Studies on stable formulations for a hydrophobic cytokine [Elektronische Ressource] / Andrea Hawe

Andrea Hawe

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München




Studies on Stable Formulations
for a Hydrophobic Cytokine







Andrea Hawe
aus München


Juni 2006

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Wolfgang Frieß betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.


München, am 26. Juni 2006




(Andrea Hawe)








Dissertation eingereicht am 26. Juni 2006
1. Gutachter: Prof. Dr. Wolfgang Frieß
2. Gutachter: Prof. Dr. Gerhard Winter
Mündliche Prüfung am 21. Juli 2006


Acknowledgements


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 want to express my gratitude to my supervisor Prof. Dr. Wolfgang Frieß for
the possibility to join his research group and especially for his professional and
enthusiastic guidance of my work, as well as all the scientific input and advice he gave
me. Furthermore, I very much appreciate that he offered me great opportunities to
present the work at numerous congresses all over the world. Thank you for the pleasant
working climate that made the development of this thesis not just possible, but a fulfilling
and exciting time.

I want to thank Prof. Dr. Gerhard Winter for his dedicated leadership of the chair, his
commitment to enable us these outstanding working conditions and numerous social
activities, like skiing in winter, barbecue in summer and many more. Thank you a lot for
the scientific and personal advice and taking over the co-referee.

Many thanks to all colleagues from research group Prof. Frieß and from research group
Prof. Winter likewise who shared the time here in Munich with me, for support and
numerous activities in daylight and night time. Special thanks to my direct lab-colleagues
Matthias Ganz and Stefanie Schüle for the overall nice time we had together in our lab.
Thanks also to my indirect lab neighbor Sandra Herrmann for the daily doorway chats.
For all the support by taking over practical work, helping in the student lab and by
literature supply, I want to thank our technical assistants Ingrid Hiltman, Imke Leitner
and Patricia Settele.

I am indebted to Boehringer Ingelheim in Biberach for the general support of the work
with material and the possibility to use their lab equipment. Special thanks to
Dr. Karoline Bechtold-Peters who managed the delicate organization of the material and
to Franz Nothelfer, who advised us in preparative chromatography.

I want to acknowledge the companies Schott AG for providing divers glass vials and
Becton Dickinson GmbH for providing pre-filled syringes for my work.

Many thanks to Prof. Dr. Geoffrey Lee at the Department of Pharmaceutical Technology
at the Friedrich-Alexander University of Erlangen-Nuernberg for the possibility to use the
modulating DSC, Prof. Dr. Udo Bakowsky from the Department of Pharmaceutical
Technology and Biopharmaceutics at the Philipps University in Marburg for performing
the AFM measurements and Dr. Stefan Wittmer form LOT Oriel in Darmstadt for
performing the disc centrifugation experiments.

From the Department of Chemistry and Pharmacy of the LMU in Munich I want to thank
Dr. Svetlana Mintova and Dr. Norbert Stock for taking the SEM pictures. Further,
Wolfgang Wünschheim for his help with the powder diffractometer and Dr. Sascha Correll
for analyzing my samples with the Low Temperature X-ray Diffractometer.

The student assistants Frank Schaubhut, Katja Schmid and Sarah Mikisch are
acknowledged for the good job they have done. It was a pleasure to work with you.

Outside the university I want to thank my flat mates Andrea, Kerstin, Johanna and Seval
for their ongoing friendship, Julia for the great time we had during studying and for all
the advice. Katrin, some special thanks for your friendship over the last years.

My parents, my sister Martina, my brothers Simon and Benno and my grandmother, I
want to thank for all the encouragement and support they gave me in all the years.

Finally, I want to thank Michael for being a great colleague here at the university and for
all your help and for my work, especially the proof-reading of the thesis. Most important I
want to thank you for your love.




























For my parents


Table of Contents

Chapter 1
Introduction and Objectives of the Thesis
1. INTRODUCTION...........................................................................................2
2. FORMULATION OF HYDROPHOBIC PROTEINS ...................................................3
2.1 Hydrophobicity of Proteins ...........................................................................3
2.2 Hydrophobic Proteins used as Pharmaceuticals................................................4
2.3 Solubility of Hydrophobic Proteins .................................................................4
2.4 Protein Adsorption ......................................................................................6
3. HUMAN SERUM ALBUMIN AS STABILIZER FOR PROTEINS ..................................9
3.1 HSA as Excipient in Protein Formulation....................................................... 11
3.2 Development of HSA-free formulations ........................................................ 13
3.2.1 Lyo- and Cryoprotection ................................................................................................................... 14
3.2.2. Protein Adsorption ............................................................................................................................ 14
3.2.3. Protein Solubility.................. 16
4. CONCLUSIONS .......................................................................................... 17
5. OBJECTIVES OF THE THESIS ....................................................................... 18
6. REFERENCES............................................................................................. 18












Chapter 2
Characterization of Cytokine Solubility and Particle Formation in Presence of
Human Serum Albumin
1. INTRODUCTION......................................................................................... 26
2. MATERIALS AND METHODS ......................................................................... 27
2.1 Materials ................................................................................................. 27
2.2 Methods.................................................................................................. 27
2.2.1 Turbidity Measurement..................................................................................................................... 27
2.2.3 Light Obscuration.............................................................................................................................. 27
2.2.4 Dynamic Light Scattering (DLS) ...................................................................................................... 28
2.2.5 Zetapotential...................................................................................................................................... 28
2.2.6 SDS-PAGE........................................................................................................................................ 28
2.2.7 Fluorescence Spectroscopy ............................................................................................................... 28
2.2.8 Attenuated Total Reflection- FTIR Spectroscopy (ATR-FTIR)........................................................ 29
2.2.9 Atomic Force Microscopy (AFM)..................................................................................................... 30
2.2.10 Disc centrifugation............................................................................................................................ 30
3. RESULTS AND DISCUSSION ....................................................................... 31
3.1 Characterization of Cytokine-HSA Formulations at Different pH and Ionic Strength
Conditions ............................................................................................... 31
3.1.1 Effect of pH and Salt on Turbidity .................................................................................................... 31
3.1.2 SDS-PAGE of the Precipitated Material ...........................................................................................38
3.1.3 Fluoresc