Designing A Fully Automated Multi-Bioreactor

ISSN 1860-6768 BJIOAM 8 (6) 633752 (2013) Vol.
8 June 2013
Systems & Synthetic Biology

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Bioreactor design for clinical-grade expansion of stem cells
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Christoph Gruber, Stefan Krahulec, Bernd Nidetzky
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Rapid screening of potential autophagic inductor agents
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Waleska K. Martins, Divinomar Severino,
Cleidiane Souza, Beatriz S. Stolf and Maurcio S. Baptista
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
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnology
Journal
DOI 10.1002/biot.201200190
Biotechnol. J. 2013, 8, 738747
Research Article
Designing a fully automated multi-bioreactor plant for

fast DoE optimization of pharmaceutical protein production
Jens Fricke1, Kristof Pohlmann1, Nils A. Jonescheit1, Andree Ellert2, Burkhard Joksch2 and Reiner Luttmann1
1 HAW-Hamburg
2 Sartorius
University of Applied Sciences, Hamburg, Germany

Stedim Biotech GmbH, Gttingen, Germany
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].
Correspondence: Prof. Reiner Luttmann, Hamburg University of Applied

Sciences, Research Center of Bioprocess Engineering and Analytical
Techniques, Lohbruegger Kirchstr. 65, D-21033 Hamburg, Germany
E-mail: reiner.luttmann@haw-hamburg.de
Abbreviations: AOX, alcohol oxidase; DoE, Design of Experiment; FDA,
United States Food and Drug Administration; MLR, multiple linear regression; PAT, process analytical technology; QbD, Quality by Design; RP,
reversed phase; SST, sum of squares total
738
A basic part of QbD is to create a process design space

(International Conference of Harmonisation (2009) ICH
Harmonised Tripartite Guideline: Q8(R2) Pharmaceutical
Development). The design space is defined by the key
and the critical process parameters identified from
process characterization studies. These parameters are
the primary focus for in-line, on-line or at-line process
analytical technology (PAT) applications [1].
Based on the ICH guidance documents, the United
States Food and Drug Administration (FDA) emphasizes
the need for improved on-line monitoring and control
methods to maintain high product quality during manufacturing operations [2]. According to Gnoth et al. [3] the
essence of the PAT initiative is primarily to monitor
process variables in order to identify critical quality attributes (CQAs). Rathore et al. [4] discussed the subject more
precisely and recommended the use of PAT in the aspects
Biotechnol. J. 2013, 8, 738747

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of process understanding, improved yield, reduction in

the production cycle time, decrease in the energy consumption, cost reduction, and real-time release of the
batches. Another aspect for improvement in product
quality, safety, and/or efficiency in the guidance of the
FDA is to improve operator safety and to reduce human
errors by implementing process automation structures
[5].
However, current pilot scale development is still inefficient, wasteful and time-consuming. The setup of many
pilot plants does not support the integration of extended
PAT and automation structures. The fact of missing PAT
prevents the application of statistical tools, which means
one variable at a time must iteratively be adopted. This
leads to a quasi-optimum with lack of information and furthermore costly delays.
In this context the use of experimental design
approaches becomes increasingly important. The FDA [5]
points out the effectiveness of methodological experiments based on statistical principles of orthogonality, reference distribution, and randomization for identifying
and studying the effect and interaction of product and
process variables. Methods such as factorial design,
response surface methodology, and DoE provide efficient
ways to optimize cultivations using a reduced number of
experiments [6].
The rising demands regarding process understanding
by using analytical, statistical tools causes in widespread
small scale DoE applications with Pichia pastoris [711].
In this view a multi-bioreactor plant was designed,
which meets all the mentioned requirements (i) extended
PAT for process observation/understanding, (ii) increasing automation, and (iii) effective determination of optimal expression conditions with experimental design
approaches.
In previous investigations the methylotrophic yeast
Pichia pastoris has been determined as suitable expression system for a high-level production with respect to the
improvement of cultivation respectively purification conditions for potential malaria vaccines [12, 13]. The production process was done with the P. pastoris strain
KM71H, phenotype methanol utilization pathway slow
(without AOX1-gen) (MutS), which was transformed with
artificial protein sequences of the Plasmodium falciparum
protein apical membrane antigen 1 (PfAMA1) obtained
from the Biomedical Primate Research Centre (BPRC) of
The Netherlands [14].
2 Material and methods
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.
2.2 Strain, media, and cultivation conditions

A recombinant Pichia pastoris MutS strain was kindly provided by Bart Faber from the BPRC of The Netherlands.
FM22 medium was used as batch and refresh medium. Fed-batch feeding solution contained of 630g/L glycerol, 2 mg/L biotin, and 10 mL/L Pichia trace minerals
4 (PTM4) stock solutions. An open loop feed control for the
glycerol feeding rate FR1 was realized to implement substrate-limited fed-batch operations. The methanol concentration was closed loop controlled to a given set point
with pure methanol. The pH-value was controlled with
12.5% ammonium hydroxide and 0.5M phosphoric acid
during bioreactor cultivation.
The culture was air-aerated with 1.5 vvm. The dissolved oxygen tension pO2 was closed loop controlled at a
set point of 25% by manipulation of agitation speed.
Investigations conducted in the current study were
performed according to our previous study [12].
2.1 Experimental setup

2.3 Measurement of potential malaria vaccine
The experimental setup shown schematically in Fig. 1
was extended by a newly developed BioPAT MFCS/win
DoE module (Sartorius Stedim Systems, Germany), which
The target protein concentration was determined by an

at-line RP-HPLC method as described previously [12].
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Biotechnol. J. 2013, 8, 738747
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.
2.4 Determination of cell-specific alcohol oxidase

content
One milliliter of cell suspension was centrifuged for
mechanical lysis. The pellet was resuspended to a cell
concentration of 100g/L with PBS-buffer. For cell disruption 1g of glass beads (0.40.6mm, Sartorius AG, Germany) were added to 100L of cell suspension and 900L
of PBS buffer in a 2-mL microtube. Cells disruption took
place by using VXR basic Vibrax (IKA-Werke GmbH &
Co. KG, Germany) for 20min at 2000rpm. Cellular debris
was removed by centrifugation (30min, 14000rpm, 4C).
Stock solution for AOX assay includes 10.5L peroxidase
(POD) (250U/mL), 33mg 2,2-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) in 20mL 100mM potassium phosphate buffer (pH 7.5) and 1050L diluted hydrogen peroxide (1L of 30% w/w H2O2 in 100mL sterile filtrated DI-H2O). The reaction was started for each sample
by the addition of 50 L 1.0% v/v methanol solution to
200L stock solution and 50L sample in each well of the
96 well plate. The reaction was monitored by the
microplate reader Sunrise (Tecan Group Ltd., Switzerland) at 405 nm over 4.5min. One unit of AOX was defined
as the amount of enzyme that induces the oxidation of
740
1M of ABTS per minute under the above experimental

conditions. Measurements were executed in triplicates.
This assay is based on refs. [16, 17].
2.5 Bioreactor instrumentation

All cultivations were performed with a 5-L BIOSTAT
Bplus bioreactor and a sixfold 1L bioreactor system BIOSTAT Qplus (both Sartorius Stedim Biotech, Germany).
The reactors are highly instrumented (turbidity, MeOH
concentration, O2 and CO2 offgas component analysis, 36
scales for accurate process balancing, and automated
processing). The detailed instrumentation is described in
Fricke et al. [12].
2.6 Sequential/parallel cultivation strategy for

process optimization
For the optimization procedures conducted in the multibioreactor plant a sequential/parallel cultivation strategy
was developed and implemented. The cell-breeding procedure followed a typical three step Pichia pastoris cultivation strategy with a batch, fed-batch and induction
phase [18]. The process started with a cell density cXL0 of
Biotechnol. J. 2013, 8, 738747

Figure 2. Time courses of two

potential malaria vaccine production runs with Pichia pastoris. The
culture conditions were only varied in pH-values. AP1M: at-line signal of UV absorption (P), cS2M:
methanol concentration, cXL: cell
density reconstructed from turbidity signal, pO2: dissolved oxygen
tension, PRDk: target protein productivity of run k.
5g/L. In the batch phase on glycerol cS1M, the cells grew

with the maximum specific growth rate 1max of 0.23 h1.
After an on-line batch end detection, a substrate-limited fed-batch phase on glycerol was started with an
exponential increase of glycerol feed FR1 for controlling
the cell-specific growth rate. The glycerol feed automatically stopped when reaching a previously defined celldensity, estimated on-line from the in-line turbidity measurement. In the induction phase the methanol concentration cS2M was controlled to a set point of 0.5g/L for about
12h. The resulting metabolic change of the cells allows to
avoid expression delays in the subsequent production
phases in the sixfold Qplus system.
In the following a defined volume of cell suspension
was transferred consecutively into the six screening reactors (Q+). After a 1:1 dilution with refresh medium an initial cell density of approximately 18.5g/L was adjusted.
The time courses of two optimization runs are shown
in Fig. 2. Depicted are cell density cXL, estimated from turbidity signal, methanol concentration cS2M, pH-value, dissolved oxygen tension pO2, and target protein absorption
AP1M. The experiments were only varied in settings for
pH. AP1M was determined at-line with the HPLC-method,
mentioned in Section 2.3.
With respect to reproducibility, nearly equal start values for cXL0 could be obtained, as demonstrated in Fig. 2.
By means of global monitoring, regarding time courses of
cXL, respectively, AP1M, an extended process analysis
could be carried out.
2.7 Quality criterion for the evaluation of

recombinant protein expression in Pichia
pastoris
For the evaluation of the screening experiments, the
mean value of target protein secretion productivity PRDk
in screening step k was used as performance index yk,
y k = PRDk = (Z (tkn tk0 ) VLkn )1
[IAP1Mkn VLkn (Z Z/X cXLkn )
n
+ IAP1Mkj VSkj (Z Z/X cXLkj)

j =1
IAP1Mk0 VLk0 (Z Z/X cXLk0 )],
(1)
and was based on off-line measurements of the amount of

active target protein IAP1M. Data taken at the end tkn and
at the beginning tk0 of a screening run, and the amount of
produced protein taken out by each sample time tkj in
between, was considered. The productivity was determined for the statistically designed experiments with
nearly identical initial conditions of cell density cXLk0 and
target protein concentration IAP1Mk0 (Table 1).
2.8 Automated DoE applications with the

sequential/parallel cultivation approach
The principle of DoE is to vary simultaneously a number
of factors, which potentially influence the outcome,
respectively, response of the process. The experiments
are distributed in a rectangular design space and performed at low and high levels of each factor, augmented with at least three additional center points. Therewith
741
Biotechnology
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Biotechnol. J. 2013, 8, 738747
Table 1. Data of performed cultivation runs
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.
DoE provides an organized approach which requires a

limited number of experiments leading to significantly
faster process optimization [19].
In this DoE study, cultivations were varied in settings
for pH-value, cultivation temperature L and inductor
concentration cS2M in the optimization procedure, respectively, pH-value, and L for the investigations relating
robustness of the biological production system. The pO2
was kept constant at 25% in all cultivations.
A central composite circumscribed (CCC) approach
was chosen for the design of the optimization experimental setup. The use of the CCC-design allows the identification of interactions between the factors and a cubic
response behavior. Table 1 shows the experimental
design of the conducted optimization. In cultivations with
a pH-value above six precipitations of media components
were observable, therefore the upper limit for varying the
pH-value was given in this case.
The goal of the optimization procedure was to approximate the determined response y,
742
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
by a cubic quadratic polynomial model using multiple linear regression (MLR).

A two-level full factorial design was used for the experimental design of the robustness testing study, shown in
Table 1.
The DoE procedures were conducted by combining
the BioPAT MFCS/win DoE module with the professional DoE software MODDE [19].
Since the DoE module is linked to the process control
system MFCS/win, the respective set-points could automatically be assigned to the parameter phase MFCSDOE_Factors, as shown in Fig. 3.
Shown are the ANSI/ISA-88.01 recipe structure with
the operations STANDBY, INOCULATION, PRODUC-
Biotechnol. J. 2013, 8, 738747

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.
TION, and TERMINATION, as well as a section of the

sequential function chart used for automatic initialization
of methanol and pO2 control as well as automatic adjustment of DoE parameters.
The sterilization, inoculation, cultivation, harvest, and
refresh operations ran automatically by use of S88
recipes derived from the SCADA-system BioPAT
MFCS/win (Sartorius Stedim Systems, Germany). This
was realized by piloting pressure valves and controlling
pumps, remote-controlled via the local digital control unit
DCU4.
3 Optimization of recombinant protein

expression
3.1 Verification of the sequential/
parallel cultivation approach
In the methylotrophic yeast Pichia pastoris, AOX is a key
enzyme involved in the dissimilation of methanol [20].
AOX catalyzes the first step in the methanol utilization
pathway, the oxidation of methanol to formaldehyde and
hydrogen peroxide [21].
Therefore the cell-specific internal AOX content gP2/X
was chosen for investigations relating changes in Pichia
pastoris metabolism during long-term cyclic cell-breeding cultivations.

A start up, inoculated from shake flasks, and three cellbreeding cycles, following the strategy described before,
are shown in Fig. 4, by means of cell density cXL, methanol
concentration cS2M, specific cell internal AOX content
gP2/X, and specific methanol uptake rate qS2/X. Each cycle
consists of a glycerol (S1) batch, a substrate-limited fed
batch on glycerol and a pre-induction phase on methanol
(S2).
As shown in Fig. 4, gP2/X was not observed during
glycerol phase in the start up cultivation. The AOX promoter is repressed by unlimited growth on glycerol, which
is well known from literature [2224]. After methanol supply, gP2/X and qS2/X began to increase rapidly. During harvest/refresh operations and after initialization of a further
batch process on glycerol (S1), associated with a fully
depletion of methanol (S2), the gP2/X decreased rapidly.
This has been reported from Jungo et al. [20] as well.
The recurring enrichment to the same level and the
subsequent decrease of gP2/X in the glycerol batch phase
of gP2/X show reproducible metabolic turnovers of the
Pichia pastoris cells during cyclic substrate change. Combined with final cell densities cXL, equally in each cycle,
the functionality of the parallel/sequential cell-breeding
approach is proven.
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Figure 4. Cyclically recurring cell-breeding

cultivations with Pichia pastoris. Every cycle
consists of a glycerol (S1) batch (u: S1
unlimited), a fed-batch phase (l: S1 limited),
and a pre-induction phase on methanol (S2).
cXL: cell density reconstructed from cell dry
weight (A) (determined in duplicate); cS2M:
methanol concentration in media phase;
gP2/X: cell internal cell-specific AOX content
( ) (determined in triplicate); qS2/X: specific
methanol uptake rate (P).
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.
3.2 Optimization of malaria vaccine expression

using the multi-bioreactor plant
The measured product concentrations were used to calculate the related performance index in Eq. (1). Data,
shown in Table 1, was used to generate the coefficients a0
to a123 by fitting the model (Eq. (2)) to the measurements
with MLR.
The goodness of fit R2 of 0.98 shows that only 2% of the
total variation is not explainable by the model. The Prediction Error Sum of Squares (PRESS) value of 0.59
(AUs/h)2 related to the Sum of Squares Total (SST) value
of 3.96 (AUs/h)2 indicates a low predicted variation. The
goodness of prediction Q2, calculated from PRESS and
SST, of 0.85 is close to the determination coefficient R2.
The reproducibility RP,
RP =1
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.
744
High significance of the model can be claimed after

consideration of ANalysis Of Variance (ANOVA) for a significance level of 95%. The p-value for regression (<0.001)
supports the analysis. The model shows no lack of fit with
a plof-value of 0.76.
Figure 5 illustrates the results of the optimization via
a response surface plot by fitting the model to the experimental data.
The conclusive result indicates optimal productivity
PRD for a methanol concentration cS2Mopt of 1.0 g/L,
a pHopt of 5.55 and a cultivation temperature Lopt of
25.8C.
As known from literature, optimum protein production
varies in accordance to the used Pichia pastoris strain,
especially relating to the geno-, respectively, phenotype of
organism (e.g. [2527]) and to the secreted foreign protein, which are directly influenced by cultivation conditions (e.g. [2830]).
Gasser et al. [27] reported that cultivation temperature
strongly impacts the regulation of specific genes. Many
important cellular processes, including the central carbon
metabolism, stress response, and protein folding are
affected by changing the growth temperature [31]. However, Cos et al. [32] observed less protein expression above
32C with Pichia pastoris, which matches the results
obtained in this report. The resulting optimum temperature Lopt is specific for the expression of the potential
malaria vaccine protein but commonly used in Pichia pastoris cultivations as well.
The ability of Pichia pastoris to grow across a relatively broad pH range [33] is well known. In contrast, the
recombinant protein stability is closely coupled to the pH-
Biotechnol. J. 2013, 8, 738747

value, controlled in cultivations. Different investigations

in this field relating to optimal protein production have
shown a wide range in adjusted pH-values [34, 35].
Compared to our results, pH-values have been fixed
around 5.5 to reduce protease effects, reported in several
works [11, 36].
The stability of the target protein was tested in culture
supernatant at different temperatures and pH-values
(data not shown). The investigations have shown a significant increase of protease effects at temperatures
27C. The degradation of the product increases with
lowering the pH-value from 6.0 to 4.8.
In summary, the optimal target protein productivity
seems to be a sensitive steady state between accelerating cellular processes and decreasing protease effects in
the culture supernatant.
3.3 Contemplation of system sensitivity

In the robustness testing procedure the sensitivity around
a response variable optimum was investigated by varying
the factor levels in a small range.
In this context L was varied by 1C and the pH-value
by 0.15 pH-units around the center point with the factor
settings L =25.8C and pH =5.55. A full factorial design
in two levels was applied as regression model. The
methanol concentration was kept constant in the optimum at 1 g/L. The statistical insignificance of small
methanol concentration changes has been proven in
pretests (data not shown). The experimental set up, the
start values for cXL, respectively IAP1M, as well as the calculated results are shown in Table 1.
The productivity PRD varies around the mean of the
center points within a given tolerance limit of 2 SDs.
The calculated Q2-value of 0.13 for the chosen model
indicates an extremely weak relationship between the
factors and the response.
The ANOVA of the regression model demonstrates
the insignificance of the robustness test model.
The low predictive power of the selected model and
the high p-value verify robustness of the response secretion productivity for small variations in factor settings
around the response variable optimum.
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.
constant conditions for investigations regarding protein

expression.
Several implemented automation structures enable
fully automated operations, e.g. piping sterilization, cultivation, and refresh, by using BioPAT MFCS/win recipes
complying with the ANSI/ISA-88.01 standard.
In addition, the implementation of experimental DoE
designs into the BioPAT MFCS/win recipe structure,
and furthermore the statistical evaluation of cultivation
data has been easily conducted by combining the new
BioPAT MFCS/win DoE module with the DoE software
MODDE.
An optimum in product expression was found in two
cultivation procedures, screening for significant parameters/search space [12] and optimization of parameter setting in at least five 24h cultivation cycles via DoE applications.
Robustness in consistent productivity was observed
by varying the factors in a small range. In respect of ICHregulations the ability of the process to tolerate changes
in the process without negative impact on quality was
demonstrated.
Regarding the incentives, pointed out in PAT initiative of the FDA [5], the highly instrumented multi-bioreactor plant fulfills the requirements in (i) process observation and understanding perfectly. In the sense of ICHs
guideline Q8R2, where process supervision is understood
as continuous process verification, improvements by
monitoring the target protein and controlling the critical
parameters could be highlighted.
745
Biotechnology
Journal
Biotechnol. J. 2013, 8, 738747
Automation structures (ii) for easier process handling

were implemented and maintained reproducible research
results. Hence, the conducted fully automated cultivation
process reduces human errors and improves the plausibility and reliability in process optimization.
The assembled statistical tools for DoE (iii) improved
plant automation and biological process understanding in
terms of interaction effects of the investigated process
parameters. An effective determination of optimum
expression conditions was observed by using an experimental design approach. At last, the multidimensional
combination and interaction of process parameters has
been demonstrated to provide assurance of constant productivity.
In summary the described multi-bioreactor plant
enables fast process developments. Product quality is
proven if operated within the design space. The smallscale system already allowed seamless scale-up into lab
scale (15 L) and into pilot scale (40 L) and could be an
interesting tool for pharmaceutical protein production
stakeholder to improve manufacturing scale process.
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)
cell-specific biomass reaction rate (g/(gh))

Indices
at
C, CO2
G
j
in
k0
kn
L
M
max
MCi
746
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
The authors thank the BMBF German Federal Ministry

of Education and Research for financial support, Project
Malariavakzine FKZ 1756X09, as well as Umetrics AB,
Sartorius Stedim Systems GmbH, HAMILTON Bonaduz
AG, and Stubli Tec-Systems GmbH for providing softand hardware components. The malaria vaccine candidate described here were developed under auspices of the
European Malaria Vaccine Development Association,
grant number LSHP-CT-2007-037506.
The authors declare no conflict of interest.
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-
Biotechnol. J. 2013, 8, 738747

[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
toris: the influence of culture conditions, biomass and induction

regime. Microb. Cell Fact. 2009, 8, 35.
Loegering, K., Mueller, C., Voss, J.-P., Luttmann, R. et al., An integrated scale-down plant for optimal recombinant enzyme production by Pichia pastoris. Biotechnol. J. 2011, 6, 428436.
Fricke, J., Pohlmann, K., Lang, R., Luttmann, R. et al., A multi-bioreactor system for optimal production of malaria vaccines with Pichia
pastoris. Biotechnol. J. 2011, 6, 437451.
Martens, S., Borchert, S.-O., Faber, B. W., Cornelissen, G. et al., Fully
automated production of potential Malaria vaccines with Pichia
pastoris in integrated processing. Eng. Life Sci. 2011, 11, 429435.
Remarque, E. J., Faber, B. W., Kocken, C. H. M., Thomas, A. W., A
diversity-covering approach to immunization with Plasmodium falciparum apical membrane antigen 1 induces broader allelic recognition and growth inhibition responses in rabbit. Infect. Immun.
2008, 76, 26602670.
Pohlmann, K., Fricke, J., Tatge, F., Luttmann, R. et al., OnlineBeobachtung rekombinanter Malariavakzine in einer Multibioreaktoranlage, 10. Dresdner Sensor-Symposium, TUDpress, ISBN 978-3942710-53-4, 317-320.
Ptter, J., Becker, R., in: Bergmeyer, H. U. (Ed.), Methods of Enzymatic Analysis, Vol. III, Verlag Chemie, Deerfield Beach, FL 1983, pp.
286293.
Keesey, J., Biochemica Information, 1st Edn., Boehringer Mannheim
Biochemicals, Indianapolis, IN 1987, pp. 58.
Cornelissen, G., Bertelsen, H. P., Lenz, K., Luttmann, R. et al.,
Production of recombinant proteins with Pichia pastoris in integrated processing. Eng. Life Sci. 2003, 3, 361370.
Eriksson, L., Johansson, E., Kettaneh-Wold, N., Wikstrm, C. et al.,
Design of Experi-ments Principles and Applications, MKS Umetrics AB, Umea 2008.
Jungo, C., Marison, I., von Stockar, U., Regulation of alcohol oxidase
of a recombinant Pichia pastoris Mut+ strain in transient continuous
cultures, J. Biotechnol. 2007, 130, 236246.
Cereghino, J. L., Cregg, J. M., Heterologous protein expression in the
methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 2000, 24,
4566.
Tschopp, J. F., Brust, P. F., Cregg, J. M., Stillman, C. A. et al., Expression of the lacZ gene from two methanol regulated promoters in
Pichia pastoris. Nucl. Acids Res. 1987, 15, 38593876.
Higgins, D. R., Cregg, J. M., Introduction to Pichia pastoris. in: Pichia
Protocols, Higgins, D. R., Cregg, J. M. (Eds.), Methods Mol. Biol., Vol.
103, Humana Press, Towata, NJ 1999 pp. 115.
[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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ISSN 1860-6768 BJIOAM 8 (6) 633752 (2013) Vol.

Systems & Synthetic Biology

Special issue: Biochemical Engineering Sciences

Biotechnology Journal list of articles published in the June 2013 issue.

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Biotechnol. J. 2013, 8, 738747