mass transfer, mixing, chinese hamster ovary cell growth and antibody production characterization

135 Pages·2009·1.38 MB·English

by  Sandeepa Sandadi

MASS TRANSFER, MIXING, CHINESE HAMSTER OVARY CELL GROWTH AND
ANTIBODY PRODUCTION CHARACTERIZATION USING RUSHTON TURBINE
AND MARINE IMPELLERS

By
SANDEEPA SANDADI
A Dissertation submitted to the
Graduate School-New Brunswick
Rutgers, The State University of New Jersey
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Graduate Program in Chemical and Biochemical Engineering
written under the direction of
Professor Henrik Pedersen
and approved by
________________________
________________________
________________________
________________________
New Brunswick, New Jersey
October 2009
ABSTRACT OF THE DISSERTATION

MASS TRANSFER, MIXING, CHINESE HAMSTER OVARY CELL GROWTH AND
ANTIBODY PRODUCTION CHARACTERIZATION USING RUSHTON TURBINE
AND MARINE IMPELLERS

By SANDEEPA SANDADI

Dissertation Director:
Professor Henrik Pedersen

Large scale production of monoclonal antibodies has been carried out over the
years using bioreactors with different length to diameter ratios, and diverse impeller and
sparger designs. The differences in these physical attributes often result in dissimilar
mass transfer, shear dynamics and mixing inside the bioreactor, that lead to disparities in
cell growth and antibody production. A rational analysis of impeller design parameters on
cell growth, protein expression levels and subsequent antibody production is needed to
understand such differences and has not been investigated in depth before. The purpose
of this study was to examine the impact of Rushton turbine and marine impeller designs
on Chinese hamster ovary (CHO) cell growth and metabolism, and antibody production
and quality. Experiments to evaluate the mass transfer and mixing characteristics were
conducted to determine if the nutrient requirements of the culture would be met. The
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analysis of mixing times indicated significant differences between marine and Rushton
turbine impellers at the same power input per unit volume of liquid (P/V). However, no
significant differences were observed between the two impellers at constant P/V with
respect to oxygen and carbon dioxide mass transfer properties. Experiments were
conducted with CHO cells to determine the impact of different flow patterns arising from
the use of different impellers on cell growth, metabolism and antibody production. The
analysis of cell culture data did not indicate any significant differences in any of the
measured or calculated variables between marine and Rushton turbine impellers. A
deterministic model was constructed to describe culture behavior under the different flow
patterns and the parameters were solved for both marine and Rushton turbine impellers.
This work was intended to bridge the gap in understanding the relationship between
impellers with different flow patterns and their effect on CHO cells and productivity.
Literature in this area has been inadequate and therefore this work is a significant
advancement in this area. More importantly, this study was able to demonstrate that the
quality of the antibody was not altered with a change in the impeller geometry, which is
of critical importance to the biopharmaceutical industry today.
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Acknowledgement

I wish to express my sincere gratitude to my research advisor Prof. Henrik
Pedersen for his guidance and support during the course of this work. I am grateful to
Schering-Plough for giving me the opportunity to conduct my research. I am indebted to
Dennis Rendeiro and Semsi Ensari for their extraordinary support and encouragement
throughout this work. I would like to acknowledge my colleagues from Schering-Plough
for their support during the course of my research, in particular John Bowers, Mark
Wittman, Nicholas Abaroa, Cesar Cornejo, and the Cell Culture Pilot Plant personnel.
I could not have progressed this far without the unwavering encouragement from
my parents, Hema and Jagadeshwar Reddy Sandadi who stand by me through all the
peaks, trials and tribulations that life has to offer. Their unconditional love and support
inspire me to aspire and achieve new heights every day of my life. I wish to thank my
brother, Sirish Sandadi, and the rest of my family and friends for all their love and
affection, which I absolutely cannot do without.

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Table of Contents

ABSTRACT OF THE DISSERTATION...........................................................................ii
Acknowledgement.............................................................................................................iv
Table of Contents................................................................................................................v
List of tables.....................................................................................................................viii
List of illustrations.............................................................................................................ix
1. Introduction.....................................................................................................................1
1.1 Mammalian Cell Culture...........................................................................................3
1.2 Mixing Characteristics...............................................................................................5
1.3 Mass Transfer Characteristics...................................................................................7
1.4 Mathematical Models for Antibody Production........................................................9
1.5 Antibody Purification and Quality..........................................................................11
2. Objectives.....................................................................................................................20
2.1 Objective 1...............................................................................................................20
2.2 Objective 2...............................................................................................................21
2.3 Objective 3...............................................................................................................21
3. Mixing and Mass Transfer Characterization.................................................................22
3.1 Materials and Methods............................................................................................22
3.1.1 Bioreactor set-up...............................................................................................22
3.1.2 Mixing time determination................................................................................23
3.1.3 Mass transfer coefficients determination..........................................................24
3.2 Results.....................................................................................................................25
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3.2.1 Mixing time determination................................................................................25
3.2.2 Mass transfer coefficients determination..........................................................26
3.3 Conclusions.............................................................................................................30
4. Cell culture and Antibody Quality................................................................................43
4.1 Materials and Methods............................................................................................43
4.1.1 Cell culture experiments....................................................................................43
4.1.2 Antibody quality assessment.............................................................................44
4.2 Results.....................................................................................................................46
4.2.1 Cell culture experiments....................................................................................46
4.2.2 Antibody quality assessment.............................................................................49
4.3 Conclusions.............................................................................................................51
5. Mathematical Model for Antibody Production.............................................................69
5.1 Theory......................................................................................................................69
5.1.1 Unstructured kinetic model...............................................................................69
5.1.2 Structured kinetic model for antibody production............................................73
5.1.2 Sensitivity analysis............................................................................................75
5.2 Results.....................................................................................................................76
5.2.1 Model simulation...............................................................................................77
5.2.1 Sensitivity analysis............................................................................................78
5.3 Conclusions.............................................................................................................80
6. Conclusions.................................................................................................................101
7. Nomenclature..............................................................................................................108
8. Bibliography...............................................................................................................113
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Appendix.........................................................................................................................119
Curriculum Vita..............................................................................................................122

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List of tables

Table 3.1: Dimensions of bioreactors used in the study...................................................32
Table 3.2: Relationship between mixing times (sec) and P/V (W/L) for marine and
Rushton turbine impellers obtained from the log linear regression fits to the data...33
Table 3.3: Results obtained for the mass transfer characterization of the reactors for both
marine and Rushton turbine impellers.......................................................................34
Table 3.4: Analysis of Variance for the mass transfer coefficients calculated for oxygen
and carbon dioxide gases as a function of the variables tested..................................35
Table 3.5: Parameter estimates for the mass transfer coefficients for oxygen and carbon
dioxide gases..............................................................................................................36
Table 4.1: Description of the operating conditions used for cell culture experiments.....54
Table 4.2: Estimation of parameters for cell culture experiments using marine and
Rushton turbine impellers..........................................................................................55
Table 4.3: Size-exclusion chromatography results...........................................................56
Table 4.4: Ion-exchange chromatography results.............................................................57
Table 5.1: Comparison of parameter predictions with Berkeley Madonna software for
values reported in the literature [36]..........................................................................81
Table 5.2: Initial conditions used to solve the model equations.......................................82
Table 5.3: Parameter estimates for marine and Rushton turbine impellers......................83
Table 5.4: Average sensitivity indices for all parameters for antibody production..........84

viii
List of illustrations
Figure 1.1: Axial and radial flow patterns in the bioreactors [6]......................................15
Figure 1.2: Schematic diagram of steps involved in transport of oxygen from a gas bubble
to inside a cell [28].....................................................................................................16
Figure 1.3: Classification of models for prokaryotic and eukaryotic cell culture systems
[39].............................................................................................................................17
Figure 1.4: Schematic representation of the hybrid model, showing both unstructured and
structured elements [36].............................................................................................18
Figure 1.5: Structure of monoclonal antibody and antibody-antigen interaction.............19
Figure 3.1: Schematic representation of Biostat C-DCU 20L bioreactor.........................37
Figure 3.2: Pictorial representation of the bioreactors used in the study..........................38
Figure 3.3: Bioreactor set-up for the mixing time measurement experiments.................39
Figure 3.4: Relationship between mixing times and power per unit volume for marine and
Rushton turbine impellers..........................................................................................40
Figure 3.5: Linear regression fits for the mass transfer coefficient for oxygen gas with
sparge rate of gases....................................................................................................41
Figure 3.6: Linear regression fits for the mass transfer coefficient for carbon dioxide gas
with sparge rate of gases............................................................................................42
Figure 4.1: Results for viable cell density using marine and Rushton turbine impellers. 58
Figure 4.2: Results for total cell density using marine and Rushton turbine impellers....59
Figure 4.3: Results for dead cell density using marine and Rushton turbine impellers....60
Figure 4.4: Results for final antibody concentration with marine and Rushton turbine
impellers.....................................................................................................................61
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Figure 4.5: Glucose profiles for the cell culture experiments with marine and Rushton
turbine impellers.........................................................................................................62
Figure 4.6: L-glutamine profiles for the cell culture experiments with marine and Rushton
turbine impellers.........................................................................................................63
Figure 4.7: Lactate profiles for the cell culture experiments with marine and Rushton
turbine impellers.........................................................................................................64
Figure 4.8: Ammonia profiles for the cell culture experiments with marine and Rushton
turbine impellers.........................................................................................................65
Figure 4.9: Dependence of specific antibody production rate (q ) on specific growth rate
Ab
(µ) of the cells............................................................................................................66
Figure 4.10: Dependence of specific lactate production rate (q ) on specific growth rate
L
(µ) of the cells............................................................................................................67
Figure 4.11: Dependence of specific ammonia production rate (q ) on specific growth
N
rate (µ) of the cells.....................................................................................................68
Figure 5.1: Experimental and predicted values for viable cell density with the use of
marine impellers.........................................................................................................85
Figure 5.2: Experimental and predicted values for total cell density with the use of marine
impellers.....................................................................................................................86
Figure 5.3: Experimental and predicted values for dead cell density with the use of
marine impellers.........................................................................................................87
Figure 5.4: Experimental and predicted values for antibody concentration with the use of
marine impellers.........................................................................................................88
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