characterizing binding affinity of somatropin and derived structures to hghab by surface acoustic

113 Pages·2013·7.38 MB·English

by  Evelyn Buyst

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutical Analysis
Drug Quality and Registration
Academic Year 2012-2013

CHARACTERIZING BINDING AFFINITY OF SOMATROPIN AND DERIVED STRUCTURES
TO HGHAB BY SURFACE ACOUSTIC WAVES AND SIZE EXCLUSION
CHROMATOGRAPHY

Apr. Evelyn Buyst
Master of Industrial Pharmacy

Promotor
Prof. dr. Apr. Bart De Spiegeleer

Mentor
Nathalie Bracke

Board of commissioners
Prof. Erwin Adams
Prof. Roger Kemel
Prof. Ann Van Schepdael
Prof. Sandra Apers
Prof. Ann Van Eeckhaut
Prof. Yvette Michotte

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutical Analysis
Drug Quality and Registration
Academic Year 2012-2013

CHARACTERIZING BINDING AFFINITY OF SOMATROPIN AND DERIVED STRUCTURES
TO HGHAB BY SURFACE ACOUSTIC WAVES AND SIZE EXCLUSION
CHROMATOGRAPHY

Apr. Evelyn Buyst
Master of Industrial Pharmacy

Promotor
Prof. dr. Apr. Bart De Spiegeleer

Mentor
Nathalie Bracke

Board of commissioners
Prof. Erwin Adams
Prof. Roger Kemel
Prof. Ann Van Schepdael
Prof. Sandra Apers
Prof. Ann Van Eeckhaut
Prof. Yvette Michotte
COPYRIGHT

"The author and the promoters give the authorization to consult and to copy parts of this
thesis for personal use only. Any other use is limited by the laws of copyright, especially
concerning the obligation to refer to the source whenever results from this thesis are cited."

April 22, 2013

Appreciations

First of all,
I would like to enounce my appreciations towards Prof. dr. Apr. Bart De Spiegeleer.
He gave me the opportunity to work in the DruQuaR laboratory. He arranged an internship at
the very last moment, offered me a very interesting and defiant topic to work on, and he was
always available for questions and his professional and expansive view on many things.

Secondly, I want to thank tremendously my mentor, Nathalie Bracke,
who was always and at every time available for questions and good advice.
It was really a pleasure to work with her.

Another word of thanks is for all other colleagues in the laboratory for their daily help and
the positive atmosphere they created to work in.

Thanks to all other students for the good company
and pleasant leisure time during this last year.

Last but not least,
I thank my parents and my family for all the opportunities
they gave me, their never ending support and encouraging words.
TABLE OF CONTENTS

1. INTRODUCTION ....................................................................................................... 1
1.1. BIOLOGICAL FUNCTION OF HUMAN GROWTH HORMONE AND ITS ROL
IN CANCER .............................................................................................................. 1
1.1.1. Neuro-endocrine tumors ....................................................................... 1
1.1.2. Growth hormone ................................................................................... 2
1.2. PROTEIN AND PEPTIDE THERAPEUTICS .................................................................. 3
1.2.1. Protein scaffolds .................................................................................... 3
1.2.2. Peptide therapeutics .............................................................................. 4
1.3. BIOSENSORS IN DRUG DEVELOPMENT .................................................................. 5
1.3.1. What are biosensors? ............................................................................. 5
1.3.2. Surface acoustic waves biosensors ......................................................... 6
1.3.3. Sensorgram of surface acoustic wave biosensors .................................... 7
1.3.4. Other tools for the characterization of binding events ........................... 8
1.3.4.1. Surface plasmon resonance............................................................. 8
1.3.4.2. Quarz crystal microbalance ............................................................ 8
1.3.4.3. Isothermal titration calorimetry ..................................................... 9
1.3.5. Comparison of biosensors and other techniques .................................... 9
2. OBJECTIVES ............................................................................................................ 11
3. MATERIALS AND METHODS .................................................................................... 12
3.1. SENSOR CHIP .......................................................................................................... 12
3.1.1. Cleaning sensor chips ............................................................................. 12
3.1.1.1. Pre-cleaning .................................................................................... 12
3.1.1.2. Chemical etching with AMP ............................................................ 12
3.1.2. Coating sensor chip with dextran ........................................................... 12
3.2. LIGAND IMMOBILIZATION ...................................................................................... 13
3.3. SAM5 BINDING EXPERIMENTS ............................................................................... 14
3.3.1. Screening for a regeneration condition .................................................. 14
3.3.2. Analytes (modified) somatropin ............................................................ 14
3.3.2.1. Binding experiment between hGHAb and (modified) somatropin with
regeneration ................................................................................... 14
3.3.3. Somatropin derived peptides ................................................................. 15
3.3.3.1. QC analysis of somatropin derived peptides ................................... 15
3.3.3.2. Binding experiments with somatropin derived peptides ............... 15
3.3.4. Data processing of SAW binding experiments for (modified) somatropin and
peptides ................................................................................................ 16
3.4. SIZE EXCLUSION CHROMATOGRAPHY .................................................................... 18
4. RESULTS AND DISCISSION ....................................................................................... 19
4.1. LIGAND IMMOBILIZATION ...................................................................................... 19
4.2. SOMATROPIN ......................................................................................................... 21
4.2.1. Screening for regeneration conditions ................................................... 21
4.2.2. SAM5 binding experiments .................................................................... 22
4.2.2.1. Binding experiment between hGHAb and somatropin .................. 22
4.2.3. SEC experiments .................................................................................... 24
4.2.3.1. Calibration curve of the SEC column .............................................. 24
4.2.3.2. SEC analysis of hGHAb .................................................................... 25
4.2.3.3. SEC analysis of somatropin ............................................................. 25
4.2.3.4. SEC analysis of mixtures of hGHAb and somatropin ...................... 27
4.3. MODIFIED SOMATROPIN ........................................................................................ 30
4.3.1. 1:1 NOTA-somatropin ............................................................................ 30
4.3.1.1. SAM5 binding experiment with regeneration ................................ 30
4.3.1.2. SEC experiments ............................................................................. 31
4.3.1.2.1. SEC analysis of 1:1 NOTA-somatropin ............................ 31
4.3.1.2.2. SEC analysis of mixtures of hGHAb and
1:1 NOTA-somatropin ..................................................... 32
4.3.2. 1:3 NOTA-somatropin ............................................................................ 33
4.3.2.1. SAM5 binding experiment with regeneration ................................ 33
4.3.2.2. SEC experiments ............................................................................. 34
4.3.2.2.1. SEC analysis of 1:3 NOTA-somatropin ............................ 34
4.3.2.2.2. SEC analysis of mixtures of hGHAb and
1:3 NOTA-somatropin ..................................................... 35
4.3.3. 1:10 NOTA-somatropin .......................................................................... 36
4.3.3.1. SAM5 binding experiments ............................................................. 36
4.3.3.1.1. Binding experiment with regeneration ........................... 36
4.3.3.1.2. Duplication of binding experiment with regeneration ... 37
4.3.3.2. SEC experiments .............................................................................. 38
4.3.3.2.1. SEC analysis of 1:10 NOTA-somatropin .......................... 38
4.3.3.2.2. SEC analysis of mixtures of hGHAb and
1:10 NOTA-somatropin ................................................... 39
4.3.4. Comparison of the modified somatropin structures ............................... 41
4.4. SOMATROPIN DERIVED PEPTIDES .......................................................................... 45
4.4.1. Choise of peptides ................................................................................. 45
4.4.2. QC analysis somatropin derived peptides .............................................. 45
4.4.3. SAM5 binding experiments .................................................................... 46
4.4.3.1. Feasability calculations ................................................................... 46
4.4.3.2. Binding experiments ....................................................................... 47
4.4.4. SEC experiments .................................................................................... 50
4.4.4.1. SEC analysis of P0320 ...................................................................... 50
4.4.4.2. SEC analysis of mixtures of hGHAb and P0320 ............................... 52
5. CONCLUSION AND PERSPECTIVES ............................................................................ 53
6. REFERENCES ........................................................................................................... 54