Académique Documents
Professionnel Documents
Culture Documents
8 June 2013
6/2013
Biofuels
Bioreactor design
Antibody purification
Biochemical
engineering
sciences
www.biotechnology-journal.com
http://dx.doi.org/10.1002/biot.201200373
Review
Host cell protein analysis in therapeutic protein
bioprocessing methods and applications
Anne Luise Tscheliessnig, Julita Konrath, Ron Bates
and Alois Jungbauer
http://dx.doi.org/10.1002/biot.201200328
Review
Large-scale production of diesel-like biofuels process design
as an inherent part of microorganism development
Maria C. Cuellar, Joseph J. Heijnen
and Luuk A.M. van der Wielen
http://dx.doi.org/10.1002/biot.201200319
Research Article
Acoustic detection of cell adhesion to a coated quartz crystal
microbalance implications for studying the biocompatibility
of polymers
Ana-Carina Da-Silva, Sandra S. Soares
and Guilherme N. M. Ferreira
Research Article
Harnessing Candida tenuis and Pichia stipitis in whole-cell
bioreductions of o-chloroacetophenone: Stereoselectivity,
cell activity, in situ substrate supply and product removal
Christoph Gruber, Stefan Krahulec, Bernd Nidetzky
and Regina Kratzer
http://dx.doi.org/10.1002/biot.201200322
Research Article
Stimuli-Responsive magnetic nanoparticles for monoclonal
antibody purification
Lus Borlido, Leila Moura, Ana M. Azevedo, Ana C. A.
Roque, Maria R. Aires-Barros and Jos P. S. Farinha
http://dx.doi.org/10.1002/biot.201200329
Regular Articles
Research Article
Organic co-solvents affect activity, stability and
enantioselectivity of haloalkane dehalogenases
Veronika Stepankova, Jiri Damborsky
and Radka Chaloupkova
http://dx.doi.org/10.1002/biot.201200378
Technical Report
Rapid screening of potential autophagic inductor agents
using mammalian cell lines
Waleska K. Martins, Divinomar Severino,
Cleidiane Souza, Beatriz S. Stolf and Maurcio S. Baptista
http://dx.doi.org/10.1002/biot.201200306
Research Article
Designing a fully automated multi-bioreactor plant for fast
DoE optimization of pharmaceutical protein production
Jens Fricke, Kristof Pohlmann, Nils A. Jonescheit,
Andree Ellert, Burkhard Joksch and Reiner Luttmann
http://dx.doi.org/10.1002/biot.201200190
http://dx.doi.org/10.1002/biot.201200320
www.biotechnology-journal.com
Biotechnology
Journal
DOI 10.1002/biot.201200190
Research Article
The identification of optimal expression conditions for state-of-the-art production of pharmaceutical proteins is a very time-consuming and expensive process. In this report a method for rapid
and reproducible optimization of protein expression in an in-house designed small-scale
BIOSTAT multi-bioreactor plant is described. A newly developed BioPAT MFCS/win Design of
Experiments (DoE) module (Sartorius Stedim Systems, Germany) connects the process control
system MFCS/win and the DoE software MODDE (Umetrics AB, Sweden) and enables therefore
the implementation of fully automated optimization procedures. As a proof of concept, a commercial Pichia pastoris strain KM71H has been transformed for the expression of potential malaria
vaccines. This approach has allowed a doubling of intact protein secretion productivity due to the
DoE optimization procedure compared to initial cultivation results. In a next step, robustness
regarding the sensitivity to process parameter variability has been proven around the determined
optimum. Thereby, a pharmaceutical production process that is significantly improved within
seven 24-hour cultivation cycles was established. Specifically, regarding the regulatory demands
pointed out in the process analytical technology (PAT) initiative of the United States Food and
Drug Administration (FDA), the combination of a highly instrumented, fully automated multibioreactor platform with proper cultivation strategies and extended DoE software solutions opens
up promising benefits and opportunities for pharmaceutical protein production.
Received
Revised
Accepted
Accepted
article online
07 MAY 2012
10 JAN 2013
26 FEB 2013
28 FEB 2013
Keywords: BioPAT MFCS/win Design of Experiments (DOE) Fully automated multi-bioreactor plant Pichia pastoris Sequential/parallel DoE cultivations
1 Introduction
The application of Quality by Design (QbD) has been
receiving more and more attention in the pharmaceutical
community. QbD requires a thorough understanding of its
manufacturing process, requiring an upfront investment
in time and resources for the development of a product [1].
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is linked to the DoE software MODDE (Umetrics, Sweden) for an automatic setup of designed experiments as
well as a fast and reliable data analysis. These tools support the concept of a fully automated sequential/parallel
DoE cultivation strategy, which was performed in a cellbreeding BIOSTAT Bplus and a sixfold screening BIOSTAT Qplus reactor system [12]. The plant was set up
with an industrial conformed sterile design including
sterilizable transfer valves (GEM GmbH & Co. KG, Germany) and quick connectors (Stubli Tec-Systems GmbH,
Germany).
Using a 5-L cell-breeding bioreactor, the same inoculation conditions for six 1-L screening bioreactors were
provided in a cyclic manner. The need of preparing inoculation material cyclically within a 24-h timeframe has
been fulfilled by a fully automated process. This was
achieved despite the requirement of switching the carbon source from glycerol for cell breeding to methanol for
induction.
Different approaches for PAT applications were implemented in terms of product quality. For simultaneous
quantification of secreted recombinant proteins, the plant
was equipped with an at-line reversed phase (RP)-HPLC
[12] and an at-line sequential injection analysis (SIA) with
Immobilized Metal Affinity Chromatography (IMAC)Bead Injection [15]. Quality assessment and reproducibility of breeding cycles was performed via off-line measurement of cell-internal alcohol oxidase (AOX) content.
The instrumentation allows direct scale-up of the
developed cultivation methods into pilot scale, as well as
small scale commercial production processes.
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Figure 1. Set up of multi-bioreactor plant for a fully automated sequential/parallel cultivation strategy. Schematically shown are cell-breeding (B+) and
screening (Q+) reactors as well as piping with valves and sterilizable quick connectors.
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(1)
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Optimization
Run k
cS2Mk (g/L)
Lk (C)
pHk ()
cXLk0 (g/L)
IAP1Mk0 (AUs)
PRDk (mAUs/h)
0.4
1.0
0.4
1.0
0.4
1.0
0.4
1.0
0.2
1.2
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
27
27
33
33
27
27
33
33
30
30
25
35
30
30
30
30
30
30
4.8
4.8
4.8
4.8
5.6
5.6
5.6
5.6
5.2
5.2
5.2
5.2
4.5
5.9
5.2
5.2
5.2
5.2
18.55
16.35
15.65
17.05
15.55
17.45
17.45
16.95
17.25
17.55
17.00
15.75
16.65
22.10
16.60
15.85
15.75
18.40
0.1263
0.0455
0.1685
0.1050
0.1655
0.1220
0.1556
0.1485
0.0565
0.1090
0.1232
0.1438
0.0434
0.1634
0.1151
0.1242
0.1217
0.1202
10.35
7.68
0.41
1.19
3.94
20.07
9.20
1.58
5.74
4.88
11.91
0.50
2.21
5.86
13.81
***a)
10.68
7.33
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Robustness testing
Run k
1
2
3
4
5
6
7
8
pHk ()
Lk (C)
cXLk0 (g/L)
IAP1Mk0 (AUs)
PRDk (mAUs/h)
5.55
5.40
5.55
5.70
5.40
5.70
5.55
5.55
25.5
26.5
25.5
24.5
24.5
26.5
25.5
25.5
17.90
19.20
19.20
19.80
18.35
18.00
19.55
19.40
0.351
0.339
0.347
0.322
0.297
0.306
0.278
0.271
17.54
12.25
15.56
13.99
11.15
16.93
12.74
14.54
a) *** No result.
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i =1
3
i =1
y = a0 + ai x i + aii x i2
+ aij x i x j + a123 x1 x 2 x 3,
(2)
1 i< j
Figure 3. S88 program structure of the DoE recipe. Shown are the recipe operations, a section of the SFC with different phases and the content of the
MFCSDOE_Factors phase.
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The developed multi-bioreactor plant met the requirement of reproducibility in cell breeding and ensures stable initial conditions in the screening cultivations, which
is achieved by implementing complex automation structures. By using linked DoE tools and extended PAT, investigations regarding process optimization in pharmaceutical protein production become more manageable and reliable.
SSCP ntot 1
,
SST nCP 1
(3)
could be calculated to 0.92 with the Sum of Squares Center Points (SSCP), the SST, the number of experiments ntot
and the number of independent Center Point experiments
nCP.
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4 Concluding remarks
In this report a fast optimization of malaria vaccine
expression with Pichia pastoris via Design of Experiments
in a multi-bioreactor plant is presented. The whole DoE
procedure comprised 38 experiments, consisting of
screening [12], optimization, and robustness testing, and
was conducted in 7 sequential/parallel approaches.
The developed multi-bioreactor plant equipped with
sterilizable piping, transfer valves and quick connectors is
based on industrial oriented sterile design and ensures
Figure 5. Response surface plot of the DoE optimization results as a function of cultivation temperature L and pH-value at a methanol concentration cS2M of 1.0 g/L. Model data was fitted to the data determined experimental by using MLR. A p-value for regression of < 0.001 claims high
model significance. The model shows no lack of fit.
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5 Nomenclature
Variables (unit)
AP1M
UV absorption of target protein P1 in media
phase M (AU)
cIK
mass concentration of component I in subsystem K (g/L)
FK
flow rate in or out of subsystem K (L/h)
IAP1M
integral of UV absorption of target protein P1
in media phase M (AUs)
mi
mass of component i (g)
NSt
stirrer agitation speed (rpm)
pO2
dissolved oxygen tension (%)
PRD
target protein secretion productivity (AUs/h)
qI/X
cell-specific reaction rate of component I
(g/(gh))
t
cultivation time (h)
VK
volume of subsystem K (L)
K
temperature in subsystem K (C)
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at-line
carbon dioxide
gaseous phase
sample index
inlet
first sample in screening run k
final sample in screening run k
liquid (media and cell) phase
media phase
maximum
Media component i
O, O2
opt
P1
R1
R2
R3
S1
S2
T1
T2
w
X
Z
oxygen
optimal
target protein
glycerol reservoir
methanol reservoir
refresh medium reservoir
substrate glycerol
substrate methanol
titration (acid)
titration (base)
set point
bio dry mass
bio wet mass
6 References
[1] Rathore, A. S., Winkle, H., Quality by design for biopharmaceuticals.
Nat. Biotechnol. 2009, 27, 2634.
[2] Glassey, J., Gernaey, K. V., Clemens, C., Schulz, T. W. et al., Process
analytical technology (PAT) for biopharmaceuticals. Biotechnol. J.
2011, 6, 369377.
[3] Gnoth, S., Jenzsch, M., Simutis, R., Lubbert, A., Control of cultivation processes for recombinant protein production: A review. Bioprocess Biosyst. Eng. 2008, 31, 2139.
[4] Rathore, A. S., Bhambure, R. Ghare, V., Process analytical technology (PAT) for biopharmaceutical products. Anal. Bioanal. Chem. 2010,
398, 137154.
[5] United States Federal Food and Drug Administration (USA), Guidance for industry, PAT A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance FDA,
2004.
[6] Mandenius, C. F., Brundin, A., Bioprocess optimization using
Design-of-Experiments methodology. Biotechnol. Prog. 2008, 24,
11911203.
[7] Pritchett, J., Baldwin, S. A., The effect of nitrogen source on yield
and glycosylation of a human cystatin C mutant expressed in Pichia
pastoris. J. Ind. Microbiol. Biotechnol. 2004, 31, 553558.
[8] Berdichevskya, M., dAnjoub, M., Mallemb, M. R., Shaikhb, S. S. et
al., Improved production of monoclonal antibodies through oxygenlimited cultivation of glycoengineered yeast. J. Biotechnol. 2011,
155, 217224.
[9] Jafari, R., Sundstrm, B. E., Holm, P., Optimization of production of
the anti-keratin 8 single-chain Fv TS1-218 in Pichia pastoris using
design of experiments. Microb. Cell Fact. 2011, 10, 34.
[10] Holmes, W. J., Darby, R. A. J., Wilks, M. D. B., Smith, R. et al., Developing a scalable model of recombinant protein yield from Pichia pas-
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24] Egli, T., van Dijken, J. P., Veenhuis, M., Harder, W. et al., Methanol
metabolism in yeasts: Regulation of the synthesis of catabolic
enzymes, Arch. Microbiol. 1980, 124, 115121.
[25] Files, D., Ogawa, M., Scaman, C. H., Baldwin, S. A., A Pichia pastoris
fermentation process for producing high-levels of recombinant
human cystacin-C. Enzyme Microb. Technol. 2001, 29, 335340.
[26] Clare, J. J., Rayment, F. B., Ballantyne, S. P., Sreerkrishna, K. et al.,
High-level expression of tetanus toxin fragment C in Pichia pastoris
strains containing multiple tandem integrations of the gene.
BioTechnology 1991, 9, 455460.
[27] Gasser, B., Maurer, M., Rautio, J., Sauer. M. et al., Monitoring of transcriptional regulation in Pichia pastoris under protein production
conditions. BMC Genomics 2007, 8, 179.
[28] Jahic, M., Gustavsson, M., Jansen, A. K., Martinelle, M. et al., Analysis and control of proteolysis of a fusion protein in Pichia pastoris fedbatch processes. J. Biotechnol. 2003, 102, 4553.
[29] Li, Z., Xiong, F., Lin, Q., dAnjou, M. et al., Low temperature increases the yield of biologically active herring antifreeze protein in Pichia
pastoris. Protein Exp. Purif. 2001, 21, 438445.
[30] Cregg, J. M., Cereghino, L., Shi, J., Higgins, D. R., Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 2000, 16, 2352.
[31] Dragosits, M., Stadlmann, J., Albiol, J., Baumann, K. et al., The effect
of temperature on the proteome of recombinant Pichia pastoris. J.
Proteome Res. 2009, 8, 13801392.
[32] Cos, O., Ramon, R., Montesinos, J. L., Valero, F., Operational strategies, monitoring and control of heterologous protein production in
the methylotrophic yeast Pichia pastoris under different promoters:
A review. Microbial Cell Factories 2006b, 5, 17.
[33] Macauley-Patrick, S., Fazenda, M. L., McNeil, B., Harvey, L. M., Heterologous protein production using the Pichia pastoris expression
system. Yeast 2005, 22, 249270.
[34] Soyaslan, E. S., Calik, P., Enhanced recombinant human erythropoietin production by Pichia pastoris in methanol fed-batch/sorbitol
batch fermentation through pH optimization. Biochem. Eng. J. 2011,
55, 5965.
[35] Batra, G., Gurramkonda, C., Nemani, S. K., Jain, S. K. et al., Optimization of conditions for secretion of dengue virus type 2 envelope
domain III using Pichia pastoris. J. Biosci. Bioeng. 2010, 110,
408414.
[36] Kobayashi, K., Kuwae, S., Ohya, T., Ohda, T. et al., High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. J. Biosci. Biotechnol. 2000, 89, 5561.
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