Bioreactor Operation Drug Delivery & Tissue Engineering Laboratory Sogang University Hyuncheol Kim, PhD

Bioreactor Operation

Drug Delivery & Tissue Engineering Laboratory

Sogang University

Hyuncheol Kim, PhD

Drug Delivery & Tissue Engineering lab.Sogang University

Bioreactor Process Steps

• The set up of the

bioreactors and the

process steps are per-

formed in a specific

order


Bioreactor Process Steps

• 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


CIP – Clean in Place


CIP – Clean in Place

• 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 prod-uct-contact surfaces free of any dirt and or-ganic debris left in the vessel from the pre-vious batch.

• The CIP cycle consists of two washes:– CIP 100 – Phosphate-free alkaline liquid

detergent containing potassium hydrox-ide (base)

– CIP 200 - Acidic liquid detergent con-taining phosphoric acid

CIP Skid

CIP Solutions


CIP – Clean in Place• 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 Man-

way LidSpray ball mounted on

manway lid



Bioreactor Set Up

• While setting up the bioreactor, the three main activi-ties are to:

– Install inlet and exhaust filters

– Prepare and install pH, DO, and CO2 probes

Calibrate as necessary

– Install addition and sample port valves


Set-up – Air Filter Housing

• The inlet and exhaust filters on a bioreac-tor 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.

• 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

Bioreactor

RingSparge

Overlay

MicroSparge

Exhaust 1

Exhaust 2

Sterile Boundary

Filters in housing


Set-up – Probes

• DO, pH and CO2 probes are im-portant 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

(Multiple types of ports are used for probes.)


Set-up – Addition & Sample Valves

• 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.

These valves are used to control flow through the ports

Nova Septum Sample Port on the bioreactor

Addition Port on the bioreactor



Pressure Hold Test

• 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


Pressure Hold Test

• 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 al-lowed 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.


Pressure Hold TestTroubleshooting a leaking pressure hold test:

1. Isolate the vessel by closing automatic valves. This tests the vessel, head plate con-nections, 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.


Pressure Hold Test



SIP – Steam in Place

• The last step in the preparation of a bioreactor, prior to media filling and in-oculation, is SIP (steam in place).

• SIP sterilization is a time-proven and economical process for killing micro-organisms 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


SIP – Steam in Place

An SIP cycle consists of these stages: Heat, Hold, and Cool Down



Media Fill

• 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.


Media Fill

Bioreactor Monitoring


Process 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 concentra-

tion, 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.


Bioreactor Control Screen

Contamination


Growth Issues

• Sometimes cells just don’t grow. Low or no growth can be at-tributed 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.


Mechanical Issues

• 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:


Troubleshooting – During the Cell Culture Run

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.


Poor Growth in a RunC

ells

Time


Troubleshooting – Cell Culture Run• 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


Signs of Contamination


Genzyme and Viral Contamination

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

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)


Genzyme and Viral Contamination

• 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


Where Do Contaminations Come From?

• Raw materials

• People

• Equipment

• Environment


Ways to Mitigate Contamination Risk


Points Where Filters are used in a Generic Process


Filtration


Filter Integrity Testing

• Filter integrity testing is performed after us-ing the media filters to ensure that the filters were not damaged and they properly filtered the media.

Filter Integrity Test

DestructiveTesting

Non Destructive Testing

Bubble PointTest

DiffusionTest

Water IntrusionTest

Forward FlowTest

PressureDecay

Pressure Hold


Pasteurization

• Pasteurization performed to minimize contamination in raw materials.

• HTST – High Temperature Short Time• UV - Ultraviolet


Pasteurization

• Need to understand the chemical make up of your

raw materials

• Size is an issue

• Timing and Cost


Viral and Bacterial Control

• Ultimate Goal is to protect the product in order to pro-tect the patient!

Thank You

Documents

Bioreactor Operation Drug Delivery & Tissue Engineering Laboratory Sogang University Hyuncheol Kim, PhD