MicroFunnel™ ST Filter Funnel Validation of Suitability for Sterility Testing Applications

Sterility testing is a microbiological analysis to determine if any living and viable organisms are present in a given sample. The product claiming to be sterile must be absolutely free of living and viable organisms. The pharmaceutical industry is required to perform sterility testing on parenteral pharmaceuticals, materials intended to be applied to large area wounds or deep incisions, and disposable plastic medical devices designed to come into contact or administer parenteral pharmaceuticals. The objective of sterility testing is to induce any organisms potentially surviving on or in the pharmaceutical to grow in a non-selective nutrient medium. Detection is by presence or absence of growth over a 14-day period.

Sterility testing can be accomplished in two ways, direct inoculation or membrane filtration method. Direct inoculation involves placing the pharmaceutical article directly into the nutrient broth––obviously the method of choice for solid items such as medical devices. Membrane filtration method is stated in the various Pharmacopeias as the method of choice for any sample that permits membrane filtration. Membrane filtration provides the following valid and important benefits: 1) it concentrates a sample on a membrane filter, 2) it separates inhibitory substances from potentially viable organisms, and 3) it provides additional rinsing and allows for the selection of different types of membrane filter media.

When performing the membrane filtration method, the sample is filtered through the membrane and rinsed. If one filter is used, the membrane is aseptically removed and cut in two equal halves. Each half is then submerged in two different nutrient broths––Fluid Thioglycollate for aerobic and anaerobic bacteria and Fluid Soybean Casein Digest Medium for aerobic bacteria and fungi. If two or more filters are used, then equal number or portions are submerged in the two nutrient broths. Fluid Thioglycollate is incubated for 14 days at 30-35 ºC and Fluid Soybean Casein Digest is incubated at 20-25 ºC for 14 days. During that time, the broths are inspected for turbidity (cloudy growth) which would indicate the sample was not sterile.

Recent Pharmacopeial guidelines have recommended validating the performance of the filter or device used for the membrane filtration method in addition to validation of the rinse fluid and volumes. The format of the MicroFunnel ST filter funnel is suitable for sterility testing because of its easily accessed and removable membrane. This validation guide will demonstrate that the MicroFunnel ST filter funnel is suitable for use in sterility testing applications and for providing maximum confidence in your testing results.

Membrane Selection

GN-6 Metricel® membrane: A 0.45 µm mixed cellulose ester membrane ideal for use in most applications.

Supor® 450 membrane: A 0.45 µm polyethersulfone membrane ideal for low binding of antibiotics or other inhibitory substances.

Materials of Construction

Filter Media:
PN 4750, 4751:	Supor (polyethersulfone) membrane
PN 4811, 4812:	GN-6 Metricel (mixed cellulose ester) membrane
Support Pad:	Cellulose
Funnel and Base:	Polypropylene
Funnel Cover:	Polystyrene
Bag and Overpack Bag:	Polyethylene
Adapter:	Polyethylene

Effective Filtration Area:
13.46 cm²

Maximum Vacuum:
635 mm Hg (25 in. Hg) (Vacuum use only)

Sterilization:
Sterilized by gamma irradiation

Each box of product is supplied with a signed product certification. The certification includes Product Number, Lot Number, Expiration Date, and Date of Manufacture of the product. Sample of the certification:

Expiration Date

MicroFunnel ST filter funnels are packaged individually bagged within an additional overpack bag, both of which are of durable polyethylene construction. The product will remain sterile as long as the packaging remains intact (free from opening or damage). When stored over time under normal conditions (15 - 30 ºC with 50 - 80% relative humidity), the package will keep its integrity. To meet international labeling regulations, an expiration date is assigned three years from the date of manufacture. Expiration Date is clearly marked on each individually labeled bag, the box label, and the Product Certification supplied with each box of product (see Product Certification in this validation guide for more details). Expiration Date is represented by year and month as follows:

Lot Number

Each batch of MicroFunnel ST filter funnels receives a unique, traceable lot number. The lot number can be found on each individually labeled bag, each box, and the Product Certification supplied with each box. Lot Number is designated by the international symbol shown below:

MicroFunnel ST filter funnels are sterilized by a contract sterilizer that is registered to be compliant with ISO9002/EN 46002 and EN 552, as well as being subject to periodic review by the U.S. Food and Drug Administration (FDA). The contract sterilizer is audited annually. MicroFunnel ST filter funnels are sterilized at a minimum dosage of 15 kGy and a maximum of 30 kGy. This dosage level is set during validation of the sterilization process. The subcontractor performs dose mapping whenever new isotope is loaded or as new products are introduced into the system.

Validation of the sterilization process is performed according to AAMI/ANSI/ISO 11137, Sterilization of healthcare products––Requirements for validation and routine control - Radiation Sterilization, Method B1. Validation of sterilization efficiency is accomplished by evaluating the least and most dense products including a product representative of the majority of products sterilized by this methodology. The validation is performed by determining the average bioburden of three lots for each product. The bioburden levels are used to determine the sub-lethal dosage level (10-2 SAL). One hundred pieces of each product are sterilized at this "sub-lethal" dose. The samples are then tested for sterility. The sub-lethal dose is accepted if < 2 positives per 100 pieces are observed. Once the sub-lethal dose is accepted, the routine dosage level is set at the 10-6 SAL level. Routine control of this system is based on quarterly dose audits and bioburden monitoring of the product lines. Quarterly dose audits are performed on a product rotation basis using the same product types from the initial validation. Bioburden levels are monitored on a routine basis and trends are analyzed. In the event that bioburden significantly increases, product is again subject to a dose audit in which the bioburden is quantified and one hundred samples are irradiated at a sub-lethal dose. All one hundred samples are then tested for sterility.

Once the validation dose parameters have been established through validation, the product is released if the dosage range falls within the established minimum and maximum specifications. This is based on dosimetric readings and certification provided to us by the contract sterilization company.

Each box of MicroFunnel ST filter funnels is labeled with a gamma irradiation detection sticker that turns red when exposed. Each individually labeled bag and each box carries the following symbols indicating the product is sterilized by gamma irradiation and intended for one time use:

Pressurized Leak Test

Purpose:

To ensure an integral seal between the funnel cylinder and base.

Procedure:

1. Place test unit onto manifold.
2. Pour 100 mL of aqueous green dye solution into test unit.
3. Attach proprietary design test cap.
4. Pressurize to 0.5 psi and hold for 15 seconds.
5. Observe for leakage and record results.
6. Relieve pressure, remove funnel from manifold, and dispose.

Results:

Lot Number/Membrane Type	Assembly Integrity (sec)
1022/Supor 0.45 µm	Integral
1022/Supor 0.45 µm	Integral
1022/Supor 0.45 µm	Integral
1022/Supor 0.45 µm	Integral
1022/Supor 0.45 µm	Integral
1026/Supor 0.45 µm	Integral
1026/Supor 0.45 µm	Integral
1026/Supor 0.45 µm	Integral
1026/Supor 0.45 µm	Integral
1026/Supor 0.45 µm	Integral
1027/Supor 0.45 µm	Integral
1027/Supor 0.45 µm	Integral
1027/Supor 0.45 µm	Integral
1027/Supor 0.45 µm	Integral
1027/Supor 0.45 µm	Integral
1647J/GN-6 Metricel® 0.45 µm	Integral
1647J/GN-6 Metricel 0.45 µm	Integral
1647J/GN-6 Metricel 0.45 µm	Integral
1647J/GN-6 Metricel 0.45 µm	Integral
1647J/GN-6 Metricel 0.45 µm	Integral
1643J/GN-6 Metricel 0.45 µm	Integral
1643J/GN-6 Metricel 0.45 µm	Integral
1643J/GN-6 Metricel 0.45 µm	Integral
1643J/GN-6 Metricel 0.45 µm	Integral
1643J/GN-6 Metricel 0.45 µm	Integral
1698J/GN-6 Metricel 0.45 µm	Integral
1698J/GN-6 Metricel 0.45 µm	Integral
1698J/GN-6 Metricel 0.45 µm	Integral
1698J/GN-6 Metricel 0.45 µm	Integral
1698J/GN-6 Metricel 0.45 µm	Integral

Diffusional Flow Test

Purpose:

To determine the integrity of the membrane and seal of the membrane within the device.

Procedure:

1. Place test unit on the manifold.
2. Wet test unit with 10% isopropyl alcohol in water.
3. Attach proprietary design test cap.
4. Apply 20 in. Hg vacuum and allow unit to stabilize.
5. Record diffusional flow value.
6. Turn off vacuum, remove unit from manifold, and discard.

Results:

Lot Number/Membrane Type	Diffusional Flow (cc/min)
1022/Supor 0.45 µm	<1.0
1022/Supor 0.45 µm	<1.0
1022/Supor 0.45 µm	<1.0
1022/Supor 0.45 µm	<1.0
1022/Supor 0.45 µm	<1.0
1026/Supor 0.45 µm	<1.0
1026/Supor 0.45 µm	<1.0
1026/Supor 0.45 µm	<1.0
1026/Supor 0.45 µm	<1.0
1026/Supor 0.45 µm	<1.0
1027/Supor 0.45 µm	<1.0
1027/Supor 0.45 µm	<1.0
1027/Supor 0.45 µm	<1.0
1027/Supor 0.45 µm	<1.0
1027/Supor 0.45 µm	<1.0
1647J/GN-6 Metricel® 0.45 µm	<1.0
1647J/GN-6 Metricel 0.45 µm	<1.0
1647J/GN-6 Metricel 0.45 µm	<1.0
1647J/GN-6 Metricel 0.45 µm	<1.0
1647J/GN-6 Metricel 0.45 µm	<1.0
1643J/GN-6 Metricel 0.45 µm	<1.0
1643J/GN-6 Metricel 0.45 µm	<1.0
1643J/GN-6 Metricel 0.45 µm	<1.0
1643J/GN-6 Metricel 0.45 µm	<1.0
1643J/GN-6 Metricel 0.45 µm	<1.0
1698J/GN-6 Metricel 0.45 µm	<1.0
1698J/GN-6 Metricel 0.45 µm	<1.0
1698J/GN-6 Metricel 0.45 µm	<1.0
1698J/GN-6 Metricel 0.45 µm	<1.0
1698J/GN-6 Metricel 0.45 µm	<1.0

Liquid Flow Test

Purpose:

To characterize a typical flow rate for each membrane type.

Procedure:

1. Place a test unit onto a manifold.
2. Pour 100 mL of 0.2 µm filtered water into test unit.
3. Open valve on manifold to apply 15 in. Hg vacuum and simultaneously start the stop watch.
4. Record the amount of time required for the water to flow through the membrane.
5. Remove the test unit from the manifold and discard.

Results:

Lot Number/Membrane Type	Flow Rate(sec)
1022/Supor 0.45 µm	16.6
1022/Supor 0.45 µm	17.9
1022/Supor 0.45 µm	16.7
1022/Supor 0.45 µm	15.9
1022/Supor 0.45 µm	16.8
1026/Supor 0.45 µm	18.1
1026/Supor 0.45 µm	17.1
1026/Supor 0.45 µm	20.0
1026/Supor 0.45 µm	19.9
1026/Supor 0.45 µm	17.4
1027/Supor 0.45 µm	16.6
1027/Supor 0.45 µm	16.5
1027/Supor 0.45 µm	15.7
1027/Supor 0.45 µm	16.3
1027/Supor 0.45 µm	16.8
Average Standard Deviation	17.23 1.27
1647J/GN-6 Metricel® 0.45 µm	10.8
1647J/GN-6 Metricel 0.45 µm	10.8
1647J/GN-6 Metricel 0.45 µm	11.4
1647J/GN-6 Metricel 0.45 µm	10.6
1647J/GN-6 Metricel 0.45 µm	13.1
1643J/GN-6 Metricel 0.45 µm	11.9
1643J/GN-6 Metricel 0.45 µm	11.8
1643J/GN-6 Metricel 0.45 µm	10.7
1643J/GN-6 Metricel 0.45 µm	12.2
1643J/GN-6 Metricel 0.45 µm	12.1
1698J/GN-6 Metricel 0.45 µm	11.6
1698J/GN-6 Metricel 0.45 µm	13.4
1698J/GN-6 Metricel 0.45 µm	12.7
1698J/GN-6 Metricel 0.45 µm	12.0
1698J/GN-6 Metricel 0.45 µm	11.7
Average Standard Deviation	11.79 0.86

Purpose

To ensure that the polyethylene bag material used to individually package MicroFunnel filter funnel products is effectively protecting the product from intrusion by the most common sanitizing and sterilizing agents, for example, Peracetic Acid and Hydrogen Peroxide.

Procedure A

Direct Contact with Liquid Peracetic Acid

1. Dilute 30% Peracetic Acid to 3.5% using distilled water.
2. Submerge 5 test units in the 3.5% peracetic acid and observe bag integrity at 30 minutes, 45 minutes, 2 hours, and 24 hours.
3. Record results.

Results

Test Unit	Time
	0 Minutes	30 Minutes	45 Minutes	2 Hours	24 Hours
1	Integral	Integral	Integral	Integral	Integral
2	Integral	Integral	Integral	Integral	Integral
3	Integral	Integral	Integral	Integral	Integral
4	Integral	Integral	Integral	Integral	Integral
5	Integral	Integral	Integral	Integral	Integral

Procedure B

Direct Contact with Liquid Hydrogen Peroxide (H₂O₂.)

1. Swab bag directly with liquid H₂O₂.
2. Allow bag to dry.
3. Submerge in water and observe for leaks.
4. Record results.
5. Submerge test unit 30% H₂O₂ for 24 hours.
6. Observe for leaks.
7. Record results.

Results

Concentration of H2O2	Exposure	Results
3%	Swab bag and allow to dry	Integral
5%	Swab bag and allow to dry	Integral
10%	Swab bag and allow to dry	Integral
20%	Swab bag and allow to dry	Integral
30%	Swab bag and allow to dry	Integral
30%	Submerged 24 hours	Integral

Procedure C

Bag Compatibility with Vapor Hydrogen Peroxide (VHP)

1. VHP Indicator NB305 Chemical Indicator Strips, lot number 201343, expiration date 2002-APR-10, were sealed into polyethylene bags (with no overpack bag) with MicroFunnel filter funnels and exposed to gamma irradiation.
2. The 15 individually-sealed bags, each containing a test strip and filter funnel, were exposed to the H₂O₂ Steris* VHP Sterilization Cycle 1000 within a La Calhène* transfer isolator at different critical control points. The transfer isolator and cycle were chosen to simulate a worst case scenario with respect to exposure time according to the parameters shown below.
3. Observe strips and record results.

CYCLE 1
Volume:	0.6 m3 = 21 ft3
Minimum temperature:	24 °C = 75 °F
Load:	Medium
SAL:	6 Log reduction
Dehumidification:
Airflow rate:	567 L/min/cmh (34 m3/h)
Time:	5 minutes
Limit target of absolute humidity:	2.3 mg/L (10 %)
Conditioning:	Conditioning phase of cycle not used in transfer isolator.
Sterilization:
Airflow rate:	567 L/min/cmh (34 m3/h)
Time: (D value = 10 minutes x 6)	60 minutes
Injection H2O2 volume:	3.3 g/minute
Aeration:
Residual of H2O2 to reach:	< 1 ppm
Airflow rate:	567 L/min/cmh (34 m3/h)
Time:	60 minutes
Total cycle:	135 minutes
H2O2 (35% used):	198 grams

Sample Number Test Strips Sealed in Bag:	Color Before Exposure	Color After Exposure
1	Blue-gray	Blue-gray
2	Blue-gray	Blue-gray
3	Blue-gray	Blue-gray
4	Blue-gray	Blue-gray
5	Blue-gray	Blue-gray
6	Blue-gray	Blue-gray
7	Blue-gray	Blue-gray
8	Blue-gray	Blue-gray
9	Blue-gray	Blue-gray
10	Blue-gray	Blue-gray
11	Blue-gray	Blue-gray
12	Blue-gray	Blue-gray
13	Blue-gray	Blue-gray
14	Blue-gray	Blue-gray
15	Blue-gray	Blue-gray
Positive Control Test Strips
16	Blue-gray	Beige
17	Blue-gray	Beige
18	Blue-gray	Beige
19	Blue-gray	Beige
20	Blue-gray	Beige

Conclusion

Test strip positive controls placed within the isolator during the cycle described above turned beige indicating that they had been exposed to VHP. All of the test strips protected within the individually-sealed polyethylene bags remained blue-gray indicating that the strips had been adequately protected from the VHP and that the bags are completely resistant to penetration of VHP.

Note: 100 mL funnels are packaged with 10 individually bagged funnels within an overpack bag; 4 overpack bags per box. 300 mL funnels are packaged with 5 individually bagged funnels within an overpack bag; 4 overpack bags per box. The polyethylene overpack bags are sized to fit within the most restrictive portal into an isolator. Overpack bags are easy to handle when working with multiple filter units and provide a smooth, consistent surface to facilitate charging of the isolator and thorough contact with the sterilizing agent.

Procedure D

Bag Compatibility with Vapor Hydrogen Peroxide (VHP)

1. VHP* Indicator NB302 Chemical Indicator Strips, Batch No. 069901, expiration date 02/2001, were sealed into individual polyethylene bags with MicroFunnel filter funnels and exposed to gamma irradiation.
2. The 15 individually-sealed bags, each containing a test strip and filter funnel, were exposed to the H₂O₂ Steris* VHP Sterilization Cycle 1000 within a La Calhène* workstation at different critical control points. The workstation and cycle were chosen to simulate a worst case scenario with respect to exposure time according to the parameters shown below.
3. Observe strips and record results.

CYCLE 2
Volume:	4.5 m3 = 159 ft3
Minimum temperature:	24 °C = 75 °F
Load:	Medium
SAL:	6 Log reduction
Dehumidification:
Airflow rate:	567 L/min/cmh (34 m3/h)
Time:	5 minutes
Limit target of absolute humidity:	4.6 mg/L (20 %)
Conditioning:
Airflow rate:	567 L/min/cmh (34 m3/h)
Time:	5 minutes
Injection H2O2 volume:	8.2 g/minute
H2O2 inlet:	4.5 mg/liter
Sterilization:
Airflow rate:	567 L/min/cmh (34 m3/h)
Time: (D value = 10 minutes x 6)	90 minutes
Injection H2O2 volume:	3.7 g/minute
Aeration:
Residual of H2O2 to reach:	< 1 ppm
Airflow rate:	567 L/min/cmh (34 m3/h)
Time:	270 minutes
Total cycle:	380 minutes
H2O2 (35% used):	400 grams

Results:

Sample Number Test Strips Sealed in Bag:	Color Before Exposure	Color After Exposure
1	Yellow	Yellow
2	Yellow	Yellow
3	Yellow	Yellow
4	Yellow	Yellow
5	Yellow	Yellow
6	Yellow	Yellow
7	Yellow	Yellow
8	Yellow	Yellow
9	Yellow	Yellow
10	Yellow	Yellow
11	Yellow	Yellow
12	Yellow	Yellow
13	Yellow	Yellow
14	Yellow	Yellow
15	Yellow	Yellow
Positive Control Test Strips
16	Yellow	Violet
17	Yellow	Violet
18	Yellow	Violet
19	Yellow	Violet
20	Yellow	Violet

Conclusion

Test strip positive controls placed within the isolator during the cycle described above turned Violet/pink indicating that they had been exposed to VHP. All of the test strips protected within the individually-sealed polyethylene bags remained yellow indicating that the strips had been adequately protected from the VHP and that the bags are completely resistant to penetration of VHP.

Note: 100 mL funnels are packaged with 10 individually bagged funnels within an overpack bag; 4 overpack bags per box. 300 mL funnels are packaged with 5 individually bagged funnels within an overpack bag; 4 overpack bags per box. The polyethylene overpack bags are sized to fit within the most restrictive portal into an isolator. Overpack bags are easy to handle when working with multiple filter units and provide a smooth, consistent surface to facilitate charging of the isolator and thorough contact with the sterilizing agent.

Purpose

To validate that the MicroFunnel ST filter funnel is suitable for use in sterility testing applications and will recover the pharmacopeial organisms at levels exceeding 70%.

Procedure

1. Prepare challenge suspension of test organism.
2. Perform filtration in triplicate.
3. Plate one filter on agar medium, submerge second filter in Fluid Thioglycollate Broth, and submerge third filter in Fluid Soybean Casein Digest Medium.
4. Perform a total of three replicates.
5. Incubate plates and broth media.
6. Count colony growth on membrane filters plated on agar medium.
7. Observe for growth in broth media.

Results

Lot Number/Membrane Type	Escherichia coli ATCC 8739
	% Recovery	P/A
		SCD	THG
1022/Supor 0.45 µm	102	Growth	Growth
1026/Supor 0.45 µm	100	Growth	Growth
1027/Supor 0.45 µm	98	Growth	Growth
1647J/GN-6 Metricel 0.45 µm	98	Growth	Growth
1643J/GN-6 Metricel 0.45 µm	112	Growth	Growth
1998J/GN-6 Metricel 0.45 µm	108	Growth	Growth

Lot Number/Membrane Type	Staphylococcus aureus ATCC 6538P
	% Recovery	P/A
		SCD	THG
1022/Supor 0.45 µm	100	Growth	Growth
1026/Supor 0.45 µm	93	Growth	Growth
1027/Supor 0.45 µm	98	Growth	Growth
1647J/GN-6 Metricel 0.45 µm	98	Growth	Growth
1643J/GN-6 Metricel 0.45 µm	98	Growth	Growth
1998J/GN-6 Metricel 0.45 µm	110	Growth	Growth

Lot Number/Membrane Type	Bacillus subtilus ATCC 6633
	% Recovery	P/A
		SCD	THG
1022/Supor 0.45 µm	100	Growth	Growth
1026/Supor 0.45 µm	96	Growth	Growth
1027/Supor 0.45 µm	90	Growth	Growth
1647J/GN-6 Metricel 0.45 µm	90	Growth	Growth
1643J/GN-6 Metricel 0.45 µm	92	Growth	Growth
1998J/GN-6 Metricel 0.45 µm	92	Growth	Growth

Lot Number/Membrane Type	Pseudomonas aeruginosa ATCC 9027
	% Recovery	P/A
		SCD	THG
1022/Supor 0.45 µm	96	Growth	Growth
1026/Supor 0.45 µm	98	Growth	Growth
1027/Supor 0.45 µm	98	Growth	Growth
1647J/GN-6 Metricel 0.45 µm	94	Growth	Growth
1643J/GN-6 Metricel 0.45 µm	96	Growth	Growth
1998J/GN-6 Metricel 0.45 µm	100	Growth	Growth

Lot Number/Membrane Type	Candida albicans ATCC 10231
	% Recovery	P/A
		SCD	THG
1022/Supor 0.45 µm	104	Growth	Growth
1026/Supor 0.45 µm	99	Growth	Growth
1027/Supor 0.45 µm	100	Growth	Growth
1647J/GN-6 Metricel 0.45 µm	98	Growth	Growth
1643J/GN-6 Metricel 0.45 µm	103	Growth	Growth
1998J/GN-6 Metricel 0.45 µm	102	Growth	Growth

Lot Number/Membrane Type	Clostridium sporogenes ATCC 19404
	% Recovery	P/A
		SCD	THG
1022/Supor 0.45 µm	85	Growth	Growth
1026/Supor 0.45 µm	86	Growth	Growth
1027/Supor 0.45 µm	93	Growth	Growth
1647J/GN-6 Metricel 0.45 µm	92	Growth	Growth
1643J/GN-6 Metricel 0.45 µm	81	Growth	Growth
1998J/GN-6 Metricel 0.45 µm	84	Growth	Growth

Lot Number/Membrane Type	Clostridium sporogenes ATCC 11437
	% Recovery	P/A
		SCD	THG
1022/Supor 0.45 µm	88	Growth	Growth
1026/Supor 0.45 µm	85	Growth	Growth
1027/Supor 0.45 µm	90	Growth	Growth
1647J/GN-6 Metricel 0.45 µm	117	Growth	Growth
1643J/GN-6 Metricel 0.45 µm	83	Growth	Growth
1998J/GN-6 Metricel 0.45 µm	81	Growth	Growth

Lot Number/Membrane Type	Aspergillis niger ATCC 16404
	% Recovery	P/A
		SCD	THG
1022/Supor 0.45 µm	95	Growth	Growth
1026/Supor 0.45 µm	77	Growth	Growth
1027/Supor 0.45 µm	81	Growth	Growth
1647J/GN-6 Metricel 0.45 µm	92	Growth	Growth
1643J/GN-6 Metricel 0.45 µm	93	Growth	Growth
1998J/GN-6 Metricel 0.45 µm	79	Growth	Growth