Physical properties of protein formulations [Elektronische Ressource] / Virginie Le Brun

Le Brun, Virginie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 29
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

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









PHYSICAL PROPERTIES
OF
PROTEIN FORMULATIONS








VIRGINIE LE BRUN

aus

Le Mans (Frankreich)








2009

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 selbständig, ohne unerlaubte Hilfe erarbeitet.



München, am 22. Juni 2009.









.............................................................................

Virginie Le Brun
















Dissertation eingereicht am 22. Juni 2009

1. Gutacher: Prof. Dr. Wolfgang Frieß

2. Gutachter: Prof. Dr. Gerhard Winter

Mündliche Prüfung am 27. Juli 2009 ACKNOWLEDGMENTS



I wish to express my gratitude to my supervisor Prof. Dr. Wolfgang Frieß for his
valuable advice and guidance through this study. I would also like to include my
gratitude to Dr. Stefan Bassarab, Dr. Patrick Garidel, Dr. Silke Mühlau, Heidrun
Schott and Torsten Schultz-Fademrecht at Boehringer Ingelheim Pharma GmbH &
Co. KG for their fundamental support of this work.

Boehringer Ingelheim Pharma GmbH & Co. KG is acknowledged for the supplying of
materials and for the financial support.

I am deeply indebted to Andrea Eiperle, Matthias Heggy and Inge Miller at H & Co. KG and Dr. Armin Giese and Tobias
Högen at the Center for Neuropathology and Prion Research of the Ludwig-
Maximilians-University for helping with the experimental studies.

I am extremely grateful to all members of our research group for the friendly
atmosphere and the numerous French breakfasts, especially to: Cornelius Pompe,
Miriam Printz, Johannes Mathes, Frank Schaubhut, Lars Schiefelbein, Katja Schmid
and the members of the “Bubble lab” Klaus Freitag, Stephan Schultes and Steliyan
Tinkov.

I would specially like to thank Magali Delais, Melanie Hübner, Claudia Lewerenz and
Nantharat Pearnchob for their friendship and support.

Finally I want to thank my parents, my sister Hélène, and Matthias for their love,
encouragement and continuous support in all circumstances. TABLE OF CONTENTS


CHAPTER 1
APPLICATIONS OF THE OSMOTIC SECOND VIRIAL COEFFICIENT IN PROTEIN
FORMULATIONS.......................................................................................................................... 1
1. CHALLENGES IN LIQUID PROTEIN FORMULATIONS.................................................................................1
2. PROTEIN SOLUBILITY ............................................................................................................................2
3. PROTEIN ASSOCIATION AND AGGREGATION ..........................................................................................4
3.1. Self-association.............................................................................................................................4
3.2. Aggregation ...............................................................................................................5
3.3. Factors inducing protein aggregation / association.....................................................................7
3.3.1 External factors in protein formulation inducing protein aggregation / association .............................. 7
3.3.2. Internal factors in protein formulation inducing protein aggregation / association............................... 7
3.4. Prevention of protein aggregation................................................................................................9
4. PROTEIN-PROTEIN INTERACTIONS .........................................................................................................9
4.1. Osmotic second virial coefficient.........9
4.2. Nature of protein-protein interactions........................................................................................10
4.2.1. Excluded volume....................... 10
4.2.2. Electrostatic interactions .................................................................................................................... 11
4.2.3. Van der Waals forces.................................................................................................... 13
4.2.4. Hydrogen bonding.............................................................................................................................. 13
4.2.5. Solvation forces.......................... 14
4.3. Analytical methods for the determination of protein-protein interactions .................................15
4.3.1. Methods adapted to the determination of B ..................................................................................... 15 22
4.3.1.1. Membrane osmometry................................................................................................................ 16
4.3.1.2. Scattering methods..................................................................................................................... 17
4.3.1.3. Sedimentation equilibrium by analytical ultracentrifugation...................................................... 18
4.3.1.4. Self-interaction chromatography................................................................................................ 19
4.3.2. Other techniques adapted to the analysis of protein-protein interactions............................................ 21
4.3.2.1. Dynamic light scattering ............................................................................................................ 21
4.3.2.2. Surface plasmon resonance ........................................................................................................ 21
4.3.2.3. Ultrasonic storage modulus measurements ................................................................................22
4.3.2.4. Concluding remarks ................................................................................................................... 23
5. APPLICATIONS OF B IN PROTEIN FORMULATIONS..............................................................................23 22
5.1. Protein crystallization ................................................................................................................23
5.2. Protein solubility ........................................................................................................................24
5.3. Protein stability ..........................................................................................................................25
5.4. Protein viscosity................27
6. OBJECTIVES OF THE THESIS .................................................................................................................29
7. REFERENCES .......................................................................................................................................30


CHAPTER 2
INSIGHTS IN LYSOZYME-LYSOZYME SELF-INTERACTIONS AS ASSESSED BY THE
OSMOTIC SECOND VIRIAL COEFFICIENT ............................................................................. 39
1. INTRODUCTION....................................................................................................................................39
2. MATERIALS AND METHODS .................................................................................................................41
2.1. Materials...................41
2.2. Methods ......................................................................................................................................42
2.2.1. Lysozyme immobilization.................................................................................................................. 42
2.2.2. ATR-FTIR adsorption spectra and second derivates .......................................................................... 42
2.2.3. Determination of the osmotic second virial coefficient B ................................................................ 43 22
2.2.4. Determination of the net lysozyme charge ......................................................................................... 44
2.2.5. Determination of protein solubility .................................................................................................... 44
3. RESULTS AND DISCUSSION..................................................................................................................44
3.1. Establishment of B measurement via SIC for lysozyme ...........................................................44 22
3.1.1. Lysozyme characterization after binding on chromatography particles.............................................. 44
3.1.2. Chromatogram analysis: retention volume determination .................................................................. 45
3.1.3. Influence of chroma