Thermo Fisher Scientific HyPerforma APPLICATION NOTE
APPLICATION NOTE HyPerforma Single-Use Fermentors
End-user evaluation of 30 L and 300 L HyPerforma
Single-Use Fermentors and scale-down model
Summar y
In moving processes from bench to pilot and ultimately
production fermentors, it is critical to have a reliable
scaledown model. The purpose of a scale-down model is
to demonstrate at the benchtop level what can be achieved
in production-scale fermentors. Scale-down models
can vary depending on the media, strain, and process
conditions. In this case, a customer wanted a scale-down
model of the Thermo Scientific™ HyPerforma™ Single-Use
Fermentor (S.U.F.) in order to establish what fermentation
processes could be scaled into the S.U.F., in particular
for the production of plasmid DNA. The 300 L S.U.F.
exceeded the customer’s expectations. The customer
anticipated that the test process using E. coli with high
oxygen consumption would reach a final OD of ~80 and
the HyPerforma S.U.F. exceeded expectations by reaching
an OD of 147.
This application also was a successful demonstration of
media preparation through culture harvest, using a jacketed
50 L Thermo Scientific™ HyPerforma™ Single-Use Mixer
(S.U.M.) (60 L working volume and heated to 67°C), 200 L
Thermo Scientific™ HyPerforma™ DS 300 Single-Use Mixer
(S.U.M.), Thermo Scientific™ Powdertainer™ BioProcess
Container (BPC) system, 2 L—50 L Thermo Scientific™
2D Labtainer™ BPC systems, pre-irradiated filters, sterile
funnel BPC, seed BPC, 30 L and 300 L HyPerforma
S.U.F.s, and Thermo Scientific™ CentriPAK™ harvest BPCs.
Purpose
To evaluate the 300 L HyPerforma S.U.F. for GMP plasmid
production process using a customer’s standard evaluation
procedure and test strain.
Procedure
The E. coli culture was maintained at 37°C ± 0.1,
pH 7 ± 0.05, maximum pressure of 0.55 psi, and a
minimum dissolved oxygen (DO) of 30%. DO control
cascaded at 200–375 rpm, 200–500 slpm air, and
0–400 slpm of oxygen as needed to maintain the minimum
set point of 30% DO.
After scaling up the model procedure to the 300 L S.U.F.,
the power-to-volume input, kLa, and gassing rates were
scaled down into the 1 L glass fermentors. The same
original procedure was then followed but using the scaleddown power-to-volume input, kLa, and gassing rates for
S.U.F. comparison. The scale-down model could then be
used for estimating performance with all production strains
for this customer.
The procedure was then repeated with the 30 L S.U.F. and
showed scalability within the HyPerforma product line.
After evaluating additional leading single-use fermentors
from two other suppliers, the customer purchased the
Thermo Scientific 300 L HyPerforma S.U.F..
E. coli cells were collected with the Sorvall BIOS 16
centrifuge using CentriPAK BPC singles with quick connect
at 5,373 x g for 15 minutes in 15 L batches. The 30 L S.U.F.
was harvested as closed system in two batches using
CentriPAK manifolds. The 300 L S.U.F. was harvested
within 3 hours using two BIOS 16 centrifuges and
CentriPAK singles filled to about 1.87 L each.
During this feasibility study, the off-gas analyses of the
cultures were compared. It was noticed that the off-gas
trend was comparable. The off-gas analyzer sample line
to each S.U.F. can be placed inside the exhaust filter exit
cavity or connected to a small sterile filter connected to the
S.U.F.’s filter chamber.
Equipment and supplies
Strain:
• HMS174(DE3)
Chemicals:
• Base
• Acid
• Antifoam C8840 (New London Chemicals)
• 60 L nitrogen source, 30% w/v feed solution
• 50 L carbon source, 50% w/v feed solution
• Batch medium
Bioreactor setup
The S.U.F. systems were set up according to the user guide
and controlled by Thermo Scientific™ TruBio™ Software,
powered by powered by the DeltaV™ Distributed Control
Platform from Emerson, utilizing the Thermo Scientific™
TruFluor DO single-use sensors and Hamilton or Mettler
Toledo single-use pH sensors. Operating parameters are
listed in Table 2.
Bioreactor inoculum, cultivation, and scale-up
The inoculum was cultured in an incubator at 37°C,
250 rpm, 1 inch arc, and for 16 hr.
Table 1. Equipment and materials.
Description Cat. No.
30 L S.U.F. Hardware S.U.F.0030.AAA.BAAABB0C00
30 L S.U.F. BPC SH3B11722.01
300 L S.U.F. Hardware S.U.F.0300.AAA.DAAABB0C00
300 L S.U.F. BPC SH3B11861.01
100 L S.U.M., jacketed
with touchscreen console
200 L plastic drum SH30959.03
Drum dolly SH30958.01
™
Nalgene
Nalgene cylindrical
5 gal tank
1.5 L funnel SH3B14865.01
PowderFill or funnel stand 129752
HQ incubator shaker 11-676-235
Seed BPC
(3 L working volume)
Seed BPC clip 122554
0.5, 1, 2, 5, 10, and 50 L Labtainer
BioProcess Container (BPCs)
Powdertainer BPCs SH30737.01 and SH30737.02
HyPerforma
Prima
Bios 16 Sorvall
Bioprocessing Centrifuge
CentriPAK BPC Adapter
2 L bucket liner
CentriPAK BPC 6 x
1.7 L harvest manifold
CentriPAK BPC Single with
Quick connect
polyethylene 5 gal tank liner 11100-0005
™
G3 Controllers NA
™
BT o-gas analyzer NC1256292
SUM0100.9002
43050-0005
SH3B9830.01
SH30712.01-.02 and
SH30963.01-.03
2 x L85007685
4 x 75003873
2 x 75003880
14 x 75003891
Table 2. 30 L and 300 L S.U.F. operating conditions.
Parameter 30 L 300 L
Initial volume 24 L 240 L
Final volume ~30 L ~300 L
Temperature 37°C 37°C
pH 7.0 ± 0.05 7.0 ± 0.05
Agitation
300–600 RPM 200–375 RPM
DO setpoint 30% 30%
DO cascade Cascade
RPM, air, then
supplement
Cascade RPM, air,
then supplement
oxygen
oxygen
Gas flow 3–60 standard
30–500 slpm
liter per min
(slpm)
Antifoam 3 mL, more as
foam detected
30 mL initially, more
as foam detected
Loading...