evaluation of pbs 15 air-wheel® single use bioreactor for ... · • 1 pbs 15 air-wheel® system...
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The Upstream Process Development team at MassBiologics has
demonstrated successful cell culture process development utilizing
MassBiologics’s chemically defined media and feed platform in
addition to optimized DASGIP bioreactor conditions to boost mAb
expression and growth. In contrast to stirred tank bioreactors, the Air-
Wheel® Single Use Bioreactor system utilizes rising gas bubbles and
converts them into rotational mixing power, lowering the amount of
shear stress on the cells. In this work we evaluated the PBS 15 Air-
Wheel® using MassBiologics’s optimized media and feed platform, pH
control, dissolved oxygen control, temperature conditions, and a stable
CHO cell line expressing an IgG1 mAb product. We compared its titer
and growth patterns to concurrent DASGIP 1L bioreactors running
under the same optimized conditions. All three 14 day fed-batch runs
were conducted with one PBS 15 Air-Wheel®, two duplicate 1L
DASGIP bioreactors with 15µm microspargers, and 100mL shake flask
controls inoculated from each bioreactor system. However, the first
experiment showed that evaporation for the Air-Wheel® culture was
43% higher than the DASGIP culture. As a result, the second
experiment used diluted feed in order to compensate for the
evaporating water calculated from the first experiment. Using the
diluted feed reduced the evaporation rate to 27% when compared to
DASGIP cultures without diluted feed. This led to the use of a lower
Air-Wheel® agitation rate for the third run, which reduced the amount
of gassing by 60%. Lower gassing reduced the evaporation rate further
to 12%. All three experiments show that viable cell density, viability,
titer, specific growth and death rates, productivity, and metabolism are
comparable between the two bioreactor systems, and that the Air-
Wheel® system provides better CO2 stripping.
ABSTRACT
MATERIALS & METHODS
Figure 1: Comparison of viable cell density and viability between the
Air-Wheel and DASGIP bioreactor systems. As shown in the results
from the first run, the daily viable cell density and viability is very
comparable between the two bioreactor systems. These trends were
also seen in the subsequent two runs.
RESULTS
Part 2: Reducing the Overall Gassing to Prevent
Excessive Evaporation of the Air-Wheel® 15 Culture
RESULTS
CONCLUSIONS
ACKNOWLEDGEMENTS
The authors would like to thank Daniel Giroux, Joe Petrosky
(PBS Biotech), Bernie Creswick, Kristina Papa (MassBiologics)
for their assistance with this project.
Materials:
– Stable CHO cell line producing IgG1 monoclonal antibody
– Chemically defined medium and feed platform
– All three runs include the use of:
• 2 DASGIPs (900mL initial working volume (w.v.))
• 1 PBS 15 Air-Wheel® system (9L initial w.v.)
• 250 mL shake flasks (100 mL initial w.v.)
Methods:
– Cells were seeded at 0.4x106 cells/mL
– All bioreactors were run for 14 days in fed-batch mode
• DASGIP gassing via 10 µm microsparger, 0.1 – 1.2 L/hr
– Agitation initially set to 120 rpm, and increased
daily based on the increasing volume
• Air-Wheel gassing via macrosparger (air) and
microsparger (oxygen)
– Runs 1 and 2 used 150L/hr (38 rpm)
– Run 3 used 60L/hr Day 0-7, then lowered gradually
to 48 L/hr (29-26 rpm)
• Bioreactor cultures were kept in 36.5oC and 40% DO
• CO2 was sparged in order to maintain pH at a maximum
of pH 7.3 (with a lower bound of pH 6.8)
• Glucose was supplemented daily to 3.9 g/L (bolus)
• Continuous feed
– Shakers were incubated at 140 rpm, 36.5oC and 5% CO2
• Glucose was supplemented daily to 3.9 g/L (bolus)
• Bolus feed
– Cell viability and density was determined by Cedex HiRes
Cell Counter (Roche)
– Metabolites, pH, and pCO2 and osmolality determined by
Nova BioProfile 400 Analyzer (Nova Biomedical)
– Additional samples were centrifuged to remove cell debris.
The supernatant was analyzed by Octet Qk (FortéBIO) for
mAb quantitation
– Specific productivity was evaluated using IVCC
Tuhina Bhattacharya, Sadettin Ozturk MassBiologics, University of Massachusetts Medical School, Boston, MA
Evaluation of PBS 15 Air-Wheel® Single Use Bioreactor for a Fed-Batch Monoclonal Antibody Process
MassBiologics
In order to compensate for the evaporating water, the feed was
diluted 15% with dH2O for the second run (based on calculations
from the first run). The amount of nutrients being added to the
culture on a daily basis was kept the same. However, diluting our
feed is not a practical solution. Furthermore, adding back
additional water to the culture reduced the amount of evaporation
to 27% more than the DASGIP cultures, so there was still a
considerable increase in osmolality for the second run.
Part 1: Findings from Air-Wheel® 15 vs. DASGIP Run 1
and Run 2
Figure 2: Comparison of culture osmolality between the Air-Wheel and
DASGIP bioreactor systems. The osmolality of the Air-Wheel culture
increased at a higher rate than the DASGIP culture, despite the
nutrient addition into the systems being equal. This was the first
indication that there was excessive evaporation in the Air-Wheel
bioreactor system when run at 38 rpm (this agitation translates to
about 150L/hr of gassing). The actual final and initial volumes were
compared to the theoretical, and it was determined that the Air-Wheel
culture had 43% more evaporation than the DASGIP cultures.
Figure 4: Comparison of culture osmolality between the Air-Wheel and
DASGIP bioreactor systems. The osmolality of the Air-Wheel culture is
very comparable to the osmolality of the DASGIP culture throughout
this 14-day run. This indicates that running the Air-Wheel bioreactor
system at 48-60 L/hr gassing allows for sufficient culture agitation and
gassing without causing excessive evaporation.
In the third run, a reduced gassing and agitation method was
used to compensate for the evaporating water in the Air-Wheel
system. The gassing tied to the vertical agitation of the wheel
was reduced from 150L/hr to 60L/hr (38 rpm to 29 rpm). As the
culture approached it’s maximum cell density, it was further
reduced to 48 L/hr (26 rpm). This was determined to be a low
enough agitation to prevent excessive evaporation, while still
allowing for sufficient mixing and CO2 stripping.
Figure 5: The Air-Wheel culture’s evaporation rate as compared to the
control DASGIP cultures for all three experiments. Diluting the feed by
15% reduced the relative evaporation to 27%. Lowering the amount of
gassing by 60% reduced the relative evaporation rate to 12%.
RESULTS
Figure 7: Average specific productivity. There is no significant
difference between the specific productivity (amount of antibody
produced per cell per day) between the Air-Wheel system and the
DASGIP system.
Figure 6: Comparison of titer normalized to DASGIP bioreactors. All
data values were calculated with the evaporation in the Air-Wheel
taken into account in order to show a fair comparison. There is no
significant difference in titer between the Air-Wheel system and the
DASGIP system.
Figure 8: Partial pressure of carbon dioxide in culture. The Air-Wheel
system has better CO2 stripping during the first seven days of growth.
This trend was seen in all three runs.
• The daily viable cell density and viability is very comparable
between the two bioreactor systems.
• There is no significant difference in titer between the Air-Wheel
system and the DASGIP system.
• There is no significant difference between the specific
productivity (amount of antibody produced per cell per day)
between the two bioreactor systems.
• The Air-Wheel system has better CO2 stripping during the first
seven days of growth.
• Cells grown in the Air-Wheel bioreactor display similar
metabolism to cells grown in concurrent DASGIP bioreactors
(data no shown).
• Lowering the amount of gassing by 60% reduced the
evaporation rate (as compared to DASGIPs) to 12%.
• For processes that require a higher Air-Wheel agitation and
gassing rate, the authors advise adding a chiller to prevent
excessive evaporation.
Figure 3: The Air-Wheel® system utilizes rising gas bubbles and
converts them into rotational mixing power, lowering the amount of
shear stress on the cells. Lowering the agitation of the system
significantly reduces the amount of total air gassing. Photo courtesy of
PBS Biotech.
Agitation: 38 rpm
Gassing: 150L/hr
Feed diluted 15% to feed
back evaporating water
Agitation: 29 rpm,
decreased to 26 rpm
Gassing: 48-60L/hr
Feed not diluted
Agitation: 38 rpm
Gassing: 150L/hr
Feed not diluted