Case Study: Techniques for Increasing Yield in Mammalian and Microbial Cell Culture Applications Using Enriched and Pure Gaseous Oxygen
Author: Monica Cardona is a marketing manager at Pall Life Sciences, 2200 Northern Blvd, East Hills, NY 11548, 516.801.9526, [email protected].
Many valuable biopharmaceutical and biotechnological products are produced by aerobic fermentation, which includes mammalian and microbial cell culture applications. Worldwide demand for fermentation products has been increasing steadily, and this trend is expected to continue. To improve productivity, manufacturers are trying to run high-strength broths with higher biomass levels to achieve larger product yields. Because of this trend, and the fact that eliminating oxygen starvation phases can increase bioreactor and fermenter yields, improved aeration concepts have been recently established to satisfy additional oxygen demands of fermentation and cell culture unit operations.
Modern aeration increasingly uses enriched or pure gaseous oxygen to improve cell culture productivity. Using sterilizing grade gas filters that can be integrity tested by means of a Water Intrusion Test (WIT) is the best way to prevent spoilage of bioreactors and fermenters by organisms and contaminants in incoming and outgoing air and oxygen streams.
However, many materials such as organic matter, plastics, or metals can ignite when they come in contact with oxygen, particularly if also subjected to static discharges, high temperatures, pneumatic shocks or mechanical impact.
In addition to the aforementioned safety aspects, oxygen or enriched oxygen gases can lead to accelerated oxidation or corrosion of component materials.
The Demand for Oxygen
Microbial and Yeast Fermentation
Bacteria and yeasts can grow and multiply very quickly, leading to a very high oxygen demand, particularly during the exponential growth phase. With ongoing growth and metabolite production towards the stationary phase, viscosity of the fermentation broth increases, which may lead to an inhibition of oxygen transfer. Limited mass transfer of oxygen during the growth phase can inhibit biomass production, reducing the number of cells available to produce the desired product. During the stationary phase, limited mass transfer can also affect production of the primary metabolite pattern due to higher viscosity.
Growth Optimization of Mammalian Cell Cultures
Mammalian cells such as hybridomas or Chinese Hamster Ovary (CHO) cells are relatively big, complex structures, susceptible to damage by shear forces. They grow to moderate concentrations (typically 1 to 5 x 106 cells per mL) in comparison to higher biomasses seen in microbial fermentations, which can be several orders of magnitude higher. Due to the lower cell growth rates and biomass in mammalian cell cultures, oxygen demand is lower (typically < 0.1 VVM [volume per volume per minute]).
Mammalian cells, however, are fragile and can be damaged by fluid mechanical forces and shear generated by impellers or collapsing gas bubbles. High shear forces may result in a higher degree of cell lysis, and as a result, increased content of host cell protein (HCP) and other impurities in the supernatant. This may lead to higher associated costs of downstream processing and purification. In addition, the loss of cells as well as higher concentration of impurities can have an adverse impact on product yield.
Thus, routine techniques of increasing aeration through high agitation rates and liberal sparging (bubbling of a chemically inert gas through a liquid) are often not possible in mammalian cell cultures. From a practical point of view, only a few methods are feasible to aerate cells in bioreactors in order to increase cell density and product yield.
Traditional Aeration Methods
Headspace or surface aeration alone is incapable of supplying enough oxygen to cell cultures of moderate density and size, but is often used successively in conjunction with sparging. For larger-sized bioreactor and fermentation vessels, the ratio of surface area to volume decreases, making surface aeration alone impractical for reactors larger than laboratory scale.
Direct sparging of air into the culture medium has proven to be an effective method for supplying air to stirred cell cultures in large fermenters. In many fermentation processes, a sintered sparger is used.
Membrane tubing systems also have been evaluated in stirred vessels as well as in other bioreactor designs. An appropriate length of tubing (such as silicone tubing) is installed in the bioreactor. Gas is passed through the tubing and gaseous oxygen diffuses through the silicone (which acts as a membrane) into the culture medium. This method eliminates the need to sparge gases directly and eliminates problems inherent in direct sparging.
Air is often used as the sole oxygen source in fermentation with typical aeration rates ranging from 0.3 to 1.5 VVM. However, air contains 21% oxygen, 78% nitrogen, and other minor gases. In order to reach the cells, oxygen from the air must dissolve in the broth and be dispersed in the bioreactor or fermenter in small enough bubbles so that the oxygen can be efficiently transferred to the cells. Typically, most of the oxygen available from air remains undissolved and vents from the bioreactors or fermenters into the atmosphere, making it difficult to obtain even the minimal dissolved oxygen level required to sustain organism growth and maintain the desired production level.
There are several fermenter designs available that can be used for aerobic fermentations, including glass or stainless steel fermenters that have air spargers and impellers, which mix the broth and form dispersed air bubbles. Another type is an airlift fermenter, which has a sparger designed to form air bubbles as well as mix the broth.
The simplest way to increase the oxygen supply to an air-based fermentation system is to increase the air flow. This can only reduce the oxygen starvation problem at moderate oxygen demand. At higher oxygen uptake rates, the air will start flooding the impellers in mechanically agitated fermenters. In an airlifted fermenter, excess air can fluidize the entire fermenter, causing the broth to be blown out of the fermentation vessel.
Installing large agitators and motors may improve the oxygen transfer rate. However, this may also increase capital expenses as well as operating cost due to increased energy requirements. Additionally, excess heat may be generated and cooling may be required. Increased agitation could damage sensitive cells, leading to lower viabilities and yields and higher impurity levels in the fermentation broth. Even if increased capital and operating expenditure are not a concern, larger agitators and more powerful motors can provide only incremental oxygen transfer rate improvements.
Using Oxygen Enrichment Techniques in Cell Culturing
Depending on the cell culture phase, various well adjusted oxygen transfer rates are applied for optimum growth and production conditions. For mammalian cell culture, it is recommended that the impeller be used solely for mixing, and that a suitable and gentle aeration device be used to satisfy the oxygen demand of the cells. The addition of oxygen to the air stream (oxygen enriched air) can provide significant increases in oxygen transfer rates.
By adding oxygen directly to the air stream before the air filter is used to sterilize the air entering the sparger, higher oxygen transfer rates can be achieved without increased capital investment. Alternately, air and oxygen can be added directly to the bioreator. Because pure oxygen bubbles have an oxygen concentration approximately five times higher than that of air, and oxygen is dispersed in small bubbles, a very high rate oxygen transfer and dissolution can be achieved.
Pure or Enriched Gaseous Oxygen Filtration in Cell Culture
To maintain sterile or monoseptic conditions in a bioreactor, sterilizing filtration of incoming and outgoing air and oxygen is necessary. For this reason, liquid-validated sterilizing grade filters that can be easily and gently tested by a WIT are the best choice to sterilize air before it reaches the bioreactor.
Removing microbial and viral contaminants from the air stream protects the nutrient media and the cells in the fermenter from spoiling organisms such as molds, yeasts, bacteria, viruses, and phages. Fermentation vessels also possess sterilizing grade exhaust and vent filters, which also help protect the bioreactor from contamination. The vent filter also protects the operator and the environment, which is especially important for genetically modified microorganisms. All of these air, exhaust, and vent filters provide further protection by their ability to remove particulate contaminants. To ensure process safety, it is recommended that the sterilizing grade gas filters used for cell culturing be tested with a correlated WIT after both steaming and usage.
Supplying pure or blended gaseous oxygen into cell culture processes is achieved by mixing gaseous oxygen and air from the pressure line by means of mixing devices in the bioreactor, followed by a sterilizing gas filtration step. Additionally, other exhaust and vent filters on the bioreactor or fermenter may also be exposed to higher oxygen concentrations. Handling and, in particular, filtration of oxygen is a safety challenge, because the presence of oxygen can lead to ignition of materials that may not be a problem in air. Therefore, a filter construction capable of resisting ignition under specific test conditions is essential. These oxygen installations may also need approval by authorities such as Germany’s Bundesanstalt für Materialforschung und –prüfung (BAM), which is the Federal Institute for Material Research and Testing in Berlin.
To evaluate the suitability of filter materials for use in oxygen service, a standard pressure shock test with 100% gaseous oxygen at 10 bar g (145 psi) pressure at 60°C (140°F) should be performed. Test results should indicate that the filter materials did not react. Generic bacterial removal validation data and liquid bacteria challenge test data also should be correlated with WIT results to demonstrate a product’s efficacy as a sterilizing grade gaseous oxygen filter in microbial and mammalian cell culture processes.
A Safe Approach
Although oxygen enrichment has led to significant increases in cell culture yields, this technique poses many safety challenges. Avoiding static discharge by using low gas flow rates and minimized linear velocities is central to eliminating opportunities for combustion.
Proper oxygen filtration is perhaps the most important safeguard against ignition in oxygen enrichment applications. Minimizing organics on filters, using “clean” filters, preferably in a single-use format, and handling them with gloves are key techniques. Avoiding O-ring lubricants and being careful to remove oil, aerosol, and particles prior to oxygen introduction should also be part of a company’s strategy for safety. However, many materials such as organic matter, plastics, or metals have the potential to ignite when they come in contact with oxygen, particularly if they are subjected to static discharges, high temperatures, pneumatic shocks, or mechanical impact.
Because of the ignition risk, installations for gaseous oxygen require a dedicated risk assessment prior to use. For example, polypropylene found in the support and drainage layers and cage of filters could all be regarded as a fuel. The membrane would provide the surface for ignition and the oxygen could serve as an oxidizing agent, creating a scenario comparable to a rocket engine. If safety recommendations are not adhered to, a filter cartridge and its retained contaminants can start to burn.
The filter materials themselves play an important role in the safety of oxygen enrichment applications. Whereas PTFE material does not readily ignite, polypropylene, a material commonly used in oxygen filters, is much more combustible. This danger increases as temperatures and pressures rise.
BAM testing of a filter’s base polymer or elastomer also provides an important benchmark for evaluating safety. Because many polymers and seals contain materials other than the base polymer or elastomer, BAM performs material-specific testing. It is important that biopharmaceutical companies inquire about these tests to ensure the most accurate picture or filter safety.
In addition to the recommendations outlined in this paper, Table 1 provides additional guidelines for ensuring safety when using oxygen to increase product yield and safety in mammalian and microbial cell culture applications.
Table 1: Safety Guidelines*
Chemistry and Materials
• Dedicated risk assessment should be performed for all processes using gaseous oxygen concentrations higher than 21%
• Only non-flammable materials are allowed to be used in pure or enriched oxygen service
• The determination of the ignition temperature should be evaluated before and after oxygen alteration. There is a safety margin of 100°C (212°F) over process temperature at process pressure.
• German requirements: the total percentage of chromium and nickel in stainless steel pipes should be greater than 22%
• Housings and pipes made from 316L, 1.44xx, 1.43xx etc. are recommended
• Gaskets for systems and components also need approval for usage in oxygen
• Testing is required for fluorocarbon elastomers, silicone elastomer, and FEP encapsulated silicone seals
• Material Safety Data Sheets (MSDS) for materials used must be checked as part of the risk assessment in relation to use and in the event of accidents such as fires.
*Source: Pall Corporation Application Notes; 2005 Feb. “Oxygen in Mammalian and Microbial Cell Applications.”