This is the an excerpt for a process description of my 2010 AICHE National Student Design Competition Report. Note the detail in the piping and instrumentation diagrams coupled with the process description.

2. Temperature sensor

3. pH/DO sensor

4. Buffer inlet tube

5. Axial flow hydrofoil agitator

6. Level probe sensor

7. Inlet gas sparge tube

9. Dissolved oxygen percentage of 5-35%

10. Temperature of 36-38°C at ambient pressure

11. Maintain an overall pH of between 6.5-7.0

12. Addition of trace amount of sodium butyrate in supplemental media

13. Maintain an overall higher lactic acid concentrationMaintaining the same temperature and pressure ranges from the growth phase lead to cell metabolite production at the same optimum levels. Also, agitation speeds of around 200 RPM allow for oxygen to dissolve into the concentrated cell solution without causing severe stress on the cells, which would lead to cell rupture. This optimal agitation speed provides a high volumetric mass transfer coefficient of air to the bioreactor culture, kLa. To create a cell medium favorable to secondary metabolite production, the reactor should be held at lower dissolved oxygen levels, which will inhibit primary metabolic pathways. The pH of the reactor can also be held at slightly acidic levels to inhibit biomass production. Another inhibition to cell growth would require adding sodium butyrate in trace amounts. Sodium butyrate allows DNA to be more accessible to the RNA polymerase enzyme. Finally, by maintaining a higher lactic acid waste concentration within the solution, cells will stop producing biomass as well.<br />These operating conditions also allow for a stable medium that will hinder glycosylation or the breakdown of monoclonal antibodies. Proteokinase enzymes responsible for cleaving proteins like MAbs into constituent parts are inhibited at neutral to slightly acidic pH. Despite this fact, MAbs must be concentrated and collected as soon as the residence time in the reactor is complete because glycosylation of proteins is time dependent. The lifetime of MAbs in solution is limited to a few hours so all downstream purification processing needs to be timely and effective. The above reactor conditions lead to a MAb yield of approximately 21.5 kg/L-reactor.<br />Clean In Place Phase:<br />Once the run phase is complete and the reactor is empty, the clean in place phase can begin. Clean in place (CIP) consists of a flushing stage, a washing stage, and two rinsing stages. The CIP feed tank (T-100) fills with ambient temperature process water used for the flushing stage. This water is pumped into the reactor via P-100, and once the reactor has reached 75% liquid volume, the agitator is switched on to maximum (1,000 RPM) to shear settled particles off of the walls of the reactor. This shearing process occurs for 10 minutes, and then the effluent is drained. <br />The flushing effluent is pumped via P-101 to the clean in place effluent tank, T-101. A sample of the effluent will be taken for analysis by the quality control unit, to check if contaminants are present via biological plating. This sample represents the final sample before product extraction occurs and therefore is important to ensure the batch isn’t contaminated. In this tank, Ammonium Sulfate is added as a flocculant to help the flushed biomass accumulate into a film within the effluent. From here, the effluent is pumped into an automatic strainer (S-100), which retains the flocculated biomass and discharges it once a pressure drop of 30 psig is reached. The discharged biomass is sent to quarantine, where it will be retained for a few days to check for contaminant growth by the quality control unit and then disposed of. The leftover liquid effluent returns to the CIP feed tank where it is combined with concentrated sodium hypochlorite (bleach) to a specific dilution of between 30 and 100 ppm. From here the washing stage begins.<br />The bleach solution is heated by a pre-heater (E-101) to a temperature of 75°C and pumped into the reactor until a volume of 25% of the working volume is reached. E-101 is controlled by a programmable logic control (FOC-104) to a specific temperature set point. Then this liquid level is maintained while the bleach solution inlet changes to a ball spray nozzle port. This spray nozzle allows for wider surface area coverage of the bleach within the reactor. <br />After an effluent level is obtained in the reactor, the pH/DO controller on the reactor (SIC-100) controls the concentration of NaOH mixed with the bleach effluent to create an alkaline washing solution with a pH of 12. The effluent continues to flow to the CIP effluent tank where flocculants are added, and it returns to the CIP feed tank. To control the bleach concentration within the washing recirculation loop, an in-line conductivity meter (XE-100) will signal a controller (XIC-100), which controls the inlet flow rate of the concentrated bleach to the CIP feed tank. An alkaline solution allows for breakdown of all cellular and media components within the recirculation loop. Equation x was used to calculate the amount of NaOH necessary to reach a pH of 12.<br />1pH=log10(1C)()<br />In this equation, C represents the overall concentration of NaOH in grams per liter of solution. After two hours of recirculation, the effluent is sent to the CIP feed tank where it is drained to a waste reservoir, cooled over time, neutralized, and flushed to the sewer. Then the reactor is refilled with water from the CIP system at ambient temperatures and HCl , which is added through the buffer inlet lines to bring the pH of the solution to 5 (controlled by SIC-100). Equation x is used, but the left side of the equation is pH instead of the inverse of pH. This slight acid wash recirculates for 30 minutes to neutralize all alkaline solution leftover in the process, which is the first of two rinsing stages. Once the slight acid is drained from the CIP system (used to neutralize the alkaline solution), ambient water from the CIP system fills the reactor to 75% volume as the final rinsing step. Lastly, this volume of water is agitated at 1,000 RPM’s and drained after 10 minutes.<br />Steam In Place Phase:<br />Upon completion of the clean in place phase, the steam in place phase begins. The sparge tube inlet to the reactor is opened to 150 psig steam and all other reactor lines are closed. Steam flow is controlled by a programmable logic control (FOC-104) to a pressure of 80 psig and a temperature of 121°C, which is equivalent to an autoclave device used to kill microbes. The vent line maintains steam pressure in the reactor and after 40 minutes, the reactor is returned to ambient temperature and pressure. This extra cleaning step ensures that all reactor sensors, inlet and outlet ports, agitation seals, reactor seals, and other hard to clean areas are sterilized before the next run phase. In combination with the clean in place phase, this cleaning scheme would also reduce sensor fouling and reduce the risk of contamination significantly (see discussion of results section).<br />The P & ID schematic below (PID-100) displays all equipment and control modules discussed above. Equipment specification sheets with a P & ID symbols key are located in Appendix A.<br /> <br />