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.
Fed-Batch Biological Production Reactors<br />Description:<br />This description represents 1 of 50 biological reactors present in the overall process. All design aspects and P & ID’s are held constant for each biological production reactor. From a seed train, the concentrated cell solution travels to R-100, one of five fed-batch biological production reactor. R-100 is a 2,000 liter continuous stirred tank reactor (CSTR), which comes equipped with:<br /><ul><li>Reactor jacket
Vent gas paramagnetic analyzer</li></ul>The reactor jacket is used to dissipate the heat of reaction generated by the biomass in the reactor. The reactor jacket is controlled by a temperature controller (TIC-100) connected to the temperature sensor (TE-100) in the reactor, which also passes through a programmable logic control (FOC-104) that determines the process state of the reactor. There are three process states for the reactor, clean in place state, feed state, and steam in place state. When the reactor is in clean in place state, FOC-104 regulates the temperature of the inlet cleaning solution to the reactor. When the reactor is in feed state, FOC-104 controls the reactor temperature. When the reactor is in the steam in place state, FOC-104 controls the steam flow rate to the reactor.<br />The pH/DO sensor (SE-100) measures the pH and dissolved oxygen (in mg of O2 per liter of solution) of the reactor contents. The pH and dissolved oxygen levels in the rector are controlled by SIC-100. SIC-100 regulates the flow of basic buffer solution (0.01M NaOH) and acidic buffer solution (0.01M HCl) through the buffer inlet tube to control the pH of the reactor contents. This controller also regulates the air supply into the reactor through the inlet gas sparge tubes to control dissolved oxygen percentage within solution. The inlet gas tubes in the reactor have an in-line membrane filter cartridge to filter out any particulate matter going to the reactor. This filter has a nominal filter size of 0.2 micron and is easily substituted via disposable filter cartridges. These tubes also have a check valve to prevent reactor contents from back flowing into gas supply lines. <br />The axial flow hydrofoil agitator has a variable speed drive motor that operates between 0 and 1,000 rpm. It is located 15° off center to ensure proper mixing of the biomass with media at the wall of the vessel. The level probe sensor (LE-100) measures the liquid level of the reactor. This level measurement is transmitted to a level controller (LIC-100), which allows for easy monitoring by operation personnel and controls the liquid level of the reactor to 75% via the a cascade with the flow rate controller (FIC-101). FIC-101 controls the pneumatic valve downstream of the production reactor and effectively controls the drain rate of the reactor. The reactor level also has a high level alarm (LAH-100) that triggers to alert personnel at levels above 80% liquid level, to prevent overflow or foam fouling of the vent gas line. The reactor, the CIP effluent tank, and the CIP feed tank have pressure relief valves sized to 88 psig, to prevent pressure failure. 88 psig is 10% above the operating pressure of these vessels, but 15% lower than the maximum allowable working pressure. The gases accumulated from the vent line will pass through a paramagnetic analyzer, which measures the oxygen concentration of the gases. This measurement is used to perform a fermentation balance to estimate the amount of glucose present in the reactor, which allows for more precise additions of make-up medium to the reactor.<br />All equipment, including piping, will be constructed from 316 stainless steel to prevent chemical corrosion from the exposure to clean in place chemicals such as sodium hypochlorite and all seals/gaskets will be fluoroelastomers, such as Viton®. All pumps in the process will be screw centrifugal impeller pumps, the same pumps used for transport of activated sludge in wastewater treatment plants. All vessels containing slurry will have conical bottoms with large diameter piping (4”) to prevent solid particulates from accumulating in the vessels or plugging discharge ports. All piping will have a pitch of ¼” per foot of piping to prevent solid settling and all gauges will be placed in vertical runs of pipe only to prevent slurry fouling. All valves requiring sterilization in this process (clean in place) will be diaphragm valves, which are easily cleaned and controlled. All sensors (elements) will be constructed from corrosion resistant materials and will be able to withstand temperatures above 121°C and pressures of 100 psig. The whole reactor system will be air tight to help prevent contamination and all stainless steel surfaces in contact with cells will be electro polished to create smooth surfaces that cells won’t stick to. Lastly, all sensors for pH, dissolved oxygen, temperature, and conductivity will be contained in an overall quality monitoring system with a data logger to record all measurements. <br />Run Phase:<br />Before the concentrated cell solution is pumped into the reactor, the reactor is pressure inerted with nitrogen through the sparge tube. The nitrogen pressure in the reactor is brought to 50 psig and vented a total of two times to ensure the oxygen level is brought to below detectable levels according to the pH/DO sensor. A low oxygen level upon onset of MAb production will allow for initial detection of media characteristics due to the rate of oxygen uptake of the culture. Once the pressure in the reactor is brought back to atmospheric, the concentrated cell solution is pumped into the top of the reactor. The vent line is left open to maintain atmospheric pressure conditions within the reactor. The vent line also contains a membrane filter cartridge to 0.2 micron that ensures minimal biological agents exit through the vent gas stream.<br />Once the reactor fills to a volume of 500 liters, the agitator is turned on to begin mixing the contents. Once mixing has started, the air inlet is opened to allow oxygen to dissolve into the concentrated cell solution. The reactor contents are then brought to and maintained at a set pH level through the buffer inlet tubes. The reactor reaches quasi-steady state once the level set point of 75% is reached. From here, the concentrated cell solution is retained for a residence time of approximately 4 days; allowing for ample monoclonal antibody formation. <br />Through the feed line, make up growth medium containing vital cell nutrients is continuously fed into the reactor. On the other side of the reactor through the product line, settled cell effluent is being pulled off and sent to the biological kill tanks present in the biological waste removal unit. <br />The flow rates of both of the feed and product lines will be determined by the cybernetic model produced for monoclonal antibody production. The product line also comes with a sample port used for high pressure liquid chromatography (HPLC) analysis of cell effluent and biological plating analysis to determine if contaminants are present within the batch. The quality control unit can filter the cell effluent and use HPLC to determine the amount and type of nutrients the cell needs, as well as the amount and type of waste products the cell is producing (see quality control unit section).<br />It is assumed that concentrated cell solution enters the reactor in the beginning of stationary phase. Therefore, to increase monoclonal antibody production and prevent chemical breakdown or cellular glycosylation of this secondary metabolite, the reactor should operate under certain conditions. <br />Optimum operations condition estimates are as follows:<br /><ul><li>Agitation speed of 80 RPM
Addition of trace amount of sodium butyrate in supplemental media
Maintain an overall higher lactic acid concentration</li></ul>Maintaining 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 />