purification and effectiveness of vaccines and antiviral compounds

187 Pages·2015·3.17 MB·English

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Dissertations, Master's Theses and Master's Dissertations, Master's Theses and Master's
Reports - Open Reports
2014
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Maria F. Gencoglu
Michigan Technological University
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Copyright 2014 Maria F. Gencoglu
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Gencoglu, Maria F., "PURIFICATION AND EFFECTIVENESS OF VACCINES AND ANTIVIRAL COMPOUNDS",
Dissertation, Michigan Technological University, 2014.
https://doi.org/10.37099/mtu.dc.etds/844
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PURIFICATION AND EFFECTIVENESS OF VACCINES AND ANTIVIRAL COMPOUNDS

By
Maria F. Gencoglu

A DISSERTATION
Submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
In Chemical Engineering

MICHIGAN TECHNOLOGICAL UNIVERSITY
2014

©2014 Maria F. Gencoglu

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This dissertation has been approved in partial fulfillment of the requirements for the Degree of
DOCTOR OF PHILOSOPHY in Chemical Engineering.

Department of Chemical Engineering

Dissertation Advisor: (cid:39)(cid:85)(cid:17)(cid:3)(cid:38)(cid:68)(cid:85)(cid:92)(cid:81)(cid:3)(cid:43)(cid:72)(cid:79)(cid:71)(cid:87)

Committee Member: (cid:39)(cid:85)(cid:17)(cid:3)(cid:36)(cid:71)(cid:85)(cid:76)(cid:72)(cid:81)(cid:81)(cid:72)(cid:3)(cid:48)(cid:76)(cid:81)(cid:72)(cid:85)(cid:76)(cid:70)(cid:78)
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Committee Member: (cid:39)(cid:85)(cid:17)(cid:3)(cid:38)(cid:75)(cid:76)(cid:81)(cid:74)(cid:16)(cid:36)(cid:81)(cid:3)(cid:51)(cid:72)(cid:81)(cid:74)

Committee Member: (cid:39)(cid:85)(cid:17)(cid:3)(cid:55)(cid:75)(cid:82)(cid:80)(cid:68)(cid:86)(cid:3)(cid:58)(cid:72)(cid:85)(cid:81)(cid:72)(cid:85)

Department Chair: (cid:39)(cid:85)(cid:17)(cid:3)(cid:46)(cid:82)(cid:80)(cid:68)(cid:85)(cid:3)(cid:46)(cid:68)(cid:90)(cid:68)(cid:87)(cid:85)(cid:68)

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To my family; Aytug, Adriana and Jose, I would not have made it without your encouragement
and love

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Table of Contents
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LIST OF FIGURES ......................................................................................................................... 10
LIST OF TABLES ........................................................................................................................... 12
PREFACE ...................................................................................................................................... 13
ACKNOWLEDGEMENTS .............................................................................................................. 14
LIST OF ABBREVIATIONS............................................................................................................ 15
ABSTRACT .................................................................................................................................... 18
CHAPTER 1: INTRODUCTION AND OVERVIEW ........................................................................ 19
1.1. Introduction..................................................................................................................... 20
1.2. Overview ........................................................................................................................ 21
1.3. References ..................................................................................................................... 24
CHAPTER 2: LITERATURE REVIEW ........................................................................................... 26
2.1. Literature review ............................................................................................................. 27
2.2. Viruses and vaccines ..................................................................................................... 27
2.3. Osmolytes ...................................................................................................................... 29
2.4. Antiviral Compounds ...................................................................................................... 31
2.4.1. Synthetic antiviral compounds ............................................................................... 32
2.4.2. Natural antiviral compounds ................................................................................... 35
2.4.2.1. Natural antiviral extracts ......................................................................................... 36
2.4.2.2.Isolated natural antiviral compounds ...................................................................... 38
2.5. Vaccine manufacturing ................................................................................................... 40
2.5.1. Upstream processes .............................................................................................. 41
2.5.2. Downstream processes .......................................................................................... 42
2.6. Vaccine delivery systems ............................................................................................... 45
2.7. Conclusions……………………………………………………………………………………52(cid:3)
2.8. References ..................................................................................................................... 53
CHAPTER 3: OSMOLYTES AS ANTIVIRAL COMPOUNDS AGAINST PORCINE PARVOVIRUS
....................................................................................................................................................... 71
3.1. Introduction..................................................................................................................... 72
3.2. Materials and methods ................................................................................................... 73
3.2.1. Materials ................................................................................................................. 73
3.2.2. Cell propagation ..................................................................................................... 73
3.2.3. Virus production and titration ................................................................................. 73
3.2.4. Cytotoxicity assay .................................................................................................. 74
3.2.5. Osmolyte toxicity .................................................................................................... 75
3.2.6. Pre-infection treatment ........................................................................................... 75
3.2.7. Plaque reduction assay .......................................................................................... 75
3.2.8. Yield reduction assay ............................................................................................. 76
3.2.9. Immunohistochemical detection of virus capsid protein production ....................... 76
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3.3. Results ........................................................................................................................... 77
3.3.1. Virus cytotoxicity in the presence of osmolytes and a salt ..................................... 77
3.3.2. Osmolyte toxicity .................................................................................................... 79
3.3.3. Reduction of virus cytotoxicity with TMAO and glycine .......................................... 80
3.3.4. Plaque reduction assay .......................................................................................... 81
3.3.5. Impact of osmolytes on infectious virus yield ......................................................... 82
3.3.6. Virus capsid protein production with the addition of osmolytes ............................. 83
3.4. Discussion ...................................................................................................................... 85
3.5. Conclusions .................................................................................................................... 86
3.6. References ..................................................................................................................... 87
CHAPTER 4: PURIFICATION OF PORCINE PARVOVIRUS WITH OSMOLYTES
FLOCCULATION, FOLLOWED BY MICROFILTRATION ............................................................. 89
4.1. Introduction ..................................................................................................................... 90
4.2. Materials and methods ................................................................................................... 92
4.2.1. Materials ................................................................................................................. 92
4.2.2. Cell propagation, virus production and titration ...................................................... 93
4.2.3. Virus flocculation and filtration ............................................................................... 94
4.2.4. Shear stress effect ................................................................................................. 94
4.2.5. Ionic strength effect ................................................................................................ 94
4.2.6. Protein flocculation and filtration ............................................................................ 95
4.3. Results and discussion .................................................................................................. 95
4.3.1. PPV flocculation in the presence of osmolytes, salts, and PEG ............................ 95
4.3.2. Qualitative shear stress effect on PPV flocculation ............................................... 99
4.3.3. pH effect on PPV flocculation ............................................................................... 100
4.3.4. Ionic strength effect on PPV flocculation ............................................................. 101
4.3.5. Preferential PPV flocculation ................................................................................ 103
4.4. Conclusions .................................................................................................................. 104
4.5. References ................................................................................................................... 106
CHAPTER 5: PURIFICATION OF SINDBIS VIRUS WITH OSMOLYTES FLOCCULATION,
FOLLOWED BY MICROFILTRATION ......................................................................................... 112
5.1. Introduction ................................................................................................................... 113
5.2. Materials and methods ................................................................................................. 114
5.2.1. Materials ............................................................................................................... 114
5.2.2. Cell propagation, virus production and titration .................................................... 115
5.2.3. Flocculation and filtration ..................................................................................... 116
5.2.4. Effect of pH and ionic strength ............................................................................. 116
5.3. Results and discussion ................................................................................................ 117
5.3.1. Flocculation of SVHR with osmolytes, PEG and salts ......................................... 117
5.3.2. pH effect on SVRH flocculation ............................................................................ 120
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5.3.3. Ionic strength effect on SVHR flocculation ........................................................... 121
5.3.4. SVHR preferential flocculation with osmolytes .................................................... 123
5.4. Conclusions .................................................................................................................. 124
5.5. References ................................................................................................................... 125
CHAPTER 6: BIOCOMPATIBILITY STUDIES OF SOFT-TEMPLATE MESOPOROUS CARBONS
..................................................................................................................................................... 129
6.1. Introduction................................................................................................................... 130
6.2. Materials and methods ................................................................................................. 133
6.2.1. Materials synthesis ............................................................................................... 133
6.2.2. Material characterization ...................................................................................... 133
6.2.3. Cytotoxicity ........................................................................................................... 134
6.2.4. Cell viability .......................................................................................................... 135
6.2.5. Hemolysis test ...................................................................................................... 136
6.2.6. Protein adsorption ................................................................................................ 136
6.2.7. Imaging of cell growth of mesoporous carbon materials ..................................... 137
6.3. Results and discussion ................................................................................................ 137
6.3.1. Materials characterization .................................................................................... 137
6.3.2. In vitro cell interactions ......................................................................................... 141
6.3.3. Hemocompatibility of mesoporous carbon materials ........................................... 146
6.4. Conclusions .................................................................................................................. 148
6.5. References ................................................................................................................... 150
CHAPTER 7: CONCLUSIONS AND FUTURE WORK ................................................................ 156
7.1. Conclusions .................................................................................................................. 157
7.2. Future work .................................................................................................................. 158
7.3. References ................................................................................................................... 161
APPENDIX A: PROOFS OF PERMISSION TO REPRODUCE PUBLISHED MATERIAL .......... 164
A.1. Permission for reproduction of Figure 2.5 .................................................................... 165
A.2. Permission for reproduction of Figure 2.6 .................................................................... 167
A.3. Permission for reproduction of Figure 2.7 .................................................................... 171
A.4. Permission for reproduction of material reproduced in Chapter 3 ............................... 176
A.5. Permission for reproduction of material reproduced in Chapter 4 ............................... 181
A.6. Permission for reproduction of material reproduced in Chapter 6 ............................... 186
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