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

2. Drug Delivery & Tissue Engineering lab. Sogang University The set up of the bioreactors and the process steps are performed in a specific order

3. Drug Delivery & Tissue Engineering lab. Sogang University 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

4. Drug Delivery & Tissue Engineering lab. Sogang University

5. 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. 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 Skid CIP Solutions

6. Drug Delivery & Tissue Engineering lab. Sogang University 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 Spray ball mounted on manway lid

7. Drug Delivery & Tissue Engineering lab. Sogang University

8. Drug Delivery & Tissue Engineering lab. Sogang University While setting up the bioreactor, the three main activities are to: – Install inlet and exhaust filters – Prepare and install pH, DO, and CO 2 probes  Calibrate as necessary – Install addition and sample port valves

9. Drug Delivery & Tissue Engineering lab. Sogang University 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. The filters should be inspected for cracks and to ensure the O-ring is intact and the filter fits into the housing tightly. Sterile Boundary Filters in housing

10. Drug Delivery & Tissue Engineering lab. Sogang University DO, pH and CO 2 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 (Multiple types of ports are used for probes.)

11. Drug Delivery & Tissue Engineering lab. Sogang University 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

12. Drug Delivery & Tissue Engineering lab. Sogang University

13. 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

14. Drug Delivery & Tissue Engineering lab. Sogang University 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.

15. Drug Delivery & Tissue Engineering lab. Sogang University 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.

16. Drug Delivery & Tissue Engineering lab. Sogang University

17. Drug Delivery & Tissue Engineering lab. Sogang University

18. 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

19. Drug Delivery & Tissue Engineering lab. Sogang University An SIP cycle consists of these stages: Heat, Hold, and Cool Down

20. Drug Delivery & Tissue Engineering lab. Sogang University

21. Drug Delivery & Tissue Engineering lab. Sogang University 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.

22. Drug Delivery & Tissue Engineering lab. Sogang University

24. Bioreactor Monitoring

25. Drug Delivery & Tissue Engineering lab. Sogang University 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.

26. Drug Delivery & Tissue Engineering lab. Sogang University

27. Contamination

28. Drug Delivery & Tissue Engineering lab. Sogang University 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.

29. Drug Delivery & Tissue Engineering lab. Sogang University 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:

30. Drug Delivery & Tissue Engineering lab. Sogang University 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.

31. Drug Delivery & Tissue Engineering lab. Sogang University Cells Time

32. Drug Delivery & Tissue Engineering lab. Sogang University 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

33. Drug Delivery & Tissue Engineering lab. Sogang University

34. 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 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)

35. Drug Delivery & Tissue Engineering lab. Sogang University 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

36. Drug Delivery & Tissue Engineering lab. Sogang University Raw materials People Equipment Environment

37. Drug Delivery & Tissue Engineering lab. Sogang University

38. Drug Delivery & Tissue Engineering lab. Sogang University

39. Drug Delivery & Tissue Engineering lab. Sogang University

40. Drug Delivery & Tissue Engineering lab. Sogang University Filter integrity testing is performed after using the media filters to ensure that the filters were not damaged and they properly filtered the media. Filter Integrity Test Destructive Testing Non Destructive Testing Bubble Point Test Diffusion Test Water Intrusion Test Forward Flow Test Pressure Decay Pressure Hold

41. Drug Delivery & Tissue Engineering lab. Sogang University Pasteurization performed to minimize contamination in raw materials. HTST – High Temperature Short Time UV - Ultraviolet

42. Drug Delivery & Tissue Engineering lab. Sogang University Need to understand the chemical make up of your raw materials Size is an issue Timing and Cost

43. Drug Delivery & Tissue Engineering lab. Sogang University Ultimate Goal is to protect the product in order to protect the patient!

44. Thank You