B ioreactor development has progressed signficantly over the last few years. However it remains important to stay abreast of additional advances in cell culture research to be able to produce new drugs efficiently. Bioengineering’s (www.bioengineering.com) new aeration filter system facilitates a quick response to altered culture conditions and maintains the composition of dissolved gases and a stable pH.
For the manufacture of speciality products, such as post-translationally modified proteins, cell cultures must be used since correct glycosylation, phosphorylation, and carboxylation of proteins are not possible with microorganisms. Plant and animal cells have different properties than microorganisms—one of the most important differences is the missing cell wall. This makes cell cultures sensitive to shear stress, and the aeration and agitation systems commonly used for bacterial cultures cannot be utilized.
The slow metabolism of plant and animal cells is reflected in slow cell growth. Cell fermentations must be conducted over a significantly longer time interval than those of bacteria. Therefore cell cultures are more sensitive to contamination not only from bacteria, viruses, and phages but also from mycoplasma. Strictly defined growth conditions for production of the desired product are also essential for plant and animal cells. These include not only complex and often expensive nutrients but also the correct composition of added gases and a stable pH. Therefore bioreactors must be adapted for cell culture.
The high shear sensitivity of animal and plant cells has led to significant alterations in the construction of plants compared to bacterial fermentors. Since the gas supply for cells is dependent on aeration and agitation, both systems must be adapted for cell culture.
Adherent cells are cultivated on microcarriers either in the reactor or in a fixed bed. Therefore they are not directly affected by shear stress. The mechanical shear stress on non-adherent cells in submerged culture can be minimized either with marine impellers, which provide a more gentle agitation than common blade stirrers, or with airlift reactors where the agitation is achieved exclusively via the aeration.
Sensitive cells are damaged by agitators and by collapsing gas bubbles and foam. The simplest way to protect the cells from bubbles and foam is the addition of antifoam agents to the medium. However these substances are unsuitable for a variety of cell types since they negatively influence the growth or metabolic functions essential for the manufacture of product. In many cases they also significantly interfere with downstream processing.
Therefore sparger systems were developed for cell culture, producing gas bubbles with minimum shear stress on the cells. With extremely sensitive cells, bubble-free aeration can be achieved via gas-permeable hoses.
Gas Composition, Supply, and Demand
The design of the aeration system and gas composition are often of vital significance for cell culture. Plant cells, especially, adapt their metabolism to minor changes in gas composition and thus alter growth characteristics and the production of secondary metabolites. Aeration with oxygen-enriched air leads to improved production results with many types of animal cells.
Unlike bacterial cultures, which are aerated with air, cell cultures are aerated with oxygen, nitrogen, CO2, and air in a mixture optimized for each type of cell and fermentation. Gas composition is regulated by sensitive control systems, and an optimum gas atmosphere should be created and maintained for the fermentation and manufacture of the product.
During cell proliferation, the pH of the medium changes. A constant pH is vital for many production processes and therefore it must be controlled. pH control is not affected with acid and base dosing as it is with bacterial fermentations but instead via controlled addition of CO2.
Supply of the four gases used is normally accomplished with a gas-mixing station. In this device the dosage of each gas is controlled by a gas-flow controller. Additionally the pH controller influences the dosage of CO2. A pO2 controller regulates the aeration with air or oxygen and nitrogen. Thus the optimum gas supply for the cells is facilitated for every phase of the fermentation.
In cell culture it is essential to maintain absolutely sterile conditions because of the high contamination risk caused by slow cell growth and long fermentation times. Naturally this also applies to the gas supply. The gases used must not only be free of microorganisms but must also be free of oil and dirt particles.
In small autoclavable bioreactors, single-use filters with hydrophobic PTFE membranes are used to sterilize process gases and exhaust air. These filters are autoclaved together with the bioreactor. In situ sterilizable small bioreactors and bigger plants utilize filter cartridges integrated in filter housings to ensure a sterile gas flow. Either filters with PTFE-protected microfibers or membrane filters with synthetic PTFE-membrane are used. Depending on the reactor design, filters and filter housings are either sterilized directly with the vessel or steamed independently in a defined sequence.
The oxygen level in liquid media is dependent on the amount of dissolved oxygen, the oxygen in the gas phase above the medium, and also the gas bubbles in the medium. Because of reduced growth rates, oxygen demand is lower than for bacterial cultures. Therefore oxygen transfer rates can also be lower, allowing low agitation speed and low aeration rates. Shear stress on the cells is thus minimized. Nevertheless, gas composition is not yet under satisfactory control in many cell fermentations.
Problems with the Gas Supply
With the high gas flow and strong agitation seen in bacterial cultures, the length of the pipes from the gas supply to the vessel is hardly relevant. Gases are distributed by inlet pressure and agitation and thus reach the cells rapidly. The efficient supply of required gases to plant and animal cells, which are less aerated and agitated, is not as simple.
Plant and animal cells tend to require a highly defined gas atmosphere and a stable pH. Long pathways for the gases and relatively high retention times in large filter housings could delay pH and pO2 control in many cell cultures and thus lead to losses in productivity.
To counteract these effects, three steps must be taken—a shortening of the pipes between the gas-mixing station and reactor, a reduction in pipe diameter, and a reduction in the dead volume of filters and filter housings.
The first two design changes are relatively straightforward and depend on plant geometry. But the alteration of filters and filter housings is more problematic.
The smallest dead leg and the shortest gas flow path are to the autoclavable single-use filters. These filters can not be sterilized with in situ sterilizable bioreactors and therefore can not be integrated into the plant. If the filters are autoclaved separately and mounted to the plant after sterilization of the reactor, a source of contamination is created.
The solution to the problem, as developed by Bioengineering, is based on the utilization of single-use filters with hydrophobic PTFE membranes and optimized dead legs for in situ sterilizable systems. Thus the retention time of the gases in the filters is minimized. Gas retention during the passage through filter housings is thusly avoided, since the new system simply dispenses with additional housings. Because of the small size of the filters the pipe diameters of the aeration lines can be kept small and the gas passage short.
How can the incompatibility of autoclavable single-use filters with in situ sterilizable bioreactors be circumvented and the full integration of filters in the plant be assured?
In Bioengineering’s new aeration system, single-use filters for each of the four gases in the gas-mixing station are located in an autoclave. This autoclave is integrated into the piping system of the plant and fitted with a viewing glass. A special set of valves, which are steam permeable, is mounted in the autoclave. The steam generated by the autoclave sterilizes the aeration line to a valve in the sterile cross. The pipe from the vessel to the sterile cross is sterilized in the course of the vessel sterilization, and the single-used filters are automatically sterilized in the autoclave.
Thus full integration of the autoclave and the filters into the plant is assured. The automatic sterilization of the entire plant, including single-use filters, is rendered possible. No manipulation of the filters is conducted after sterilization, minimizing the contamination risk and ensuring conformity with applicable regulations.
The advantages of the Bioengineering system are many. Cell culture operations in bioreactors can be supplied with the required sterile gases in the shortest possible time—this creates fast control loops for pH and pO2 control and therefore efficient response to all changes in cell metabolism during fermentation. Integration and automation of the aeration system significantly minimizes the work effort with single-use filters and provides the same hygienic safety as is standard with larger filters and filter housings.