On the influence of feedstock properties and composition on process development of expanded bed adsorption [Elektronische Ressource] = Einfluss der Eigenschaften und Zusammensetzung biotechnologischer Rohlösungen auf die Prozessentwicklung im Rahmen der Fließbettadsorption / vorgelegt von Peter Jochen Brixius

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INSTITUTE OF ENZYMETECHNOLOGY PROTEIN PURIFICATION DEPARTMENTHEINRICH HEINE UNIVERSITY NOVO NORDISK A/SDÜSSELDORF/GERMANY GENNTOFTE / DENMARKOn the influence of feedstock properties and composition on processdevelopment of expanded bed adsorptionEinfluss der Eigenschaften und Zusammensetzung biotechnologischer Rohlösungen auf dieProzessentwicklung im Rahmen der FließbettadsorptionI n a u g u r a l - D i s s e r t a t i o nzurErlangung des Doktorgrades derMathematisch-Naturwissenschaftlichen FakultätDer Heinrich-Heine-Universität DüsseldorfVorgelegt vonDiplom Biologe Peter Jochen Brixiusaus Köln2003Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät derHeinrich-Heine-Universität DüsseldorfReferentin: Prof. Dr. Maria Regina KulaKorreferent: Prof. Hanns WeissTag(e) der mündlichen Prüfung: 03.06.2003DANKSAGUNGDANKSAGUNGDiese Arbeit entstand am Institut für Enzymtechnologie der Heinrich Heine UniversitätDüsseldorf im Forschungszentrum Jülich unter der Leitung von Frau Prof. Dr. Maria ReginaKula.Frau Prof. Dr. Kula gilt mein besonderer Dank für die freundliche Aufnahme in ihrem Institut,die Überlassung des Promotionsthemas sowie der stetigen Diskussionsbereitschaft undfachkundigen Unterstützung. Herr Prof. Dr. Weiss gilt mein Dank für die Übernahme desKorreferates.Ein besonderer Dank geht auch an die Firma Novo Nordisk und hier insbesondere an Frau IngerMollerup, Herr Ole E. Jensen, Herr Jesper S.
Publié le : mercredi 1 janvier 2003
Lecture(s) : 31
Source : D-NB.INFO/968548016/34
Nombre de pages : 242
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INSTITUTE OF ENZYMETECHNOLOGY PROTEIN PURIFICATION DEPARTMENT
HEINRICH HEINE UNIVERSITY NOVO NORDISK A/S
DÜSSELDORF/GERMANY GENNTOFTE / DENMARK
On the influence of feedstock properties and composition on process
development of expanded bed adsorption
Einfluss der Eigenschaften und Zusammensetzung biotechnologischer Rohlösungen auf die
Prozessentwicklung im Rahmen der Fließbettadsorption
I n a u g u r a l - D i s s e r t a t i o n
zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
Der Heinrich-Heine-Universität Düsseldorf
Vorgelegt von
Diplom Biologe Peter Jochen Brixius
aus Köln
2003Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf
Referentin: Prof. Dr. Maria Regina Kula
Korreferent: Prof. Hanns Weiss
Tag(e) der mündlichen Prüfung: 03.06.2003DANKSAGUNG
DANKSAGUNG
Diese Arbeit entstand am Institut für Enzymtechnologie der Heinrich Heine Universität
Düsseldorf im Forschungszentrum Jülich unter der Leitung von Frau Prof. Dr. Maria Regina
Kula.
Frau Prof. Dr. Kula gilt mein besonderer Dank für die freundliche Aufnahme in ihrem Institut,
die Überlassung des Promotionsthemas sowie der stetigen Diskussionsbereitschaft und
fachkundigen Unterstützung. Herr Prof. Dr. Weiss gilt mein Dank für die Übernahme des
Korreferates.
Ein besonderer Dank geht auch an die Firma Novo Nordisk und hier insbesondere an Frau Inger
Mollerup, Herr Ole E. Jensen, Herr Jesper S. Johansen, Herr Henrik Valore sowie an alle
anderen Mitarbeitern des Protein Purifikation Department sowie des hgH Projects &
Optimisation Departments für eine hervorragende Kooperation in den letzten drei Jahren sowie
für die freundlich Aufnahme und Unterstützung während meiner Arbeiten in den Labors von
Novo Nordisk Gentofte (Dänemark) und nicht zuletzt auch danke für die finanzielle und
materielle Unterstützung die diese Arbeit erst ermöglicht hat.
Ganz herzlich danken möchte ich mich auch der Arbeitsgruppe „Aufarbeitung“ unter der Leitung
von Herrn Priv.-Doz. Dr. Jörg Thömmes und seit Januar 2001 Herrn Dr. Jürgen J. Hubbuch für
eine stets angenehme und fruchtbare Arbeitsatmosphäre. Besonders erwähnt seien hier Markus
Halfar für seine praktische Unterstützung sowie Dipl. Ing. Esther Knieps-Grünhagen, Dipl. Ing.
Holger Gieren, Dr. Ute Reichert und Dr. Dong-Qiang Lin. Desweiteren möchte ich mich bei
Sina Paezold bedanken die als studentische Hilfskraft diese Arbeit tatkräftig unterstützt hat.
Außerdem gilt mein Dank allen Mitarbeitern des Instituts für Enzymtechnologie die hier noch
nicht erwähnt worden sind.1 CONTENT I
1 CONTENT
1 CONTENT .............................................................................................. I
2 FIGURES AND TABLES ...................................................................... V
3 ABSTRACT (GERMAN) ....................................................................... 1
4 SYMBOLS & ABBREVIATIONS........................................................... 4
5 INTRODUCTION................................................................................... 9
5.1 Downstream Processing 9
5.1.1 Extractive Separation.................................................................................. 12
5.1.2 Adsorptive Separations............................................................................... 12
5.1.3 Stable fluidization....................................................................................... 13
5.1.4 Chromatographic interactions..................................................................... 17
5.1.5 Ion Exchange Chromatography in Expanded Bed Adsorption................... 20
5.2 Biomass-Adsorbent Interactions ................................................................... 23
5.2.1 Quantification of biomass/adsorbent interaction ........................................ 23
5.2.2 Deep bed filtration theory – A mechanistic approach ................................ 26
5.3 Properties of biomass suspension.................................................................. 30
5.3.1 Particle size analysis ................................................................................... 30
5.3.2 Zeta Potential .............................................................................................. 32
5.4 Microbial cell wall properties........................................................................ 35
5.4.1 E. coli.......................................................................................................... 36
5.4.2 Saccharomyces cerevisiae .......................................................................... 39
5.5 Cell disruption................................................................................................. 40
5.5.1 Sonication ................................................................................................... 41CONTENT
5.5.2 Bead mill..................................................................................................... 42
5.5.3 High pressur Homogenization .................................................................... 44
5.6 Protein properities used in process design studies ...................................... 46
5.6.1 Insulin ......................................................................................................... 46
5.6.2 Human Growth Hormone (hGH)................................................................ 48
5.6.3 Formate dehydrogenase .............................................................................. 49
6 RESULTS AND DISCUSSION............................................................ 52
6.1 Feedstock properties & Biomass-Adsorbent Interactions in Expanded Bed
Adsorption....................................................................................................... 52
6.1.1 Zeta potential development of intact cells during high cell density
cultivation of Escherichia coli.................................................................................... 53
6.1.2 Impact of cell disruption on feedstock properties....................................... 57
6.1.3 Escherichia coli host strains........................................................................ 75
6.1.4 Scalability and feasibility study.................................................................. 77
6.1.5 Saccharomyces cerevisiae........................................................................... 83
6.1.6 Summary and Conclusions I....................................................................... 87
6.2 EBA & Downstream Process development .................................................. 89
6.2.1 Insulin ......................................................................................................... 89
6.2.2 Summary and Conclusions II (Insulin Precursor MI3)............................. 102
6.2.3 Human Growth Hormone (hGH)............................................................. 104
6.2.4 Anion Exchange Chromatography (EBA-mode)...................................... 105
6.2.5 Cation Exchangegraphy (EBA-mode) ..................................... 115
6.3 Aqueous two phase systems ......................................................................... 124
6.3.2 Summary and Conclusions III (hGH)....................................................... 130
7 MATERIAL & METHODS ................................................................. 132
7.1 Protein Analysis ............................................................................................ 139
7.1.1 Total Protein Determination (Bradford Assay)......................................... 139
7.1.2 SDS Page .................................................................................................. 140CONTENT
7.1.3 RP-HPLC-Analysis for Insulin Precursor MI3......................................... 140
7.1.4 FPLC-Analysis for hGH ........................................................................... 144
7.2 Protein Adsorption ....................................................................................... 149
7.2.1 Finite bath uptake experiments................................................................. 149
7.2.2 Breakthrough Analysis ............................................................................. 153
7.2.3 Modeling of Breakthrough Curves in Packed Beds.................................. 156
7.3 Biomass-Adsorbent Interactions 161
7.3.1 Pulse-Response Experiment ..................................................................... 161
7.3.2 Residence Time Distribution (RTD) (Fernández-Lahore et al., 2001)..... 163
7.4 Protein adsorption in expanded bed mode................................................. 165
7.4.1 Human insulin precursor mi 3 .................................................................. 165
7.5 Cell cultivation .............................................................................................. 166
7.5.1 Saccharomyces cerevisiae......................................................................... 166
7.5.2 E. coli........................................................................................................ 167
7.6 Cell disruption............................................................................................... 171
7.6.1 Bead Mill .................................................................................................. 171
7.6.2 French Press 172
7.6.3 Ultrasound................................................................................................. 173
7.7 Aqueous two phase system (ATPS)............................................................. 173
7.7.1 Preparation of ATPS................................................................................. 173
7.7.2 Ultrafiltration of ATPS top phases ........................................................... 173
7.8 Physical and chemical Properties of Biomass Suspension........................ 174
7.8.1 Zeta Potential ............................................................................................ 174
7.8.2 Size analysis.............................................................................................. 175
7.8.3 Viscosity ................................................................................................... 175
7.9 DNA analysis................................................................................................. 176
8 APENDIX .......................................................................................... 177CONTENT
8.1 RTD analysis ................................................................................................. 177
8.1.1 Evaluation of RTD curve:......................................................................... 177
8.1.2 Tanks in series Model ............................................................................... 179
8.1.3 Moments of RTD...................................................................................... 180
8.2 Modeling of breakthrough curves............................................................... 181
8.2.1 Transport mechanism & appropriated models.......................................... 182
8.3 Electrostatic interactions ............................................................................. 183
8.4 Particle Size Distribution Data.................................................................... 185
8.4.1 Homogenization technique ....................................................................... 185
8.4.2 Homogenization conditions in the French Press....................................... 187
8.4.3 Pilot scal cell disruption............................................................................ 194
8.4.4 Saccharomyces cerevisiae Homogenization conditions in the French Press
202
8.5 ATPS hGH..................................................................................................... 208
9 REFERENCES.................................................................................. 2092 FIGURES AND TABLES V
2 FIGURES AND TABLES
a FIGURES
FIG. 5-1: IMPACT OF THE NUMBER OF UNIT OPERATIONS EMPLOYED DURING DOWNSTREAM
PROCESSING ASSUMING A GIVEN YIELD PER STEP: 99% (); 95% (); 90% (); 85%
() (FISH AND LILLY, 1984). ................................................................................... 10
FIG. 5-2: DOWNSTREAM PROCESSING SCHEME ................................................................ 11
FIG. 5-3: PERFECTLY CLASSIFIED EXPANDED BED............................................................ 15
FIG. 5-4: CELL PULSE TRANSMISSION THROUGH AN EXPANDED BED OF STREAMLINE SP
AT PH 7.0 (FEUSER ET AL., 1999)............................................................................. 21
FIG. 5-5: CELL PULSE TRANSMISSION THROUGH AN EXPANDED BED OF STREAMLINE
DEAE (FEUSER ET AL., 1999).................................................................................. 22
FIG. 5-6: SET-UP OF A PULSE RESPONSE EXPERIMENT AND TYPICAL RESULTS OF A PULSE
RESPONSE EXPERIMENT WITH (CTI = 0.22) AND WITHOUT (CTI =1.0)
BIOMASS/ABSORBER INTERACTION. ......................................................................... 24
FIG. 5-7: GRAPHICAL DESCRIPTION OF THE PDE MODEL. (N: MASS TRANSFER BETWEEN
ZONES, PE: OVER ALL AXIAL DISPERSION, α: FRACTION OF LIQUID IN PLUG FLOW).26
FIG. 5-8: CORRESPONDING SPHERES, USING DIFFERENT TECHNIQUES FOR PARTICLE SIZE
DETERMINATION (HTTP://WWW.MALVERN.DE)......................................................... 31
FIG. 5-9: DIFFERENT DISPERSION CONDITIONS OF A COLLOID SYSTEM
(HTTP://WWW.MALVERN.DE).................................................................................... 32
FIG. 5-10: CHARGE DISTRIBUTION IN THE STERN DOUBLE LAYER MODEL ................... 33
FIG. 5-11: COMPARISON BETWEEN GRAM POS. (RIGHT) AND GRAM NEG. (LEFT) CELL
WALL (PAUSTIAN, 2001).......................................................................................... 36
FIG. 5-12: LIPOPOLYSACCARIDE (NIKAIDO, 1996). ......................................................... 37
FIG. 5-13: MAJOR PHOSPHOLIPIDS IN THE CYTOPLASMIC MEMBRANE OF ESCHERICHIA COLI
70-80 % PHOSPHATIDYLETHANOLAMINE, 15-25 % PHOSPHATIDYLGLYCEROL, 5-10
% CARDIOLIPIN. R : SATURATED FATTY ACID, R : UNSATURATED FATTY ACID1 2
(KADNER, 1996)...................................................................................................... 38
FIG. 5-14: RELATIONSHIPS AMONG COMPONENTS OF SACCHAROMYCES CEREVISIAE CELL
WALLS. (A) PROTOTYPICAL MODULE WITH COMPONENTS INDIVIDUALLY LABELED2 FIGURES AND TABLES VI
AND COLORED. THE MANNOPROTEIN POLYPEPTIDE IS BLUE, AND OLIGOSACCHARIDES
ARE SHOWN IN YELLOW, LABELED AS N OR O LINKED. ONLY A FEW OF THE BRANCH
POINTS OF THE GLUCANS ARE SHOWN. CHITIN CAN ALSO BE LINKED TO THE B1,6
GLUCAN. (B) ASSOCIATION OF MODULES TO FORM A WALL LATTICE. COLORS ARE AS
IN PANEL A. THE B1,3 GLUCAN CHAINS ARE INTERTWINED TO DESIGNATE TRIPLE
HELICES, AND CHITIN IS SHOWN AS A CRYSTALLINE MICRO-DOMAIN. CROSS-LINKING
OF MANNOPROTEINS THROUGH DISULFIDE AND OTHER BONDS IS NOT DEPICTED
(LIPKE AND OVALLE, 1998)..................................................................................... 40
FIG. 5-15: CELL DISRUPTION METHODS ........................................................................... 41
FIG. 5-16: BEAD MILL WITH HORIZONTAL GRINDING CHAMBER SYSTEM TRINEX®
(NETZSCH-FEINMAHLTECHNIK, GERMANY) ......................................................... 42
FIG. 5-17: DETAILS OF VALVE SEAT OF APV-GAULIN HIGH-PRESSURE HOMOGENIZER... 44
FIG. 5-18: ENZYMATIC PROCESSING OF INSULIN (STRYER, 1994).................................... 46
FIG. 5-19: COMPARISON OF PIG, BOVINE AND HUMAN INSULIN........................................ 47
FIG. 5-20: PRIMARY STRUCTURE OF HGH........................................................................ 48
FIG. 5-21: COFACTOR REGENERATION USING FDH.......................................................... 49
FIG. 5-22: ESCHERICHIA COLI HOMOGENATE TRANSMISSION, MEASURED BY THE PULSE-
RESPONSE EXPERIMENT FOR DIFFERENT ADSORBENTS AT PH 7,5 AND 5 MS/CM
CONDUCTIVITY (1M AMMONIUM SULPHATE ADDED FOR ADSORPTION TO
STREAMLINE PHENYL) (REICHERT ET AL., 2001) ..................................................... 51
FIG. 6-1: SURFACE CHARGE OF AN REC. L-PHE PRODUCTION ESCHERICHIA COLI STRAIN
(CELLS RECEIVED FROM NICOLE RÜFFER IBT II OF THE RESEARCH CENTRE JÜLICH,
GERMANY) DURING A FED BATCH CULTIVATION MEASURED IN 10 MM
-1NA HPO /CITRIC ACID BUFFER AT A CONSTANT CONDUCTIVITY κ= 5 MS CM2 4
ADJUSTED WITH SOLID NACL AND VARIOUS PH. AT ( ■) PH 2, ( ●) PH 3, ( ▲) PH 7 55
FIG. 6-2: SURFACE CHARGE OF ESCHERICHIA COLI JM 101 CELLS DURING A FED BATCH
CULTIVATION MEASURED IN 10 MM NA HPO /CITRIC ACID BUFFER AT A CONSTANT2 4
-1CONDUCTIVITY κ= 5 MS CM ADJUSTED WITH SOLID NACL AND VARIOUS PH. AT ( ■)
2+ -1
PH 2, ( ●) PH 3, ( ▲) PH 7. (C = 14 G L IN THE FEED MEDIA).......................... 55MG
FIG. 6-3: SURFACE CHARGE OF ESCHERICHIA COLI JM 105 CELLS DURING A FED BATCH
CULTIVATION MEASURED IN 10 MM NA HPO /CITRIC ACID BUFFER AT A CONSTANT2 42 FIGURES AND TABLES VII
-1CONDUCTIVITY κ= 5 MS CM ADJUSTED WITH SOLID NACL AND VARIOUS PH. ( ■) PH
2+ -12, ( ●) PH 3, ( ▲) PH 7. (C = 10G L IN THE FEED MEDIA)................................. 56MG
FIG. 6-4: SURFACE CHARGE OF ESCHERICHIA COLI JM 105 REC FDH CELLS DURING A FED
BATCH CULTIVATION MEASURED IN 10 MM NA HPO /CITRIC ACID BUFFER AT A2 4
-1CONSTANT CONDUCTIVITY κ= 5 MS CM ADJUSTED WITH SOLID NACL AND VARIOUS
2+ -1
PH. AT ( ■) PH 2, ( ●) PH 3, ( ▲) PH 7. (C = 10 G L MGSO IN THE FEEDMG 4
MEDIA)..................................................................................................................... 56
FIG. 6-5: VISCOSITY OF 40 % E: COLI HOMOGENATE OBTAINED FROM DIFFERENT CELL
DISRUPTION METHODS (USING A BIOMASS SUSPENSION OF C = 40 % WW) ............. 58B
FIG. 6-6: 0,8 % AGAROSE GEL ANALYSIS OF HOMOGENATES OBTAINED FROM VARIOUS
CELL DISRUPTION METHODS..................................................................................... 59
FIG. 6-7: VISCOSITY OF ESCHERICHIA COLI BIOMASS SUSPENSION (C = 40% WW) DURINGB
MULTIPLE PASSAGE HOMOGENIZATION IN A FRENCH PRESS AT TWO DIFFERENT
OPERATING PRESSURES ( ▼) 482 BAR AND () 965 BAR. ......................................... 60
FIG. 6-8: 1,5 % AGAROSE GEL ANALYSIS ETHIDIUMBROMIDE STAINED FRENCH PRESS
HOMOGENATE AT DIFFERENT HOMOGENIZATION CYCLES AND A OPERATION
PRESSURE OF 965 BAR.............................................................................................. 60
FIG. 6-9: CTI USING BEAD MILL HOMOGENATE WITH & WITHOUT BENZONASE™
TREATMENT (ESCHERICHIA COLI JM105). ................................................................ 61
FIG. 6-10: AVERAGE SIZE DISTRIBUTION OBTAINED BY DIFFERENT CELL DISRUPTION
METHODS USING 40 % WW ESCHERICHIA COLI JM 101, SUSPENSION....................... 63
FIG. 6-11: SURFACE CHARGE OF CELL DEBRIS OBTAINED BY DIFFERENT CELL DISRUPTION
METHODS. USING 40 % WW ESCHERICHIA COLI JM 101. MEASUREMENT IN 100 MM
-1TRIS/HCL PH 8.0 κ = 10 MS CM ............................................................................ 63
FIG. 6-12: AVERAGE SIZE DISTRIBUTION OF ESCHERICHIA COLI HOMOGENATE TREATED
WITH DIFFERENT PRESSURE AND CYCLES IN THE FRENCH PRESS () 965 BAR, ( ●) 482
BAR.......................................................................................................................... 65
FIG. 6-13: SURFACE CHARGE OF ESCHERICHIA COLI HOMOGENATE TREATED WITH
DIFFERENT PRESSURE AND CYCLES IN THE FRENCH PRESS. () 965 BAR, ( ●) 482 BAR.
................................................................................................................................65
FIG. 6-14: IMPACT OF PARTICLE SIZE ON THE SURFACE CHARGE OF ESCHERICHIA COLI
CELL DEBRIS............................................................................................................. 66

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