Development of an aqueous suspension of recombinant human bone morphogenetic protein-2 (rhBMP-2) [Elektronische Ressource] = Entwicklung einer wässrigen Suspension des recombinanten menschlichen Knochenwachstumsproteins-2 (rhBMP-2) / vorgelegt von Daniel Hans Schwartz

Daniel Schwartz

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• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

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


Development of an Aqueous Suspension of
Recombinant Human Bone Morphogenetic
Protein-2 (rhBMP-2)


Entwicklung einer wässrigen Suspension des recombinanten
menschlichen Knochenwachstumsproteins-2 (rhBMP-2)












vorgelegt von
Daniel Hans Schwartz
aus Pegnitz

München, 2005
ERKLÄRUNG
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsord-
nung vom 29. Januar 1998 von Herrn Prof. Dr. W. Frieß betreut.



EHRENWÖRTLICHE VERSICHERUNG
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe angefertigt.



München, am 18.03.2005
...............................................................
Daniel Hans Schwartz












Dissertation eingereicht am: 5.4.2005
1. Gutachter: Prof. Dr. W. Frieß
2. Gutachter: Prof. Dr. G. Winter
Tag der mündlichen Prüfung: 31.5.2005



TO MY MOTHER
In love and perpetual gratefulness












„Geschrieben steht: “Im Anfang war das Wort!”
Hier stock ich schon! Wer hilft mir weiter fort?
Ich kann das Wort so hoch unmöglich schätzen,
ich muß es anders übersetzen,
Wenn ich vom Geiste recht erleuchtet bin.
Geschrieben steht: Im Anfang war der Sinn.
Bedenke wohl die erste Zeile,
das deine Feder sich nicht übereile!
Ist es der Sinn, der alles wirkt und schafft?
Es sollte stehn: Im Anfang war die Kraft!
Doch auch indem ich dieses niederschreibe,
Schon warnt mich was, dass ich dabei nicht bleibe.
Mir hilft der Geist! Auf einmal seh ich Rat
Und schreibe getrost: Im Anfang war die Tat!“

Goethe, Faust, 1224-1237 Acknowledgements
The present thesis has been acquired at the Institute for Pharmacy and Food
Chemistry, Department of Pharmaceutical Technology, at the Friedrich-Alexander
University of Erlangen-Nuernberg and the Department of Pharmacy, Pharmaceutical
Technology and Biopharmaceutics at the Ludwig-Maximilians-University of Munich.
I am mostly indebted to my supervisor Prof. Dr. W. Frieß, to give me the opportunity to
join his working group and to prepare this thesis. I very much appreciated his scientific
advice and his ongoing interest in the progress of my work. His great personality and
his soft skills make it a pleasure to work with him.
I also want to express my thanks to Prof. Dr. J. Lee, the chairman of the Department of
Pharmaceutical Technology in Erlangen, who accorded me an excellent technological
education and granted relief during my first time as a PhD student. From the kind and
collegial working group in Erlangen, I want to set apart PhD Monika Geiger, my “pre-
decessor”, who introduced me in the secrets of BMP and provided ongoing mentor-
and friendship beyond her time at the university – thanks a lot!
At the University of Munich, the chairman and co-referee, Prof. Winter, is greatly
acknowledged. By his personal leadership, he managed to create a very pleasant
working atmosphere, and his door was always open for both scientific and personal
advice.
I would like to thank all my colleagues who warmly welcomed me in Munich and who
greatly contribute to the excellent atmosphere (mirrored, e.g. in the legendary “Weiß-
wurstfrühstück” in the “Fürstenegger”, in trips through the “Schickeria” of Munich with
Richard Fuhrherr, or in the dancing contest in the “Soul” between our group and the
group of Prof. E. Wagner). I want to express my gratitude to Iris Metzmacher and
Andreas Rutz who shared the lab with me. Thanks for the pleasant time and your
friendship – the indefatigable helpfulness of Andreas is greatly acknowledged.
Outside the university, great thanks are addressed to Wyeth BioPharm, Andover, MA,
USA for funding and supporting the work with material. Special thanks go to Susan
Sofia for her engagement, her ongoing support across the ocean and for critical
proofreading the final work. I am also indebted to all whom I meet and worked with
during my visit in the USA and to the working groups who performed the animal testing
and the cell culture studies for me. I am also much obliged to Andrea Schmitt from
Brucker Optics for her support with FTIR-measurements, and to Klaus Hellerbrand,
Scill Biomedicals, for the opportunity to use the µDSC.
Within the period of almost 4 years at two different locations, I had the pleasure to
collaborate with a lot of people, who to mention all would go beyond the scope of this
acknowledgement. This is particularly true for all who did a great job in the background
– the secretary, the technical staff, the cleaners and engineers. So ‘thanks’ to all who
contributed in one way or another to this work, but were not explicitly listed. I
Table of Contents
Table of Contents................................................................................................ I
List of Abbreviations........................................................................................... V
1 Introduction ................................................................................................. 1
1.1 Bone Morphogenetic Proteins.............................................................. 1
1.1.1 Milestones in History..................................................................... 1
1.1.2 Biochemistry ................................................................................. 2
1.1.2.1 BMP-2 and its Family ............................................................ 2
1.1.2.2 Features of rhBMP-2 ............................................................. 3
1.1.2.3 Signaling Pathway ................................................................. 5
1.1.2.4 Pharmacokinetics of rhBMP-2 ............................................... 7
1.1.3 Biologic Activity: Mechanism of Ossification ................................. 7
1.1.4 Delivery Systems.......................................................................... 8
1.1.5 Applications of BMPs.................................................................... 9
1.1.5.1 Use of rhBMP-2 in Spinal Fusion Applications ...................... 9
1.1.5.2 Use of rhBMP in the Treatment of Non-unions.................... 14
1.1.5.3 Further Potential Applications.............................................. 14
1.2 Formation of Protein Particles by Precipitation .................................. 17
1.2.1 Formation of Dry Particles by Standard Methods ....................... 17
1.2.2 Protein Precipitation: Definition and Principles ........................... 19
1.2.3 Precipitation Methods ................................................................. 19
1.2.3.1 Crystallization ...................................................................... 20
1.2.3.2 Isoelectric Precipitation........................................................ 20
1.2.3.3 Salt-induced Precipitation: Salting-out................................. 21
1.2.3.4 Miscible Organic Solvent Precipitation ................................ 24
1.2.3.5 Precipitation by Non-ionic Polymers .................................... 25
1.2.3.6 Precipitation by Metal Ions................................................... 26
1.2.3.7 Protein Binding Dyes ........................................................... 27
1.2.3.8 Precipitation by Ionic (Poly)-Electrolytes ............................. 27
1.2.3.9 Affinity Precipitation ............................................................. 29 II Table of Contents
1.2.4 Precipitate Formation .................................................................. 29
1.2.5 Precipitation Process Conditions................................................. 30
2 Objectives of the Work .............................................................................. 31
3 Materials and Methods 32
3.1 Materials............................................................................................. 32
3.1.1 rhBMP-2...................................................................................... 32
3.1.2 Reagents and Substances .......................................................... 32
3.2 Methods 35
3.2.1 Focus on the Protein ................................................................... 35
3.2.1.1 Dialysis of Bulk Formulation................................................. 35
3.2.1.2 Ultrafiltration......................................................................... 35
3.2.1.3 Concentration Determination ............................................... 36
3.2.1.4 High Molecular Weight Species and Aggregation ................ 36
3.2.1.5 Monitoring of Oxidation and Isomerization........................... 37
3.2.1.6 Peptide Mapping .................................................................. 37
3.2.1.7 Proteins’ Secondary Structure Integrity................................ 38
3.2.1.8 Differential Scanning Calorimetry......................................... 3