Bioreactor Operation
Hyuncheol Kim, PhD
Drug Delivery & Tissue Engineering Laboratory
Sogang University
• The set up of the
bioreactors and the
process steps are
performed in a
specific order
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• Focus on the steps for preparing the bioreactor for use.
These steps include:
1. Clean in Place (CIP)
2. Set up of probes and valves
3. Pressure hold
4. Steam in Place (SIP)
5. Media fill
Drug Delivery & Tissue Engineering lab.
Sogang University
Drug Delivery & Tissue Engineering lab.
Sogang University
•
CIP (clean in place) is always the first step in
preparing a bioreactor for use.
•
CIP consists of a series of detergent washes,
followed by water rinses that leave all produ
ct-contact surfaces free of any dirt and organ
ic debris left in the vessel from the previous
batch.
CIP Skid
•
The CIP cycle consists of two washes:
– CIP 100 – Phosphate-free alkaline liquid
detergent containing potassium
hydroxide (base)
– CIP 200 - Acidic liquid detergent
containing phosphoric acid
CIP Solutions
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•
•
•
•
•
CIP solution is injected into the bioreactor through both:
– the ring sparger, at the bottom of the vessel
– one or more spray balls, which are typically mounted near the top of the bioreactor
In order to clean the vessel properly, the CIP solution must reach all areas of the vessel,
including the headplate, addition valves and piping, sample valves, impeller blades, and
exhaust piping.
Spray balls are made of stainless steel. Holes are drilled in the spray ball to ensure the
inside of the vessel gets complete spray coverage.
Some bioreactors or tanks have multiple spray balls.
Following the CIP solution step, the liquid is drained and the bioreactor is rinsed with
water (WFI).
Spray ball
under
Manway Lid
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Spray ball mounted on
manway lid
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• While setting up the bioreactor, the three main
activities are to:
– Install inlet and exhaust filters
– Prepare and install pH, DO, and CO2 probes
 Calibrate as necessary
– Install addition and sample port valves
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Sogang University
Sterile Boundary
•
•
•
•
The inlet and exhaust filters on a
bioreactor system form an important part
of the sterile boundary.
These filters are essential for removal of
bacteria from the gas streams that enter
the bioreactor.
These hydrophobic gas inlet and exhaust
filters must be installed on the bioreactor.
Overlay
Bioreactor
Exhaust
1
Ring
Sparge
Exhaust
2
Micro
Sparge
The filters should be inspected for cracks
and to ensure the O-ring is intact and the
filter fits into the housing tightly.
Jacket of
Bioreactor
Filters in housing
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• DO, pH and CO2 probes are
important tools that monitor the
cell growth environment
• Installation:
– Be careful not to damage the
probes when putting them
into the bioreactor ports (pH
probes are glass and will
break)
– Ensure they are screwed (or
clamped) into the ports well
to ensure sterility of the
bioreactor is maintained
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(Multiple types of ports are used for
probes.)
•
Addition and sample valves need to be installed on the vessel prior to the
pressure hold and the SIP cycle.
•
The pressure hold will check to ensure there are no leaks in the piping and the
SIP cycle will sterilize the valves to prevent contamination.
Addition Port on the
bioreactor
Nova Septum Sample Port
on the bioreactor
These valves are
used to control flow
through the ports
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Sogang University
Drug Delivery & Tissue Engineering lab.
Sogang University
• A pressure hold test is performed prior to SIP and adding media
to the bioreactor.
• Its purpose is to check the integrity of the sterile boundary. The
areas that are checked include:
– Bioreactor vessel / head plate connections
– Gas inlet and exhaust filter housings
– All piping between the inlet and exhaust filters
– Addition and sample valves added to the vessel
– Probe connections
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• Procedure
– Bioreactor is pressurized to a certain set-point
– Pressure is allowed to stabilize for a number of minutes
 A small pressure drop is expected during the stabilization period
– Pressure is then held for a number of minutes, and pressure drop is
measured
– The total pressure drop cannot exceed a predetermined criteria
• At a constant temperature, any pressure drop above the maximum
allowed can be attributed to a leak in the system.
– If a leak is detected, the bioreactor should not be used, it should be
checked for the leak source.
– The bioreactor must pass pressure hold prior to use.
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Troubleshooting a leaking pressure hold test:
1.
Isolate the vessel by closing automatic valves. This tests the vessel, head plate
connections, and probe connections.
•
If the leak continues, check the connections on the head plate and ensure the
probes are installed correctly.
2.
If the leak stops, open the automatic valves one by one and allow for the system to
stabilize. When the pressure drops, you have found the area of the leak.
Drug Delivery & Tissue Engineering lab.
Sogang University
Drug Delivery & Tissue Engineering lab.
Sogang University
Drug Delivery & Tissue Engineering lab.
Sogang University
• The last step in the preparation of a bioreactor, prior to media filling and
inoculation, is SIP (steam in place).
• SIP sterilization is a time-proven and economical process for killing
microorganisms through the application of moist heat in the form of saturated
clean steam under pressure.
• The rate by which microbial organisms are thermally inactivated depends on
the temperature and duration of heat exposure.
• The amount of time the bioreactor is exposed to the desired SIP temperature
set-point is called the hold time or exposure time.
– - This time and temperature is determined during the validation of the
vessel
– - Typical hold times range from 30 – 45 minutes
– - Typical SIP temperature set-point is ≥ 121 °C
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An SIP cycle consists of these stages: Heat, Hold, and Cool Down
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Drug Delivery & Tissue Engineering lab.
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•
After the bioreactor has been sterilized, the vessel is ready to receive the
batch media needed for the cells to grow.
•
The batch media must be sterilely added to the sterile bioreactor. This is
done through sterile media filters.
•
Media filters are placed in stainless steel housings and are then SIP’d.
•
While filtering media into the bioreactor, the operator must watch the
weight, as well as the pressure gauges before and after the media filters.
•
Media filters can clog and by watching these two trends (the vessel weight
and the pressure gauges) the operator can identify if fouling (clogging) is
occurring.
•
Media filters are single use and must be disposed of properly after use.
Drug Delivery & Tissue Engineering lab.
Sogang University
Drug Delivery & Tissue Engineering lab.
Sogang University
Bioreactor Monitoring
• Cell culture processes need to be monitored to ensure the cells and
the equipment are performing as expected.
• Daily sampling of the cell culture for cell count, glucose
concentration, pH, etc. is a type of process monitoring.
• Additional process monitoring is performed with the computer.
Control systems log all the data it receives from the reactor, such
as temperature, pH, DO, agitation, into process trending software.
• The user can view the value of a data source for the entire history
of the run, i.e. the temperature of a vessel during SIP can be
mapped.
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Drug Delivery & Tissue Engineering lab.
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Contamination
• Sometimes cells just don’t grow. Low or no growth can be
attributed to a number of reasons, including:
– Media not correctly formulated, missing a component
– Cell density too low after inoculation
– Agitation or gassing turned off
– pH of media too high or too low
• Daily process monitoring and sampling enables the operator to
be alerted to growth issues.
• The bioreactor is also equipped with alarms to notify operators
of mechanical issues that will affect the cells.
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• Sometimes mechanical equipment malfunctions.
Common mechanical equipment issues include;
– Leak in a steam valve
– Broken pump
– Leak in a mechanical seal
• Process monitoring should take place to watch out for
issues during all manufacturing activities:
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Probes (DO and pH) on the bioreactor can fail.
• Each bioreactor has two DO and pH probes in case one fails during a
run.
• If you suspect a probe has a bad reading, take a sample of the media to
check the reading. There is always backup equipment to check pH
• Also, check the data trends of the probes and inspect the probe cables.
Cells may not grow or may grow slower than typical.
• You need to watch out for contamination, incorrect media formulation,
incorrect media pH, incorrect media osmolality, vessel temperature,
and gassing.
• Low inoculation density can also lead to poor growth.
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Cells
Time
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• Some indications of contamination include:
– pH is above or below set point
– Oxygen flow rate is higher than normal
– Culture viability and cell density are lower than expected
Possible contaminants: Mycoplasma & Yeast
Drug Delivery & Tissue Engineering lab.
Sogang University
Drug Delivery & Tissue Engineering lab.
Sogang University
July 2009 – Headlines in Boston
Globe
• Genzyme halts production on
2 key drugs
• Virus shuts Genzyme plant,
holds up drugs for 8,000
• Genzyme struggles to recover
from virus
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Pipes are wiped during decontamination in the
cell-culture area at Genzyme Corp.'s facility in
Allston. A virus was discovered at the plant.
(Wendy Maeda / Globe Staff)
• Lost Plant Time / Salaries
– 100s of workers working on the cleanup
• Lost Production
– Halted production on the drug
– Lost over $300 million in revenue
– Had to ration the drug to the patients
• Clean up Costs
– Threw away everything that couldn’t be bombed
– Vaporized the facility
– Removed all walls, insulation
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• Raw materials
• People
• Equipment
• Environment
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Drug Delivery & Tissue Engineering lab.
Sogang University
Drug Delivery & Tissue Engineering lab.
Sogang University
Drug Delivery & Tissue Engineering lab.
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Filter Integrity
Test
•
Filter integrity testing
is performed after
using the media filters
to ensure that the
filters were not
damaged and they
properly filtered the
media.
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Destructive
Testing
Bubble Point
Test
Forward Flow
Test
Non Destructive
Testing
Diffusion
Test
Pressure
Hold
Water Intrusion
Test
Pressure
Decay
• Pasteurization performed to minimize contamination in raw
materials.
• HTST – High Temperature Short Time
• UV - Ultraviolet
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• Need to understand the chemical make up of your
raw materials
• Size is an issue
• Timing and Cost
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• Ultimate Goal is to protect the product in order to
protect the patient!
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Thank
You