Oxygen-Safe Culture Applications

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 thistrend is expected to continue.

To improve productivity, manufacturers are trying to run high-strength broths with higher biomass levels to achieve larger product yields. Due to the 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. Sterilizing grade gas fi lters that can be integrity-tested by means of a Water Intrusion Test (WIT) are 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 potentially ignite when they come in contact with oxygen, particularly when subjected to static discharges, high temperatures, pneumatic shocks, or mechanical impact.

Oxygen or enriched oxygen gases can also lead to accelerated oxidation or corrosion of component materials.

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. This 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 that are susceptible to damage by shear forces. They grow to moderate concentrations (typically 1 to 5 x 10’ 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 are fragile and can be damaged by fl uid mechanical forces and shear generated by impellers or collapsing gas bubbles. High shear forces may result in a higher degree of cell lysis. This increases the content of host cell protein (HCP) and other impurities in the supernatant. This may lead to higher associated costs of downstream processing and purifi cation. In addition, the loss of cells and 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. It 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 is proven to be an effective method for supplying air to stirred cell cultures in large fermenters. In most fermentation, a sintered sparger is used.

Membrane tubing systems also have been evaluated in stirred vessels and 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 effi ciently 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 diffi cult to obtain even the minimal dissolved oxygen level required to sustain organism growth and to 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 and to 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 fl ooding the impellers in mechanically agitated fermenters. In an airlifted fermenter, excess air can fl uidize 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 costs 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. Despite increased capital and operating expenditure, larger agitators and more powerful motors can only provide incremental oxygen transfer rate improvement.

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. Oxygen to the air stream (oxygen-enriched air) can also provide signifi cant increases in oxygen transfer rates.

By adding oxygen directly to the air stream before the air fi lter is used to sterilize the air entering the sparger, higher oxygen transfer rates can be achieved without more capital investment (See Figure 1). Alternately, air and oxygen can be added directly to the bioreator. Pure oxygen bubbles have an oxygen concentration that is approximately fi ve times higher than that of air, and oxygen is dispersed in small bubbles, achieving a very high rate of oxygen transfer and dissolution.

Pure or enriched gaseous oxygen filtration in cell culture
To maintain sterile or monoseptic conditions in a bioreactor means sterilizing fi ltration of incoming and outgoing air and oxygen is necessary. For this reason, liquid-validated sterilizing grade fi lters 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 fi lters, which also help protect the bioreactor from contamination. The vent fi lter also protects the operator and the environment, which is especially important for genetically modifi ed microorganisms.

These air, exhaust, and vent fi lters provide further protection by their ability to remove particulate contaminants. To ensure process safety, it is recommended that the sterilizing grade gas fi lters 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 fi ltration step.

Additionally, other exhaust and vent fi lters on the bioreactor or fermenter may also be exposed to higher oxygen concentrations. Handling and, in particular, fi ltration 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 fi lter construction capable of resisting ignition under specifi c test conditions is essential. These oxygen installations may also need approval by authorities such as Germany’s Bundesanstalt für Materialforschung und -prüfung (Federal Institute for MaterialResearch and Testing [BAM]) in Berlin.

To evaluate the suitability of fi lter 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 fi lter 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 effi cacy as a sterilizing grade gaseous oxygen fi lter in microbial and mammalian cell culture processes.

A safe approach
Although oxygen enrichment has led to signifi cant increases in cell culture yields, this technique poses many safety challenges. Avoiding static discharge by using low gas fl ow rates and minimized linear velocities is central to eliminating opportunities for combustion.

Proper oxygen fi ltration is perhaps the most important safeguard against ignition in oxygen enrichment applications. Minimizing organics on fi lters, using “clean” fi lters, preferably in a single-use format, and handling them with gloves is important.

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 (See Figure 2).

Due to ignition risk, installations for gaseous oxygen require a dedicated risk assessment prior to use (See Figure 3). For example, polypropylene found in the support and drainage layers and cage of fi lters 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 fi lter cartridge and its retained contaminants can start to burn.

The filter materials play an important role in the safety of oxygen enrichment applications. Polytetrafl uoroethylene (PTFE) material does not readily ignite, but polypropylene, a material commonly used in oxygen fi lters, is much more combustible. This danger increases as temperatures and pressures rise.

BAM testing of a fi lter’s base polymer or elastomer also provides an important benchmark for evaluating safety. BAM performs material-specifi c testing as many polymers and seals contain materials other than the base polymer or elastomer. Biopharmaceutical companies should inquire about these tests to ensure the most accurate picture or fi lter safety. PA

Figure 1

Figure 2

Figure 3

Safety Guidelines Chemistry and materials • Dedicated risk assessment should be performed for all processes using gaseous oxygen concentrations higher than 21%. • Only non-fl ammable 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 UW/9: the total share of chromium and nickel in stainless steel pipes should be above 22%. • Housings and pipes made from 316L, 1.44xx, 1.43xx, etc. are recommended. • Gaskets for systems and components need approval for usage in oxygen. • Testing is required for fl uorocarbon 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. Operation of gaseous oxygen systems • Sparged air often contains oil from the compressor which, if mixed with oxygen in a common transfer pipe, presents a potential fi re hazard. Therefore, air from compressors must be oil-free or use oxygen-suitable oils. • If any oils or liquid hydrocarbons are present and collect on the fi lter, they can ignite spontaneously (especially in a hot system), or be ignited by a spark. Oils are available specifi cally for use in oxygen service. • Single use of sterilizing grade gas fi lters are recommended. Special instructions on fi lter installation and operation required • Oil, aerosol, and particle-free pressurized air lines can be supported by using coalescers and stainless steel, and particle filters. • The risk of static discharge at low fl ow rates is small. However, with high fl ow rates in dry gases (or low humidity), static risks are higher. Grounding the housing is recommended in these cases. • Avoid static discharging that may lead to membrane damage, ignition and combustion of filters. • Precautions must be taken to avoid oxygen back-fl ow into the air line. • Pressure shocks and pulses should be avoided. • Valves should only be operated and actuated slowly during opening and closing. • Automated systems should be used to prevent operator errors.