This article was written by the engineering staff of B. Braun Biotech.
Every day we hear more about the dramatic advances in genome engineering. Researchers believe their work will lead to cures for the most deadly and debilitating diseases. B. Braun Biotech in Allentown, Pa., makes some of the tools used in this research. The company manufactures bioprocessing reactors and cell fermenters, and has customized multiplex fermentation systems to enhance cell production throughput.
At the heart of these multiplex systems is a traditional industry product, the solenoid valve. Typically, Pneutronics/General Valve, a division of Parker Hannifin Corp., applies its solenoid valve technologies for such applications as the piloting of air cylinders, controlling deflation rates in blood pressure cuffs, or the introduction of solvent gradient mixes into liquid chromatographs. But now the engineers at B. Braun have expanded the role of its solenoid valves into the genomics arena.
In order to understand the fit between these two technologies, we first must look at the required process.
Fermentation is a term used by microbiologists to describe any process for the production of cells, naturally improved strains, or genetically engineered strains.
Batch fermentation is basically a closed system. After the sterilized solution in the fermenter is inoculated with microorganisms, nothing is added, except oxygen, an antifoam agent, and acid or base to control the pH. The microorganism growth is controlled under extremely stringent physiological conditions.
One of the most critical factors in the operation of a fermenter is the provision of adequate gas exchange. Oxygen is the most important gaseous substrate for microbial metabolism, and carbon dioxide is the most important gaseous metabolic product. When oxygen is required as a microbial substrate, it is frequently a limiting factor in fermentation.
Because of its low solubility, only 0.3 mm O2, equivalent to 9 mg/l, dissolves in one liter of water at 20ºC in an air-water mixture. An active and concentrated microbial population will deplete this amount of oxygen in a few seconds, unless oxygen is supplied continuously. In contrast, during the same period the amounts of other microbial processes are oxygen limited. This is why the concept of gas-liquid mass transfer in bioprocessing is centered on oxygen transfer, even if other gases such as carbon dioxide; hydrogen, methane, and ammonia can also be involved.
The system needs to monitor the O2 and CO2 levels to ensure that critical oxygen concentrations are with specified values. The nutrient media for production must be optimized, not only for ingredients used but also for how the medium is prepared and sterilized and the pH valve before and after sterilization. The entire process needs to be managed and monitored to ensure these activities.
The process management is concerned with:
• Setting up the initial process conditions.
• Monitoring to ascertain whether the process is following the required course.
• Facilitating manual adjustments to the process variables.
• Deciding when to terminate the process and/or to transfer or harvest the product.
• Calculating the mass and thermal balances, rates of reaction, kinetics and yields.
• Monitoring contamination and process hygiene.
B. Braun Biotech developed a system called the multiplexer to ensure proper process management while maintaining high cell production throughput. The multiplexer directs an exhaust gas sample from the fermenter to a mass spectrometer for analysis. The multiplexer can sequence up to 12 fermenter samples through one mass spectrometer. It does this by the use of General Valve 4-station Series 66 valve manifold arrays. The Series 66 valve is located between each cell fermenter and the mass spectrometer. (See schematic below).
When the valve is sequenced open, it allows the sample gas into the mass spectrometer for analysis. The analyzer then determines the molecular level of each specific gas and determines whether the ideal fermentation environment exists. The mass spectrometer analysis will provide information on the pH level, oxygen transfer and uptake rates, carbon dioxide evolution rate and sugar level for yield determinations. The results of the mass spectrometer analysis may require the operator to adjust the agitation speed, temperature, flow rates, oxygen percentage, pH level, and pressure among other variables within the cell fermenter.
The problem encountered by the company was simple, but not necessarily easy to solve. The general industry solenoid valves that they were accustomed to using were susceptible to corrosion from the high humidity levels in the exhaust gas stream. Since they currently use Parker Hannifin for all their instrumentation fittings and isolation and pilot valves, contacted a representative at that company, who then located and connected the B. Braun engineers to General Valve sale engineers Dan Whelahan and Tom Powell.
Whelahan and Powell assessed the situation and offered a manifold solenoid valve mounted onto a four-station Teflon manifold equipped with easy access ¼-28 quick connect fittings. The solenoid valve was completely impervious to corrosion, because it was constructed with an elastomeric diaphragm and an inert PEEK body that isolate the gas media from any metallic wetted components.
The Series 66 valve also offered the added advantage of very low internal and negligibly low unswept volumes. These features allowed B. Braun engineers to minimize the sample size into the mass spectrometer, thus reducing any potential negative effects to the fermenter from the loss of the volume of the O2/CO2 sample gas. The valve's low unswept volumes gave B. Braun the added flexibility of switching lines without concern of carryover from one sample gas stream to the next, thereby maximizing system efficiencies and gaining their customer's desired high cell production throughput.