Large-scale production and cost effective purification of llama single domain antibody ToxA5

196 Pages·2013·3.44 MB·English

by  Albert Parisien

CHG 9999
THÈSE DE DOCTORAT
Production à grande échelle dans Escherichia coli TG1 et purification de l’anticorps à domaine
simple ToxA5.1 provenant du lama ayant pour cible l’entérotoxine A produite par Clostridium
difficile

DOCTORAL THESIS
Large-scale production in Escherichia coli TG1 and purification of llama single domain antibody
ToxA5.1 against Clostridium difficile toxin A

A doctoral research thesis submitted in partial fulfillment of the requirements of the doctoral
program for the degree of Doctorate in Chemical Engineering
By
Albert Parisien
Supervisors
Dr. Christopher Q. Lan
Dr. Jules Thibault

Department of Chemical and Biological Engineering
University of Ottawa
May 31 2013
©Albert Parisien, Ottawa, Canada, 2013
i
Résumé
Les coûts pour le système de santé associés aux problèmes causés par des souches de
Clostridium difficile résistantes aux antibiotiques, évalués à plus d’un milliard de dollars, ne
cessent d’augmenter. Les toxines excrétées par C. difficile sont la cause et peut-être aussi la
solution à ces problèmes. En neutralisant les toxines, les effets néfastes peuvent être contrôlés. A
cet effet, cette thèse présente les résultats d’études permettant la production à grande échelle en
bio-fermenteurs et la purification d’un nouvel agent thérapeutique, le ToxA5.1, un anticorps à
domaine simple (pSJF2H-ToxA5.1) se liant à la toxine A de C. difficile. Le projet a été divisé en
quatre segments: 1) ToxA5.1 étant une protéine intracellulaire, une production de biomasse
élevée était la première étape vers la production à grande échelle. Pour obtenir une fermentation
à haute densité cellulaire (FHDC), les effets de la concentration initiale en glucose et la stratégie
d'alimentation basée sur un pH-stat furent étudiés; 2) Suite à la FHDC, les effets de l’ajout
d’extraits de levures, la température ainsi que la période d’induction furent étudiés; 3) Afin de
récupérer la protéine recombinante, un système de lyse cellulaire sélective où les effets
synergiques du Triton X-100 et de la température sur la lyse furent étudiés. Et enfin 4) La
purification de ToxA5.1 à l’aide de nanoparticules de nickel synthétisées par la méthode polyol a
été étudiée.
En combinant la stratégie FHDC avec une augmentation de la température d’induction et
l’ajout d’extrait de levure, une concentration de 127 mg/L de ToxA5.1 a été obtenue. En
utilisant la lyse synergétique à 60°C avec 1% de Triton X-100, il fut possible de récupérer 95%
de ToxA5.1 initialement présent dans les cellules en plus d’augmenter la proportion de ToxA5.1
de 27% dans le lysat. De plus, ToxA5.1 purifié ne démontrait aucune perte d’activité suite au
traitement thermo-chimique. Il fut déterminé que la concentration de la polyvinylpyrrolidone
avait un impact sur la taille des nanoparticules lors de la synthèse avec des particules ayant des
diamètres allant de 131 nm à 47 nm. Finalement, il fut possible de modéliser les interactions de
liaisons du système ToxA5.1-NNP.

ii
Abstract
Drug resistant strains of Clostridium difficile are a major health concern with over 3
million cases costing over 1 billion $ per year in the United-States. The diseases associated with
these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the
severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce
CDAD morbidity and this project was aiming at investigating the large-scale production and
recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant
Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these
objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular
recombinant protein, obtaining a high biomass production was the first step towards large-scale
production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding
strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature,
induction timing and media supplementation with complex nitrogen sources were investigated;
3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be
recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and
temperature were studied. And finally 4) Single-step purification using nickel nanoparticles
(NNP) synthesized via a modified polyol method was studied.
Combining the HCDC strategy with a temperature shift and yeast extract addition at the
time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective
cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1
was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to
27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which
proved to be highly selective and could be used up to five times. Diameter of the NNP
synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ±
20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

iii
Acknowledgements
I would like first to acknowledge my supervisors, Drs. Christopher Q. Lan and Jules
Thibault, for providing the opportunity to work in the field of fermentation and gene expression.
Their financial support through NSERC Discovery grants and the Canada Foundation for
Innovation as well as their guidance, at professional and personal levels, was really appreciated.

I would also like to acknowledge Dr. Colin Mackenzie from the NRC Institute for
Biological Sciences Antibody Engineering Group for making this project possible by providing
the bacterial strain. Also from the same organization, I would like to acknowledge Dr. Jamshid
Tanha for his assistance and guidance and Dr. Greg Hussack who has been my primary contact
and who patiently answered my many questions and performed some of the experiments reported
in this thesis (ELISA and Biacore).

I would like to acknowledge lab members: Kristin Chow, Fady Al-Zarka, Aatika Ahmed,
Kevin Nhan, Sallé Dembélé, Daniel O’Neil, and Christian Euler for the help they provided in
experiments.

I would like to acknowledge the department staff, Louis Tremblay, Gérard Nina and
Franco Ziroldo for the technical assistance as well as Francine Pétrin and Sylvie Saindon for
administrative assistance.

I also would like to acknowledge the financial support of the Ontario Government
through OGS scholarships, the Canadian Government through a NSERC PGSD and Excellence
scholarships, and the University of Ottawa through the Excellence scholarship.

Finalement, j’aimerais exprimer ma gratitude à ma femme, Annie, pour toutes ces années
de support moral ainsi qu’à mes enfants, Lauriane, Philippe et Clément pour l’inspiration dont ils
ont été la source.
iv
Table of Contents
Résumé ............................................................................................................................................ ii
Abstract .......................................................................................................................................... iii
Acknowledgements ....................................................................................................................... iv
Table of Contents ........................................................................................................................... v
List of Figures ............................................................................................................................... xii
List of Tables ................................................................................................................................ xv
Collaborator’s Contributions .................................................................................................... xvi
Nomenclature ............................................................................................................................ xviii
1. Chapter 1: Introduction........................................................................................................ 1
1.1 References ........................................................................................................................... 9
2 Chapter 2: Literature review ............................................................................................. 12
2.1 Clostridium difficile and Clostridium difficile associated diseases .................................. 12
2.2 Monoclonal antibodies and single domain antibodies ..................................................... 13
2.3 High cell density culture ................................................................................................... 15
2.3.1 Cell growth ............................................................................................................... 15
2.3.2 Protein expression at HCDC .................................................................................... 18
2.4 Cell disruption technologies ............................................................................................. 20
2.4.1 Complete cell lysis ................................................................................................... 21
v
2.4.2 Partial cell disruption ............................................................................................... 22
2.5 Protein recovery and purification using magnetic affinity adsorbents (MAA) ................. 23
2.6 Magnetic affinity adsorbents............................................................................................. 24
2.7 Nickel nanoparticle synthesis ........................................................................................... 28
2.8 References ......................................................................................................................... 29
3 Chapter 3: High cell density culture of recombinant Escherichia coli expressing
ToxA5.1 using dual-point pH-stat fed-batch fermentation ................................................................... 39
3.1 Abstract ............................................................................................................................. 39
3.2 Introduction ...................................................................................................................... 40
3.3 Materials and Methods ..................................................................................................... 43
3.3.1 Bacterial strain and plasmid ..................................................................................... 43
3.3.2 Media and feeding solution ...................................................................................... 44
3.3.2.1 Medium composition .......................................................................................... 44
3.3.2.2 Inoculum preparation .......................................................................................... 45
3.3.3 Fermentations ........................................................................................................... 45
3.3.4 Feeding strategies ..................................................................................................... 46
3.3.4.1 Single-point pH-stat (SPPS) Fed-batch .............................................................. 46
3.3.4.2 Dual-point pH-stat (DPPS) control strategy ....................................................... 46
3.3.5 Analysis of glucose and acetate ............................................................................... 47
3.4 Results ............................................................................................................................... 47
3.4.1 Batch fermentation at high initial glucose concentration ......................................... 47
3.4.2 Effect of genetic modification on cell growth .......................................................... 49
3.4.3 Single point pH-stat (SPPS) ..................................................................................... 51
vi
3.4.4 Dual point pH-stat (DPPS) ....................................................................................... 53
3.4.4.1 Low glucose feeding dosage (DPPSL) ............................................................... 53
3.4.4.2 High glucose dosages (10 g/L) (DPPSH) ........................................................... 55
3.5 Discussion ......................................................................................................................... 60
3.5.1 Effect of initial glucose concentration on HCDC .................................................... 60
3.5.2 The shortcut feeding loop with SPPS ....................................................................... 61
3.6 Effects of glucose feeding dosage in dual-point pH control ............................................. 62
3.7 Conclusion ........................................................................................................................ 63
3.8 Acknowledgements ............................................................................................................ 64
3.9 References ......................................................................................................................... 64
4 Chapter 4: Large-scale expression of a single domain antibody targeting Clostridium
difficile toxin A in Escherichia coli TG1 using pH-stat control............................................................. 67
4.1 Abstract ............................................................................................................................. 67
4.2 Introduction ...................................................................................................................... 68
4.3 Materials and Methods ..................................................................................................... 70
4.3.1 Bacterial strain and plasmid ..................................................................................... 70
4.3.2 Medium preparation ................................................................................................. 71
4.3.3 Protein quantification ............................................................................................... 71
4.3.4 Effect of temperature and media on cell growth ...................................................... 72
4.3.5 Effect of temperature on growth and expression ..................................................... 72
4.3.6 Effect of yeast extract induction timing and inducer concentration in defined
medium………………………………………………………………………………………………73
4.3.7 Fermentation in bioreactors ...................................................................................... 74
vii
4.3.7.1 Inoculum preparation .......................................................................................... 74
4.3.7.2 Dual-point pH-stat fermentations ....................................................................... 74
4.3.7.3 Glucose and acetate ............................................................................................ 75
4.4 Results ............................................................................................................................... 75
4.4.1 Effects of media and temperature on cell growth..................................................... 75
4.4.2 Protein expression .................................................................................................... 76
4.4.2.1 Protein expression in flasks ................................................................................ 76
4.4.2.2 Bioreactor protein expression ............................................................................. 78
4.5 Discussion ......................................................................................................................... 82
4.5.1 Effect of temperature and media on cell growth ...................................................... 82
4.5.2 Effect of temperature on cell growth and ToxA5.1 expression ............................... 83
4.5.3 Effect of yeast extract, induction and inducer .......................................................... 84
4.5.4 Fermentation in bioreactors ...................................................................................... 85
4.6 Conclusion ........................................................................................................................ 86
4.7 Acknowledgements ............................................................................................................ 86
4.8 References ......................................................................................................................... 87
5 Chapter 5: Enhanced recovery of single domain antibody ToxA5.1 against Clostridium
difficile toxin A using synergistic selective lysis of Escherichia coli TG1cells ...................................... 90
5.1 Abstract ............................................................................................................................. 90
5.2 Introduction ...................................................................................................................... 91
5.3 Material and Methods ....................................................................................................... 94
5.3.1 Cell growth and protein expression .......................................................................... 94
5.4 Cell Lysis........................................................................................................................... 95
viii
5.4.1 Small volume synergistic lysis ................................................................................. 95
5.4.2 Synergistic lysis in Erlenmeyer flasks ..................................................................... 95
5.4.3 Protein quantification and activity determination .................................................... 96
5.4.4 Calculations of ToxA5.1 recovery and selectivity ................................................... 97
5.5 Results .............................................................................................................................. 99
5.5.1 Synergistic lysis experiments in micro-centrifuge tubes .......................................... 99
5.5.2 Synergistic lysis in Erlenmeyer flasks ................................................................... 101
5.5.2.1 Time course profiles for the synergistic lysis ................................................... 101
5.5.2.2 Fixed length synergistic lysis ............................................................................ 103
5.5.3 Effect of synergistic lysis on ToxA5.1 activity ...................................................... 105
5.6 Discussion ....................................................................................................................... 106
5.6.1 Synergistic lysis experiments in micro-centrifuge tubes ........................................ 106
5.6.2 Synergistic lysis in Erlenmeyer flasks ................................................................... 108
5.7 Conclusion ...................................................................................................................... 110
5.8 Acknowledgements .......................................................................................................... 111
5.9 References ....................................................................................................................... 111
6 Chapter 6: Nickel nanoparticles synthesized by a modified polyol method for the
purification of histidine-tagged single domain antibody ToxA5.1 (Published in Journal of Materials
Research, 2012) ....................................................................................................................................... 114
6.1 Abstract ........................................................................................................................... 114
6.2 Introduction .................................................................................................................... 115
6.3 Materials and Methods ................................................................................................... 116
6.3.1 Synthesis of nickel nanoparticles ........................................................................... 116
ix
6.3.2 Characterization of Ni nanoparticles ...................................................................... 117
6.3.3 Protein purification and regeneration of Ni nanoparticles ..................................... 118
6.3.4 Calculations of binding capacities and ToxA5.1 recovery ..................................... 121
6.4 Results and discussion .................................................................................................... 122
6.4.1 NNP characteristics ................................................................................................ 122
6.4.1.1 XRD .................................................................................................................. 122
6.4.1.2 SEM .................................................................................................................. 123
6.4.2 Selective binding of the His-tagged ToxA5.1 by the NNP .................................... 125
6.4.3 Reusability of the NNP .......................................................................................... 129
6.5 Conclusion ...................................................................................................................... 131
6.6 Acknowledgements .......................................................................................................... 131
6.7 References ....................................................................................................................... 132
7 Chapter 7: Effect of polyvinylpyrrolidone:nickel ratio on properties and performance
of nickel nanoparticles for purification of histidine-tagged recombinant protein (ToxA5.1) .......... 134
7.1 Abstract ........................................................................................................................... 134
7.2 Introduction .................................................................................................................... 135
7.3 Material and Methods ..................................................................................................... 135
7.3.1 Cell growth and protein expression ........................................................................ 135
7.3.2 Synthesis of nickel nanoparticles (NNP) ............................................................... 136
7.3.3 Protein purification ................................................................................................. 137
7.3.4 Protein quantification ............................................................................................. 138
7.3.5 Characterization of nanoparticles ........................................................................... 139
7.3.6 Morphology and size distribution of the nickel nanoparticles ............................... 139
x