Professional Documents
Culture Documents
School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4
7ET, UK
For correspondence:
Sarah J Routledge
Tel: +44 (0) 121 204 3168
E-mail: s.j.routledge@aston.ac.uk
Abstract
Pichia pastoris is a widely-used host for recombinant protein production. The foaming
associated with culturing it on a large scale is commonly prevented by the addition of
chemical antifoaming agents or antifoams. Unexpectedly, the addition of a range of
antifoams to both shake flask and bioreactor cultures of P. pastoris has been shown to
alter the total yield of the recombinant protein being produced. Possible explanations for
this are that the presence of the antifoam increases the total amount of protein being
produced and secreted per cell or that it increases the density of the culture. Antifoaming
agents may therefore have specific effects on the growth and yield characteristics of
recombinant cultures, in addition to their primary action as de-foamers.
1. Introduction
When producing recombinant proteins on a large scale, growth in bioreactors is often the
most convenient option. One problem with such cultures, which are typically intensely
aerated and stirred, is the formation of foam. Foaming can lead to reduced process
productivity since the bursting of bubbles may damage cells and proteins (1) and result in
loss of sterility if the foam escapes the bioreactor (2). To prevent the formation of foam,
thermal methods (3), mechanical agitation, ultrasound or most often addition of chemical
antifoaming agents or antifoams (2) is employed.
Chemical antifoams can act as surfactants and generally consist of several components.
There are many different varieties which can usually be grouped as hydrophobic solids
dispersed in carrier oil, aqueous or water-based suspensions or emulsions, liquid single
component antifoams and solid antifoams (3, 4). There are thought to be several
mechanisms of foam dispersion by chemical antifoams: bridging-dewetting, spreading
fluid entrainment and bridging-stretching (5), but the precise details are not well
understood.
It is clear that antifoams are often added to bioreactors without considering the effects on
the bioprocess, the cells or the recombinant proteins being produced. This is surprising as
antifoams are known to affect the kLa or oxygen transfer rate of a system (6-14), the
ability of cells to secrete protein and can even be used at high concentrations to boost the
yield of a recombinant protein in shake flasks (15). However, different types of antifoam
at varying concentrations can have different effects. These properties should be taken into
account when choosing the most suitable antifoam for a particular process.
1.1. Antifoams
A wide variety of antifoams is available, with different compositions and properties. One
consideration when deciding on the type of antifoam to be used is whether it is suitable
for a particular process. Certain antifoams may not be suitable for some applications,
such as producing proteins for drug development, as not all are FDA approved. Some
antifoams may also affect downstream processing, such as silicone-containing antifoams,
which can coat equipment. Several antifoams should therefore be tested to determine
which is the most appropriate for the process.
1.3. kLa
The kLa, or volumetric mass oxygen transfer coefficient, is a measure of how much
oxygen is transferred into the medium over a certain amount of time (14). The kLa of a
system can be influenced by several factors such as the properties of the medium
including its viscosity, the presence of organisms and their by-products. Additions to the
medium such as antifoams also have an effect (13, 14). It has been observed that low
concentrations of antifoam can reduce the kLa but at higher concentrations the kLa may
rise (10, 12). To ensure optimum oxygen transfer within a system, the effect of differing
concentrations of the antifoam to be used should be assessed.
protein (GFP) (15). Two groups of antifoams were identified in the study, depending on
their mode of action: one that resulted in improvements in biomass yields of the cultures
and one that enhanced protein production or secretion. This finding may provide a simple
method to increase productivity in recombinant protein production experiments. It also
provides much needed insight into how antifoams interact with yeast host cells.
2. Materials
2.1. Antifoams
Antifoams from different groups can be selected depending on process requirements e.g.
1.
Culture medium required for the process e.g. BMMY medium for Pichia pastoris
cultures is composed of 1% yeast extract, 2% peptone, 100 mM potassium
phosphate pH 6.0, 1.34% YNB, 4 x 10-5% biotin, 0.5% methanol. Dissolve 10g
yeast extract and 20g peptone in 700 mL water and autoclave at 121C for 20 min.
After cooling to room temperature, add 100 mL 1 M potassium phosphate buffer
pH 6.0, 100 mL 10 YNB, 2 mL biotin and 100 mL 10 methanol. Store at 4C
with a shelf life of approximately 2 months.
2.
YNB: dissolve 134 g yeast nitrogen base with ammonium sulfate and without
amino acids in water to a total volume of 1L and filter sterilize. Store at 4C with a
shelf life of approximately 1 year.
3.
4.
10 methanol (5%): mix 5 mL methanol with 95 mL water and filter sterilize. Store
at 4C with a shelf life of approximately 2 months.
5.
6.
7.
8.
Stopwatch.
1.
2.
The medium used for kLa measurements should be the same as for a full bioreactor
run e.g. BMMY (see section 2.2).
3.
A bioreactor with control systems and equipment usually used for a full bioprocess,
including a DO probe.
An agar plate with colonies of the organism of interest e.g. P. pastoris X33 GFP
grown on YPD agar composed of 1% yeast extract, 2% peptone, 2% dextrose
(glucose), 2% agar. Dissolve 20g peptone and 10g yeast extract in water to a total
volume of 900mL and add 20g agar. Autoclave the solution at 121C for 20 min
and cool to room temperature before adding 100 mL 10 glucose and pouring
plates which should be stored at 4C.
2.
3.
5.
One 250 mL baffled shake flask and 18 100 mL non-baffled shake flasks for
antifoam evaluations, all autoclaved.
6.
7.
3. Methods
3.1. Bartsch Test
1.
2.
3.
Pipette the antifoam to be tested into the medium to 0.01% v/v, i.e. 10L (see
Notes 1 and 2).
4.
Seal the cylinder using parafilm and shake up and down ten times at ambient
temperature to induce foam formation.
5.
Record the total volume of the system (the medium and foam combined), and the
volume of the medium alone every 30 s for 15 min (see Note 3).
6.
Determine the activity by subtracting the volume of the medium from the total
volume.
7.
2.
A working volume of 1 L medium is used and the bioreactor set up for conditions
typical of the required bioprocess, e.g. 1.0 L min-1 compressed air (60% O2), pH
6.0, 30C and 700 rpm (See Note 4).
3.
Calibrate the DO probe by measuring the DO in the bioreactor at these settings for
100 % (60 % O2 in compressed air will achieve 100 % DO), then flush with
nitrogen gas instead of air to obtain a 0 % DO reading.
4.
kLa measurements are carried out by starting at 100 % DO and flushing with
nitrogen until the DO drops to 0 %. Supply the system with air and the DO
gradually rises to 100 %, whereupon it is again flushed with nitrogen to reduce it to
0 % before reconnecting the air. The time points are recorded by the data logger
and the DO can be plotted versus time, as shown by Figure 2.
5.
6.
The data generated for the upwards slope of the plot are used to calculate the kLa
with the following equation:
Where t1 and t2 are consecutively-logged time points, c1, is the oxygen saturation
concentration and c1 is the oxygen concentration at each time point. An example
calculation can be found in the Notes section (see Note 5).
7.
10
8.
Combining (C = n/V) and the universal gas law (PV=nRT) gives C=P/RT,
allowing the concentration of air in the gas phase in the head space of the vessel,
CgAir, to be calculated.
9.
10.
Dividing the gas concentration of O2 by MO2 gives the maximum liquid oxygen
saturation concentration, c1, at 100% DO.
11.
3.3 Analyzing the influence of antifoams on the yield of GFP in shake flasks
1.
Pick a single colony of P. pastoris X33 GFP from a YPD plate and inoculate a 250
mL baffled shake flask containing 50 mL of BMGY.
2.
3.
11
4.
5.
6.
7.
8.
Incubate the flasks with the desired concentration of antifoam at 30C, 220rpm. A
suggested experimental set-up is triplicate flasks for antifoams at 0%, 0.2%, 0.4%,
0.6%, 0.8% and 1.0%.
9.
10.
After 24 h add 100% sterile methanol to 1% v/v i.e. 200 L to 20 mL, and continue
the incubation.
11.
12.
Analyze the protein content. For secreted GFP, centrifuge 1 mL samples at 18,625
g for 10 min and separate the pellet and supernatant. Analyze the supernatant
samples on a fluorescence plate reader (Figure 3).
Dilute samples from section 3.3 step 11 using PBS to obtain a concentration of 106107 cells mL-1. Use a haemocytometer and a light microscope.
2.
3.
12
4.
4. Notes
1.
2.
Some antifoams are viscous and sticky and may take several seconds to be drawn
into a pipette tip of the required volume. Additionally they may stick to the inside
of the pipette tip and therefore must be dispensed fully by repeatedly drawing
medium into the tip and pipetting it out.
3.
Foam may reach different heights around the sides of the cylinder, therefore the
highest level of foam should be recorded.
4.
A lower impeller speed may be required before adding antifoam as the foam level
may become too high at higher speeds depending upon the type of medium.
5.
An example of a kLa calculation using the constants and variables from section 3.2
step 7 is given here:
a. Calculate the concentration of air in the gas phase at 100% DO:
C = P/RT
b. Using the concentration of air in the gas phase, the concentration of O2 present
in the gas phase is calculated:
13
c1, = CgO2/MO2
= 0.83/30
= 0.028 mol m-3 at 100% DO
e. Substitute the Cl values calculated at particular time points into the equation in
3.2 step 6 to calculate the kLa.
5. References
1.
Holmes, W. J., Smith R. and Bill R.M. (2006) Evaluation of antifoams in the
expression of a recombinant FC fusion protein in shake flask cultures of
Saccharomyces cerevisiae, Microb Cell Fact 5, 30.
2.
Varley, J., Brown, A., Boyd, R., Dodd, P. and Gallagher, S. (2004) Dynamic
multipoint measurement of foam behaviour for a continuous fermentation over a
range of key process variables, Biochem Eng J 20, 61-72.
3.
14
4.
Joshi, K., Jeelani, S., Blickenstorfer, C., Naegeli, I. and Windhab, E. (2005)
Influence of fatty alcohol antifoam suspensions on foam stability, Colloids Surf A
263, 239-249.
5.
6.
7.
Arjunwadkar, S.J., Sarvanan, K., Kulkarni, P.R. and Pandit, A,B, (1998) Gasliquid mass transfer in dual impeller bioreactor, Biochem Eng J 1, 99-106.
8.
Calik, P., Ileri, N., Erdinc, B.I., Aydogan, N. and Argun M: (2005) Novel
antifoam for fermentation processes: fluorocarbon-hydrocarbon hybrid
unsymmetrical bolaform surfactant, Langmuir 21, 8613-8619.
9.
Koch, V., Rffer, H., Schgerl, K., Innertsberger, E., Menzel, H. andWeis J.
(1995) Effect of antifoam agents on the medium and microbial cell properties and
process performance in small and large reactors, Process Biochem 30, 435-446.
10.
Morao, A., Maia, C., Fonseca, M., Vasconcelos, J. and Alves, S. (1999) Effect of
antifoam addition in gas-liquid mass transfer in stirred fermenters, Bioprocess
Eng 20, 165-172.
11.
15
12.
Liu, H.-S., Chiung, W.-C. and Wang, Y.-C. (1994) Effect of lard oil and caster oil
on oxygen transfer in an agitated fermentor, Biotechnol Tech 8, 17-20.
13.
14.
15.
Routledge, S.J., Hewitt, C.J., Bora, N. and Bill, R.M. (2011) Antifoam addition to
shake flask cultures of recombinant Pichia pastoris increases yield, Microb Cell
Fact 10, 17.
16.
Denkov, N.D., Tcholakova, S., Marinova, K.G. and Hadjiiski, A. (2002) Role of
oil spreading for the efficiency of mixed oilsolid antifoams, Langmuir 18, 58105817.
17.
18.
16
Figure legends
Figure 1: A Bartsch test of foam volume over time for various antifoams in BMMY
medium. Foam volume over time was recorded for 0.01% v/v of each antifoam in
BMMY medium where n = 5. All antifoams were effective at foam destruction compared
with the control and most foam was destroyed within 1 min.
Figure 2: DO over time for a 2 L glass bioreactor containing 1 L BMMY medium. The
bioreactor was flushed with nitrogen to reduce the DO, followed by air. The DO was
recorded in mV with a data logger and PicoLog software.
Figure 4: Viable cells without antifoam (A) and viable cells with 0.6% antifoam A (B).
Population A is made of events that are related to electronic and particulate noise and are
not cells. Population B are cells showing enhanced green fluorescence due to GFP and
17
quadrant C is where dead cells stained red with propidium iodide (PI) would be observed.
Antifoam A did not adversely affect the viability of the cells.
18
Control
Antifoam C
Antifoam A
SB2121
P2000
Mazu DF 204
J673A
15
13.5
12
10.5
Time (min)
9
7.5
4.5
1.5
80
60
40
20
Figure 1
120
100
(mV)
Figure 2
(ms)
Figure 3
**
500
*
40
450
35
400
GFP ( g)
300
25
250
20
200
15
150
OD595
30
350
10
100
5
50
0
0
0
0.2
0.4
0.6
0.8
% Antifoam
500
**
450
35
30
400
GFP (g)
300
20
250
15
200
150
10
100
5
50
0
0
0
0.2
0.4
0.6
% Antifoam
0.8
OD595
25
350
Figure 4