Application of hydrocyclone for cell separation in mammalian cell perfusion cultures [Elektronische Ressource] / von Elsayed Ahmed Elsayed Ahmed
150 Pages
English
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Application of hydrocyclone for cell separation in mammalian cell perfusion cultures [Elektronische Ressource] / von Elsayed Ahmed Elsayed Ahmed

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Learn all about the services we offer
150 Pages
English

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Application of Hydrocyclone for Cell Separation in Mammalian Cell Perfusion Cultures Vom Fachbereich für Biowissenschaften und Psychologie der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte Dissertation von Elsayed Ahmed Elsayed Ahmed aus Kairo, Ägypten 1. Referent: Prof. Dr. W.-D. Deckwer 2. R. Wagner eingereicht am: 28.07.2005 Mündliche Prüfung (Disputation) am: 29.09.2005 Druckjahr: 2005 Gedrückt mit Unterstützung des Deutschen Akademischen Austauschdiensts (DAAD). ii Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung des Fachbereichs für Biowissenschaften und Psychologie, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Elsayed EA, Piehl G-W, Nothnagel J, Medronho RA, Deckwer W-D, Wagner R. 2005. Use of hydrocyclone as an efficient tool for cell retention in perfusion cultures. In: Gòdia F, Fussenegger M (Eds.) Animal Cell Technology Meets Genomics. Dordrecht, Springer, p. 679-682. Patent Wagner R, Elsayed EA. 2004. Method, apparatus and system for separating eucaryotic or procaryotic cells or other particularly biological material from a suspension.

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Published 01 January 2005
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Exrait








Application of Hydrocyclone for Cell Separation
in Mammalian Cell Perfusion Cultures



Vom Fachbereich für Biowissenschaften und Psychologie
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig


zur Erlangung des Grades eines Doktors
der Naturwissenschaften
(Dr. rer. nat.)


genehmigte
Dissertation






von
Elsayed Ahmed Elsayed Ahmed
aus Kairo, Ägypten
























1. Referent: Prof. Dr. W.-D. Deckwer
2. R. Wagner
eingereicht am: 28.07.2005
Mündliche Prüfung (Disputation) am: 29.09.2005
Druckjahr: 2005

Gedrückt mit Unterstützung des Deutschen Akademischen Austauschdiensts (DAAD).
ii
Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung des Fachbereichs für
Biowissenschaften und Psychologie, vertreten durch den Mentor der Arbeit, in folgenden
Beiträgen vorab veröffentlicht:

Publikationen
Elsayed EA, Piehl G-W, Nothnagel J, Medronho RA, Deckwer W-D, Wagner R. 2005. Use of
hydrocyclone as an efficient tool for cell retention in perfusion cultures. In: Gòdia F,
Fussenegger M (Eds.) Animal Cell Technology Meets Genomics. Dordrecht, Springer, p.
679-682.

Patent
Wagner R, Elsayed EA. 2004. Method, apparatus and system for separating eucaryotic or
procaryotic cells or other particularly biological material from a suspension. World
Intellectual Property Organization, WO 2004/099362 A2. European Patent, PCT/EP
04/004976.

Tagungsbeiträge
Elsayed EA, Piehl G-W, Nothnagel J, Medronho RA, Deckwer W-D, Wagner R. 2003. Use of
thhydrocyclone as an efficient tool for cell retention in perfusion cultures. (Poster). 18 ESACT
Meeting, Animal Cell Technology Meets Genomics. May 11-14, Granada, Spain.

Elsayed EA, Piehl G-W, Nothnagel J, Medronho RA, Deckwer W-D, Wagner R. 2003. Use of
hydrocyclone as an efficient and easily scalable perfusion system for mammalian cell
bioreactors. (Vortrag). GVC/DECHEMA Vortrags- und Diskussionstagung „Zellkulturen:
Vom biologischen System zum Produktionsprozess“, May 26-28, Bad Dürkheim, Germany.

Elsayed EA, Piehl G-W, Nothnagel J, Medronho RA, Deckwer W-D, Wagner R. 2003. Use of
thhydrocyclone as an efficient tool for cell retention in perfusion cultures. (Poster). 14
National Fermentation Symposium (SINAFERM 2003). August 5-8, Florianopolis, Brazil.

iiiAcknowledgement

The following work was carried out from April 2002 to April 2005 in the Cell Culture
Technology Department, Gesellschaft für Biotechnologische Forschung GmbH (GBF),
Braunschweig, under the supervision of Prof. Dr. W.-D. Deckwer and Prof. Dr. R. Wagner.

I am greatly indebted to Prof. Dr. Deckwer for suggesting the topic of the thesis, his
constructive criticism, constant support, guidance and encouragement during the work as well
as his meticulous reading of the manuscript. My deepest gratitude and appreciation are
expressed to him.

I wish also to express my sincere gratitude to Prof. Dr. Wagner for providing the opportunity
to work in the former Cell Culture Technology group and also for his valuable instructions,
sincere help and generous support during the experimental work.

My sincere appreciation is to Prof. Dr. A.-P. Zeng for agreeing to be the second examiner.
Deepest appreciation is also to Prof. Dr. C. Müller-Goymann for agreeing to act as a chairman
of the examining committee.

This work wouldn’t have been achieved without professional support of many people.
Sincerely, I would like to thank Anja Kobold, Nadine Konisch, Christin Dangel, Gerd-Walter
Piehl, Herbert Krafft and Jürgen Nothnagel for their technical support and undeniable help
during the experimental work. Thank is also due to Joachim Hammer for amino acid analysis.
Sincere thank is to Maria Höxter for her help during the flow cytometry studies.

All the members of the former Cell Culture Technology Department are greatly appreciated
for their generous help and the friendly working atmosphere. Special thanks are due to Samira
Fargali.

Finally, I would like to acknowledge my wife Hayam, and my daughter Mariam for their
grateful support and understanding for the long nights that I had to spend in the GBF. My
parents, I wish to thank for all their support and patience.

This work was partially financed by the Deutscher Akademischer Austauschdienst (DAAD).
iv Table of Contents
Table of Contents

Symbols and Abbreviations……………………………………………………………….. ix

1. Introduction and Aim of the Work………………..……………………........................ 1

3 2. Review of Literature……………………………………………………………………..
2.1. History of animal cell culture………………………………………………………... 3
2.2. Industrial biotechnology and animal cell culture…………………………………….. 4
2.3. Bioreactors for animal cell cultivation………………………………………………. 6
2.3.1 Suspended cell bioreactors……………………………………………………... 6
2.3.2. Micorcarrier bioreactors……………………………………………………….. 7
2.4. Cultivation processes for animal cell culture………………………………………… 8
2.4.1. Batch cultivation……………………………………………………….……… 9
2.4.2. Fed-Batch cultivation……………………………………………………….…. 10
2.4.3. Continuous cultivation……………………………………………………….... 10
2.4.3.1. Chemostat culture…………………………………………………….. 11
2.4.3.2. Perfusion culture……………………………………………………… 11
2.5. Separation of mammalian cells in continuous perfusion cultivation………………… 12
2.5.1. Filtration……………………………………………………….………………. 13
2.5.2. Gravitational sedimentation…………………………………………………… 16
2.5.3. Centrifugation……………………………………………………….…………. 17
2.5.4. Ultrasonic separation……………………………………………………….….. 19
2.5.5. Dielectrophoretic separation…………………………………………………… 21
2.5.6. Hydrocyclones……………………………………………………….………… 22
2.5.6.1. Separation principle of hydrocyclones………………………………... 23
2.5.6.2. Theoretical considerations……………………………………………. 24
2.5.6.3. Separation theories of hydrocyclones………………………………… 25
2.5.6.3.1. Equilibrium orbit theory……………………………….…... 25
2.5.6.3.2. Residence time theory……………………………….……... 26
2.5.6.4. Hydrocyclone application in biological separation…………………… 27
2.5.6.5. Hydrocyclone and mammalian cell culture technology………………. 28

30 3. Materials and Methods…………………………………………………………………..
vTable of Contents
30 3.1. Materials…………………………………………………………………………......
3.1.1. Cell lines……………………………………………………………………….. 30
3.1.1.1. BHK 21pSVIL2………………………………………………………. 30
3.1.1.2. HeLa…………………………………………………………………... 30
3.1.1.3. CHO-3D6……………………………………………………………... 30
3.1.1.4. SP-2/0 AG 14…………………………………………………………. 30
3.1.1.5. CHO-ATIII…………………………………………………………… 30
3.1.2. Cultivation media……………………………………………………………… 31
3.1.2.1. SMIF-7 Medium……………………………………………………… 31
3.1.2.2. DIF-1000 Medium……………………………………………………. 31
3.1.2.3. SMIF-6 Medium……………………………………………………… 32
3.1.2.4. ZKT-I Medium……………………………………………………….. 33
3.2. Methods…………………………………………………………………………….... 33
3.2.1. Cell propagation and cultivation………………………………………………. 33
3.2.2. Cell cryo-preservation…………………………………………………………. 33
3.2.3. Cell revitalization……………………………………………………………… 34
3.2.4. Medium preparation…………………………………………………………… 34
3.2.5. Analytical methods…………………………………………………………….. 35
3.2.5.1. Sample preparation…………………………………………………… 35
3.2.5.2. Estimation of total cell concentration………………………………… 35
3.2.5.3. Estimation of viable cell concentration……………………………….. 35
3.2.5.4. Estimation of glucose and lactate concentration……………………… 35
3.2.5.5. Determination of lactate dehydrogenase activity……………………... 36
3.2.5.6. Amino acid analysis…………………………………………………... 36
3.2.5.7. Determination of antithrombin III concentration…………………….. 36
3.2.5.8. FACS-Analysis……………………………………………………….. 37
3.2.6. Bioreactor system……………………………………………………………… 38
3.2.6.1. Bioreactor inoculation and cultivation………………………………... 39
3.2.6.2. Bioreactor sampling…………………………………………………... 39
3.2.7. Hydrocyclone………………………………………………………………….. 40
3.2.7.1. Hydrocyclone geometry………………………………………………. 40
3.2.7.2. Hydrocyclone installment…………………………………………….. 41
3.2.7.3. Determination of inlet pressure drop, flow split and flow ratio of
hydrocyclone………………………………………………………….. 41
vi Table of Contents
3.2.7.4. Separation of mammalian cells using intermittent hydrocyclone
operation…………………………………………………….………... 42
3.2.7.5. Automation and control of hydrocyclone perfusion system………….. 44
3.2.7.6. Effect of continuous hydrocyclone operation on cell viability……….. 45
3.2.8. Theoretical calculations………………………………………………………... 46

4. Results and Discussion………………………………………………………………….. 49
4.1. Physical characterization of hydrocyclone operation.……………………………….. 49
4.1.1. Physical characterization of hydrocyclone operation under different operation
conditions………………………………..……………………….………….… 49
4.1.2. Effect of hydrocyclone geometry on the physical characteristics of separation. 51
4.2. Effect of different pressures generated by the hydrocyclone on cell viability and the 55
separation efficiency of the hydrocyclone....................................................................
4.2.1. BHK cells………………………………..…………………………………….. 56
4.2.2. HeLa cells………………………………..…………………………………….. 58
4.2.3. Summary of the pressure effect of the hydrocyclone system on BHK and
HeLa cells………………………..……………………………………………. 60
4.3. Effect of the continuous operation of the hydrocyclone on cell viability……………. 64
4.3.1. HeLa cells…………………………..………………………………………….. 64
4.3.2. CHO-ATIII cells....……………………………………………………………. 65
4.3.3. Summary of the effect of continuous hydrocyclone operation on cell viability… 68
4.4. Application of hydrocyclone to continuous perfusion cultivation…………………… 70
4.4.1. HeLa cells…………………………..………………………………………….. 70
4.4.2. CHO-3D6 cells....……………………………………………………………… 72
4.4.3. SP-2/0 AG 14 cells...………………………..…………………………………. 76
4.4.4. Summary of the continuous cultivations of HeLa, CHO-3D6 and SP-2/0 cells
with intermittent hydrocyclone operation...……..………….…………………. 83
4.5. Continuous cultivation of CHO-ATIII cells with intermittent hydrocyclone
operation under control of the UBICON system…………………………………… 87
4.5.1. Continuous cultivation of CHO-ATIII cells under different pressures
generated by the hydrocyclone HC 2520 system……………………………... 88
4.5.2. Continuous cultivation of CHO-ATIII cells with intermittent operation of the
hydrocyclone HC 2515 at 0.95 bar.…………………………………………… 98

viiTable of Contents
4.5.3. Comparison between the hydrocyclone perfusion and membrane perfusion
systems………………………………………………………………………… 103
4.6. Scaling up of the hydrocyclone perfusion system…………………………………… 106

5. Summary………………………………..……………………………………………….. 109

6. References………………………………..………………………………………………. 112

7. Appendix………………………………..………………………………………………... 123
viii Symbols and Abbreviations
Symbols and Abbreviations

Abbreviations
ATIII Antithrombin III
BHK Baby hamster kidney cells
CHO Chinese hamster ovary cells
Conc. Concentration
Gln. Glutamine
HC Hydrocyclone
LDH Lactate dehydrogenase
MAb Monoclonal antibody
RPM Revolution per minute

Symbols
-2b Field force intensity (m s )
d Cell diameter (m)
d Cut size (m50
D Hydrocyclone diameter (cm) c
D Inlet diameter (cm)i
D Overflow diam(cm) o
D Underflow diameter (cm)u
E Cell separation efficiency ( / )
E’ Reduced separation efficiency ( / )
E Separation efficiency of the underflow ( / ) underflow
E Separation efficiency of the overflow ( / ) overflow
-2g Gravity acceleration (m s )
L Length of cyclone (cm)
m Exponent
n
-2P Operating pressure drop (N m )
-2P Static pressure drop (N m ) s
-1Q Feed volumetric flow rate (L min )
-1Q Overflow volumetric flow rate (L min ) o
-1Q Underflow volum(L min ) u
ixSymbols and Abbreviations
r Particle radius (m)
R Fractional recovery of solids ( / )
R Flow ratio ( / ) f
T Residence time (s)
-1v Radial settling velocity (m s ) r
-1v Terminal settling velocity (m s ) t
-1v Axial settling velocity (m s ) z
V Volume of the bioreactor (L) r
V Volume of the underflow (L) u
V Volume of the overflow (L) o
x Particle or cell size (m)
-1X Cell concentration in the feed suspension (cells mL )
-1X e bioreactor after HC operation (cells mL ) after
-1X Cell concentration in the overflow (cells mL ) o
-1X Cell concentration in the underflow (cells mL ) u

Greek symbols
-3ρ Density of the liquid (kg m )
-3ρ Density of the cells (kg m ) s
η Liquid viscosity (Ps s)
-1ω Angular velocity (s )
τ Modified particle relaxation time (s)

Units
bar Pressure

d Day
h Hour L Liter
min Minute mL Milliliter
s Second µL Microliter

g Mass mm Millimeter
mg Milligram
-1µg Microgram µkat Micromol s
x