at RUC, especially Christina, Sophus, Søren, Nicolaj and Peter for making the time in Denmark so much fun. I would like to thank the Dr. August und Dr. Anni Lesmüller-Stiftung for supporting my research stay at the Roskilde-University. During my time at Prof. Frieß group I had a lot of interactions with different researchers. I would like to thank PD Dr. Armin Giese and Prof. Dr. Joachim Rädler for the help with the FCS experiments, Dr. Lars Allmendinger and Dr. Holger Lerche for giving analytical advice and performing MS and NMR experiments for me. Dr. Bernhard Kämpf, Helmut Hartl and Dr. David Stevenson were so kind to perform analytical experiments for me. Of course Dr. Marco Keller and Markus Gruber did a great job of synthesizing the trehalose esters. Also my dear colleague at Merck Dirk Schiroky gave very helpful input into MS interpretation. Some students were helping me with experiments that are mentioned here. From all of them I would like to thank Benjamin Waldmann and Marcel Lietz who were the first students and who I kept contact over the time. Also Monika Alter and Tobias Fister who convinced me to take one more group of students should be mentioned as they generated data that explained a lot. I asked friends to proof-read one part or the other of this work. Dr. Meike Römer, Dr. Katrin Kinkel, Dr. Cornelius Pompe, Torge Schiefelbein, Dr. Martin Meyer, I really thank you for the quick responses and the scientific input I got. I would like to thank my colleagues at pharmaceutical technology for the fun time, deep discussions and nice cooperation. We had a blast with all the cakes, the chocolate almonds and burning the calories again at our weekly after-work jog or at triathlons. A special thanks goes out to Kerstin Höger and Dr. Frank Schaubhut and of course his wife Susan Schaubhut who have been not only lab mates, but also became very close friends. During my time I found and rediscovered some very close friends. Susanne, Julia, Martin, and Christof have been around me, supported me and cheered me up, when Munich was too much for me again. During writing my thesis in Darmstadt also Veronika and Katrin were the best friends I could possibly imagine. I look forward to the day you swim faster than me in a triathlon. Most important, I would like to thank my family. Without you I wouldn‟t be who I am now. Thanks for all you made possible for me, for your encouragement and your support. For my parents Table of contents: Chapter 1: Introduction 1.1. Formulation Challenges with Biologicals ............................................................. 1 1.1.1. General Considerations ................................................................................ 1 1.1.2. Physical Instability ......................................................................................... 3 1.1.3. Chemical Instability: ...................................................................................... 4 1.2. Stabilizing Additives in Pharmaceutical Protein Formulation ............................... 6 1.2.1. Other Excipients than Surfactants ................................................................. 7 1.2.2. Surface Active Agents (Surfactants) in Parenteral Formulations of Protein Pharmaceuticals:..................................................................................................... 9 1.2.2.1. Physicochemical Aspects of Surfactants in Protein Formulations: ............. 9 1.2.2.2. Surface competition ................................................................................. 10 1.2.2.3. Molecular interactions between proteins and surfactants .......................... 11 1.2.2.4. Surfactants in pharmaceutical products ................................................... 12 1.2.2.5. Problems with Surfactants in Protein Formulation .................................... 13 1.2.2.6. Approaches to Understand Interaction of Proteins and Surfactants ......... 14 1.2.2.6.1. Isothermal Titration Calorimetry ............................................................ 15 1.2.2.6.2. Two-dimensional NMR spectroscopy .................................................... 17 1.2.2.6.3. Fluorescence Correlation Spectroscopy ............................................... 17 1.2.2.6.4. Electron Paramagnetic Resonance Spectroscopy ................................ 20 1.2.2.6.5. Viscometry ............................................................................................ 20 1.2.2.6.6. Small angle X-ray scattering ................................................................. 21 1.2.2.6.7. Surface tension measurements ............................................................. 21 1.2.2.6.8. Atomic force microscopy ....................................................................... 22 1.2.2.6.9. Equilibrium Dialysis ............................................................................... 23 1.2.2.6.10. Flourescence Spectroscopy ................................................................ 23 1.2.2.6.11. Other ................................................................................................... 24 1.3. Objective of this thesis ....................................................................................... 24 1.4. References: ....................................................................................................... 26 Chapter 2: Physicochemical characterization of technical mixtures of sugar based surfactants. 2.1. Introduction ........................................................................................................ 38 2.2. Materials and Methods ...................................................................................... 44 2.2.1 Materials ...................................................................................................... 44 2.2.2 Methods ........................................................................................................... 44 2.2.2.1 Solubility testing ........................................................................................ 44 2.2.2.2 Tensiometry ............................................................................................... 45 2.2.2.3 Pyrene Interaction Fluorescence ............................................................... 46 2.2.2.4 Hemolytic Activity ...................................................................................... 47 2.2.2.5 Photon Correlation Spectroscopy (PCS) ................................................... 48 2.2.2.6 Viscometry ................................................................................................ 48 2.2.2.7 Elemental Analysis .................................................................................... 48 2.2.2.8 Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) 49 2.2.2.9 Content of Peroxides ................................................................................. 49 2.2.2.10 Mass spectrometry .................................................................................. 50 2.3. Results and Discussion ..................................................................................... 50 2.3.1 Poorly Soluble Surfactants ........................................................................... 53 2.3.1.1. Poorly Soluble Sugar Esters .................................................................... 53 2.3.1.2. Hardly Soluble Alkyl Glucosides ............................................................... 57 2.3.2. Soluble Surfactants ..................................................................................... 59 2.3.2.1 Water Soluble