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Wednesday, July 16, 2008

40 L FERMENTER

AIM
· To carryout FSIP of 40 L Fermenter
· To perform fermentation of yeast culture in batch mode
· To study the effect of dosing on the growth of yeast
· Cleaning in place and ESIP

PRINCIPLE
Sterilization in place of any equipment can be done conveniently thereby eliminating the need of subsequent aseptic connections or shut down of the whole process. Typically SIP uses saturated steam at 15 psi for at least 30 minutes. In Microbial fermentation, FSIP is performed. The whole sterilization is done after charging the media into the vessel. On the other hand, in animal cell culture ESIP is carried out where the sterilization is done before charging the vessel with media. Before ESIP, integrity of vessels and filters is checked.

LIST OF MATERIALS
Pre-inoculum of Pichia pastoris culture, conical flask, TSB media powder, dextrose powder, antifoam, measuring cylinder, brown paper, spatula, cotton, rubber bands, pipette, test tubes, test tube stand, beaker, tissue, cuvette, distilled water, WFI

LIST OF EQUIPMENTS
40 liter Fermenter and other utilities, HMI (Human machine interface), weighing machine, pH meter, autoclave, laminar air flow, shaker cum incubator, spectrophotometer, media preparation vessel, harvest vessel, CIP skid.





PROCEDURE:
Ø Pre- fermentation steps
1. Prior to the first day of the fermentation process in the 40l Fermenter, the Pichia Pastoris culture inoculum was setup by inoculating 5l Fermenter with volume 3l of TSB media of pH 5.0..
2. 250 ml of 25% of antifoam (silicone oil) was prepared and autoclaved.
3. 2N NaOH (250ml) was prepared and autoclaved.
4. 20 liter of TSB media pH 5.0 prepared in a 3l concentrated form by dissolving 600g of TSB in 3 liter W.F.I. and autoclaved.
Ø Thermo siphon sterilization
1. All the utility lines were checked.
2. The mechanical seal and the thermo siphon pot were sterilized by passing steam for 20 minutes. The corresponding steam traps were opened.
3. Chilled water supply was started after closing the steam supply valve so that the condensate forms in the pot. Once the condensate was formed, the corresponding valves were opened such that the condensate reached the mechanical seal.
4. The probes (pH and DO) were attached. The calibration of pH probe was done using buffer of pH 7.0 and 4.0.
5. The 4 filters (pre-filter, surface, sparger, vent) were fixed into the respective slots and the corresponding housings were placed and tightened using TC clamps.
6. 17 L of W.F.I. from the media preparation room was transferred into the Fermenter vessel.
7. 3 liter of xxxxxx(autoclaved)xxxxxx concentrated media was poured.
Ø Perform the Pressure Hold Test
1. Adjust 2 bar pressure using the PRV and wait till the pre-filter pressure gauge attains 2 bar. Pressure hold test was performed in the surface and sparger air filter by opening the respective lines and isolating the filters. The pressure hold test was carried out from the surface filter to the vessel and then from the vessel to the exhaust filter.
2. The agitation was started at 200 RPM and the vent drain valve was opened.
Ø Draining of the jacket
1. The drain and jacket inlets were opened and closed after 2-3 min.
Ø Heating step
1. Steam was passed to the jacket and the corresponding steam trap was opened.
2. The exhaust and the 3 steam traps on the exhaust line were opened.
3. When the temperature of the vessel reached 70°C the needle valve on the exhaust line was opened for 2 minutes to remove the air traps.
4. All transfer lines and their respective steam traps were opened to remove air trap
Ø Sterilization step
1. When the temperature reached 121°C, steam was supplied to the vessel also by opening the respective valve. Steam valves and the drain valves of the surface and sparger air filters were opened to sterilize them. The sampling and the harvest port lines were also sterilized. Sterilization process was continued for 20 minutes and when the temperature shooted up, it was regulated by supplying and cutting off the steam supply into the vessel through the surface and sparger air filters.
2. Now, supply only jacket steam for 10 minutes. Close the sparger line to the Fermenter and surface line.
Ø DRying of filter
1. The surface and sparger air lines and their corresponding drain were opened for 10 minutes.


Ø Completion of sterilization
1. After total sterilization time of 30 min, the transfer lines were closed. The steam valves were also closed. The surface air valve was opened to allow air to pass through the vessel.
Ø Cooling of the fermenter
1. This was done by supplying chilled water through the open loop first. It was noted that the vessel was pressurized during this process so as to avoid collapsing of the vessel due to the sudden temperature change. When the temperature dropped down to 60ºC, the chilled water was supplied to the jacket using the closed loop.
Ø Setting of parameters
1. Aeration - 0.5 vvm (10 LPM).
2. Before agitation was started the thermo siphon was set at 2 bar pressure.
3. Temperature loop was started.
4. Condenser was switched on so that evaporation loss due to aeration is minimised.
5. Heat exchanger on the vent was switched on so that the exhaust filter does not choke.
6. Back pressure of 0.2 bar was maintained.
7. Antifoam was added into the fermenter.
8. DO probe was calibrated. The present amount of oxygen was treated as 100%.
Ø Inoculation
1. Temperature inside the vessel was brought down to a set point of 300C which was the optimum for yeast culture. The transfer ports were connected to their respective positions and were sterilized by passing steam. The inoculum was added to the vessel using a peristaltic pump and the process was started.