28

Feature

Filtration+Separation May 2006

Gas filtration:

sterile microfiltration for bioreactors

C
ontinuing with the broad theme of gas filtration, we now look at sterilising gas filters - a valuable and indispensable part of modern biotech and pharmaceutical processes. Jignesh Padia of QSV Biologics, and Paul Huntington from Cheme Engineering take us through the process options.
the microbes in the bioreactor, which can be carried over as an aerosol. Bioreactor vent gas filtration requires some attention because it is often saturated with water vapour, and this can block the vent filter with condensation on the filter membrane or in the filter pores. To prevent wetting of the filter, either a pre-heater or a heated filter housing is used to warm the vent gas to keep the water vapour above its dew point. Process tank venting: Process tanks are sterilised in place (SIP) with their vent filter installed and the vent filter is used during cool down after steaming to pressurise the tank with sterile air, which breaks the vacuum caused by the

Pharmaceuticals, biotechnology, and healthcare are high growth segments for the filter market due to the broad growth of aseptic and sterile processing. The present cartridge filter market is about US$2 billion per year with a projected growth rate of 9 percent. This article is focused on the sterile filtration of gases used for the fermentation process and fermenter/tank vent applications.

Filter applications for bioreactors and sterile vessels

Typical gas filtration applications that are seen in this type of service are: Sparger/inlet air filtration: All bioreactor processes require sterile air or air/gas mixtures as a raw material. The amount of air or gas mixture required can vary for microbial and mammalian cell culture processes due to their difference in OUR (oxygen uptake rate) or oxygen demand. Microbial cells such as E.coli have a high O2 requirement and can require a process with at least 30% DO (dissolved oxygen saturation). In order to meet this requirement it is not uncommon to sparge air through the bioreactor at 1-2 VVM (Volume of air per volume of liquid per minute). Fermenter vent gas filtration: Vent gas filtration is required for the sterility of the process and containment of
Pharmaceuticals, biotechnology, and healthcare are high growth segments for the filter market due to the broad growth of aseptic and sterile processing.

Filtration+Separation May 2006

Feature

29

Biotech/pharmaceuticals vent filter suppliers

Manufacturer Cuno Pall Millipore

Sartorius Domnick Hunter

Brand Name Microfluor II Emflon Durapore Aervent Aerex Sartofluor GA High Flow Tetpor H.T.

MOC Hydrophobic PTFE Double-layer hydrophobic PTFE Hydrophobic PVDF Hydrophobic PTFE Hydrophobic PTFE Hydrophobic PTFE Expanded PTFE

conditions. This is particularly true of vent filter sizing and the manufacturer will require further information about the tank to be vented, to determine the proper sizing for this application.

Filter housings
The size of the filter housing is determined by the nearest selection of size and number of filter cartridges required to meet gas filtration requirements. The selection of the filter housing type is restricted to the inline style or the T style which are given the position of the inlet and outlet connections in relation to the filter cartridge. The T style housing is the most common choice for two reasons it is easier to install; and to configure with a cleaning cap and spray ball for cleaning in place. If you are steaming in place, or your operating pressure is above 15 psig, the filter housing should be purchased as a registered pressure vessel built to ASME Section VIII Division 1 code.

condensing steam. During normal processing conditions the vent filter allows the tank to breathe sterile air, but precautions need to be taken to ensure that during the process steps the filter does not become wetted or blocked by powders (process materials) from process additions to the tank.

liquid challenge test with Brevundimonas diminuta ATCC 19146. This evaluation is done based on the complete removal of greater than 107 colony forming units (cfu) per cm2 of effective filtration area. Filter manufacturers will normally perform an aerosol challenge test demonstrating 100% Brevundimonas diminuta removal, as well as a viral and bacteriophage aerosol challenge test. Note that there is no standardised test procedure for these aerosol challenges. Filter manufacturers will also perform a sodium chloride aerosol test using a condensation nuclei counter (CNC) to determine the particulate removal rating in gas.

Cartridge filter construction

The most common type of vent filter used is the code 7 cartridge filter with the top spear. The filter membrane is fabricated in a circular pleat, which is bonded to the cage of the filter cartridge at the top and bottom creating an inner core. The support cage is made up of the lower mounting lock tabs, the perforated plastic cage that protects the pleated membrane and the top end cap that has the bayonet spear. In general, filter cartridges are used with the high pressure side on the outside of the filter, because the filter has the greatest structural strength in this direction of gas flow; this is referred to as the forward flow direction of the cartridge.

Integrity test methods for hydrophobic filters

To ensure that the filter is performing correctly, an integrity test (IT) is performed pre and post use. Various IT methods are used and acceptable results of these tests are

Filter sizing
The gas inlet and vent filters are sized to meet the highest flow conditions at the worst case process conditions to be effective throughout the operation of the process. The sizing should take into account the following conditions, to determine the highest flow and worst case process operating conditions before sizing the filter: Forward or reverse flow conditions Normal operating maximum flow and pressure conditions; Maximum flow and pressure conditions during SIP; Maximum and dirty clean pressure drops. The sizing of the filter is best left to the filter manufacturer who will determine the best filter selection based on all of the above operating

Hydrophobic filter removal ratings

Hydrophobic filter cartridges used in such applications have a retention rating of 0.2 microns sterilising grade. As per ASTM F838-83 and FDA guidelines, a filter with this retention rating is evaluated based on a

Features of the ideal air filter Ability to retain organisms through the full range of usage; High-flow rates at low pressure drop; Ease of installation; Ability to in situ integrity test; Can withstand multiple steaming cycles; Compatible MOC (Material Of Construction); Low extractable levels; Regulatory compliant and Validatable.

30

Feature

Filtration+Separation May 2006

exposed to a set pressure upstream of the membrane. The pressure upstream of the filter is monitored to determine the flow rate (based on a know-volume upstream of the filter). The diffusional gas flow rate across the wetted membrane is directly related to the pore size distribution in the membrane. Note that in both Bubble Point and Pressure Decay/Hold tests, thorough flushing of the filter with the alcohol/water mixture is essential to prevent false IT failures.

Water intrusion test (WIT)

This is a different methodology from the previous two tests, and the upstream volume of the membrane is filled with water. This volume is then exposed to air pressure upstream of the membrane. After an initial stabilisation phase, a continuous reduction in upstream water volume can be observed. This reduction in water volume is caused by water diffusing through the membrane, and can be measured by either monitoring the pressure drop upstream of the filter, or the gas flow required to maintain pressure upstream of the filter. It is essential that the membrane cartridge is allowed to cool down to ambient conditions before the water intrusion IT is performed on the filter and housing. Cold water contacting the warm membrane can result in localised water penetration resulting in false positive water intrusion result.

Mammalian cell culture

Water intrusion test

Sterilise in place (SIP)

Sterile vent filters need to be sterilised in place with the tank to be truly sterile. The maximum process conditions that most filters should be subjected to vary from manufacturer to manufacturer but are typically 125oC for 30 minutes at 15 psid in the forward flow direction and 7 psid in the reverse. Always confirm the suitability of the filter for the SIP conditions with the filter manufacturer before use. Steaming in the reverse flow direction requires that the number of sterilisations be reduced from the forward flow duty cycles, and filters are more sensitive to pressure variations in reverse flow. For SIP the correct rated housing, piping, and valves should be selected that meets the requirements of ASME BPE 2004, ASME Process Piping B31.3 and the ASME Boiler and Pressure Vessel Code Section VIII Div 1. Drain lines on the housings must have sanitary steam traps that can handle the

Bacterial cell culture

based on the bacterial liquid challenge test (see hydrophobic filter removal ratings).

Bubble point
In this method, a filter is wetted out using an alcohol/water mixture (used because the filter membrane is hydrophobic). The filter is exposed to increasing air pressure on the upstream side of the membrane, until the pores in the filter

membrane begin to vent, causing a distinct change in the gas flow through the filter. The pressure at which this change occurs is know as the bubble point and is related to the pore size distribution in the membrane.

Pressure decay/pressure hold

In this method, a filter is wetted out using an alcohol/water mixture. The filter is

Filtration+Separation May 2006

Feature

31

condensate load, provide ports for temperature monitoring points and, install sections to hold and recover spore strips for sterilisation studies. It is critical to review how you are going to perform a post SIP IT test without breaking sterility. This is most usually achieved by IT of the filter, once the main vessel has been sterilised and can be used as a sterile reservoir for the filter during IT.

the filter works, and is suitable for their application. The manufacturer is responsible for providing information in support of the end users requirements for details on the filters performance; materials of construction, integrity data correlations to microbial retention; data on the filters range of application; and the validation of the integrity testing of the integrity tester. The filter manufacturers produce validation guides that will include the test methods, test data and support data that you will use to support your validation. As part of your validation effort, the key points that need to be addressed are: Validation of the bacterial retentive properties of the filter on the actual product; Proving that the filter remains integral at the end of the filtration run; Validating the integrity tester used for integrity testing of the filter;

Proving that the filter does not release harmful extractables into the product; Prove that the filter does not adsorb or absorb components of the product formulation.

Summary
Membrane cartridge filters are a very costeffective way to produce sterile gas streams for bioreactors and sterile process tanks. They can be effective when applied correctly, taking into account the application and its process conditions. Finally the filter xmust be validated and this process involves the physical, chemical and biological testing of the filter under extreme process conditions to provide assurance that the filters (when correctly used and integrity tested) will provide a consistently sterile gas stream.

Clean in place (CIP)

Once vent filters housings are larger than one round, they are difficult to remove manually and consideration should be given to cleaning them in place. To obtain the best possible CIP conditions, use one of the modern cleanable filter housings that has a machine base that eliminates the tube sheet, and a spray ball that can contact all wetted parts. Ensure that there is an adequate supply of CIP fluid at sufficient flow and pressure to meet the housing spray ball requirement, and sufficient drainage to handle the CIP flow without allowing it to collect in the base of the housing.

Authors: Jignesh Padia [www.qsvbiologics.com] Process development associate Paul Huntington [Cheme Engineering Inc]

Filter material compatibility

Filter material compatibility with the process application needs to be tested before using membrane filters in a Good Manufacturing Practices (GMP) environment. Incompatibilities between the filter and the process can arise from one or all of the following chemical nature of the gas or aerosols; adsorption on the filter; temperature; or pressure. It is the responsibility of the end user to verify that the process is compatible with the materials of construction of the cartridge filter, and that the process does not cause the degradation or extraction of the filter material into their product by using an extractable testing program. Filters also need to be non pyrogenic according to USP and must meet the requirement for bacterial endotoxins of <0.25 EU/mL. They should also pass the USP Plastics Class VI Test for the injection testing of extracts and implantation of the materials used to make the filter.

Validation
The user and the filter manufacturer share the responsibility for the validation of the filter application. The user is responsible for final validation testing to ensure that