Quality Assurance Document

for
BIO-X II
Air Sterilisation Grade
Filter Cartridges

dh
domnick hunter
PROCESS DIVISION
 Contents

page no.

1 Summary

1 Introduction

2 Quality Assurance

2 Materials of Construction

3 Validation of Bacterial Removal Efficiency

Experimental Protocol - Outline Procedure

3 Experimental Conditions Summary

4 Aerosol Challenge Integrity Testing

4 Bacterial Aerosol Challenge Results Summary

5 Discussion of Bacterial Challenge Results

6 Conclusions

7-8 Product Evaluation

Reverse Steam In-Situ Sterilisation - Outline Procedure - Results
Dynamic Heat Trials - Outline Procedure - Results
Static Heat Trials - Outline Procedure - Results
Summary

9-10 Airbourne Bacterial Challenge Procedure

Apparatus - Flow meters - Aerosol Generation - Sampling Techniques

11 Bacterial Challenge Test Organism

Stock Cultures - Care & Maintenance
Organism Description
Characteristics Enabling Identification
Preparation of Bacterial Challenge Suspension

12 References

Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges
 Summary
BIO-X II filter cartridges have been comprehensively validated as sterilising grade filters for compressed air
and gas applications. BIO-X II has been shown to exceed the performance characteristics necessary to
assure reliable and cost-effective operational service.

INTRODUCTION

A considerable amount of effort has gone into validating the construction and operational efficiencies of
domnick hunter BIO-X II air and gas sterilising grade filter cartridges. It was important to precisely quantify
and qualify operational characteristics so that the performance and biosecurity of BIO-X II filter cartridges can
be assured. It was also necessary to understand how the filter cartridges perform under potentially abusive
conditions so that recommended operational guidelines (references 1 and 6) can be made.

The purpose of an air and gas sterilising filter is to remove all types of potential biological contaminants yet
allow the maximum passage of a sterile gas stream to the aseptic process. It is therefore essential that the
validation protocol should assure the effectiveness of microbial removal under conditions that represent
realistic cartridge operation. The domnick hunter bacterial aerosol challenge test apparatus (reference 2)
has been specifically designed and developed to achieve realistic and relevant filter test conditions.

The correlation between bacterial retention and non-destructive integrity test values is mandatory for many
applications of sterilising grade compressed air and gas filters.

In order to establish secure operational guidelines it was necessary to expose BIO-X II filter cartridges to non-
optimal or potentially abusive testing procedures. One of the most damaging treatments to any sterilising
grade filter cartridge is repeated exposure to high temperature steam flow combined with rapid temperature
changes inducing large thermal shock loadings. The test protocols outlined within Performance Evaluation
ensure that the cartridge is guaranteed to remain integral following the minimum number of recommended
steam sterilisation cycles.

Biological safety, chemical compatibility, non-volatile extractables and bacteriophage removal are important
parameters for some applications of BIO-X II. domnick hunter has undertaken and continues to undertake,
validation of BIO-X II for these parameters.

BIO-X II is designed to be a compressed air and gas sterilising filter.

1 Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges
 Quality Assurance

domnick hunter places the greatest importance on achieving and maintaining the highest levels of product
quality assurance through every stage of design, development and manufacture. As with all domnick hunter
products, BIO-X II must satisfy quality assurance standards. BS EN ISO 9001:1994 are amongst the
highest achievable levels of international standards for quality management systems and domnick hunter
has achieved these standards.

BIO-X II filters are manufactured in carefully controlled environmental conditions. Quality assurance
inspection and test procedures are implemented from vendor assessment, specification and receipt of raw
materials, through every stage of the manufacturing procedure to ultimate product testing prior to packing and
release.

domnick hunters responsibility as a manufacturer of quality extends beyond their site at Birtley, UK to the
actual points of filter use through the domnick hunter companies in USA, Germany, Scandinavia, France
Spain, China, Japan and the worldwide network of domnick hunter distributors.

Every BIO-X II filter cartridge module carries a serial number. This ensures full traceability of all BIO-X II
products manufactured by domnick hunter including materials, test results, quality procedures and
manufacturing process records. The quality control testing procedures carried out on components, work in
progress and completed cartridges include the following:

* Non-destructive integrity tests on 100% of cartridges

* Flow rate tests to ensure cartridge performance

* Steam testing

Materials of Construction

FILTER COMPONENTS MATERIAL

Filtration Medium Borosilicate Microfibre

Core Stainless Steel
Cage Stainless Steel
Support Layers Nomex *
End caps Stainless Steel
O Rings Silicone, EPDM, Viton
Encapsulation Method Epoxy resin bonded.

Nomex* is a registered trade mark of E.I Dupont Nemours.

Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges 2
 Validation of Bacterial Removal Efficiency
EXPERIMENTAL PROTOCOL:

The basis of this test procedure was to challenge standard production MER-AZ BIO-X II filter cartridges with
an airborne aerosol containing a highly penetrating micro-organism that can withstand the rigours of the
challenge procedure (i.e. nebulisation and desiccation). The organism used is Brevundimonas diminuta
ATCC 19146. Great care had to be taken in designing the testing protocol in order to ensure experimental
reproducibility. This included ensuring effective apparatus sterilisation, containment and viability of challenge
organism using relevant operational procedures. For example, it is important to sample the full downstream
(effluent) flow from the filter under test as very low numbers of organisms are involved.

OUTLINE PROCEDURE:

1) Steam sterilise pipework and filter housings. Chemically sterilise nebuliser and downstream slit sampler.
Dry using sterile compressed air.
2) Chill upstream challenge samplers (sterilised Porton type all-glass impingers).
3) Add 100ml of bacterial suspension to nebuliser bowl aseptically but do not pressurise yet in order to run
sterility controls.
4) Initiate main line flow through apparatus and run downstream flow slit sampler for sterility control for 2
minutes. Aseptically change control agar plate for first test plate in slit sampler.
5) Pressurise nebuliser to 50 psig and check main flow-line pressure is 26 psig.
6) Run test for 30 minutes, aseptically changing downstream (effluent) slit sample plates every 10 minutes.
Sample upstream challenge for 2 minutes in mid-run at 15 l/min flow rate through impingers.
7) Incubate all test and control plates at 30C for 48 hours after performing appropriate dilution to upstream
challenge samples.
8) Control run: carry out standard test protocol without test filter present to ensure organism viability on
downstream side which is fully sampled for the first and last minutes of a 30 minute run using fresh
sample plates. No reduction in test organism viability must be observed through the course of all 30
minutes control test runs.

Experimental Conditions Summary

Line Pressure : 26 psig
Nebuliser Pressure : 50 psig
Flow Rate : 200 Nl/min (12 Nm3/h)
Fluid : Dry aerosol
Relative Humidity Level at Ambient Temp. : 90%
Steam Sterilisation : 30 min. at 121C
Chemical Sterilisation : Formaldehyde
Test Filter Cartridge Type : MER-AZ
Test Housing : ZVA-01A-BTE

3 Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges
 Aerosol Challenge Integrity Testing
The particles generated by the domnick hunter VALAIRDATA aerosol integrity test equipment have a
particle size distribution such that the mean particle diameter lies within the 0.1 - 0.3m range. This is the
most penetrating and hence searching size range of particle with which to challenge a sterilising air filter.

It is shown within the discussion that a worst-case bacterial challenge to a BIO-X II MER-AZ filter cartridge
is 2 x 108 per year. To effect a realistic and secure aerosol integrity test this number must represent the
minimum challenge of aerosol particles.

When set up to integrity test a MER-AZ filter cartridge, the domnick hunter VALAIRDATA integrity test
equipment generates a typical minimum challenge of 4 x 1012 particles per minute. Therefore, the more
penetrating and searching aerosol challenge is at least 20,000 times the equivalent worst case bacterial
challenge but uses microgram quantities of aerosol to achieve such a secure test.

The sensitivity of aerosol challenge integrity testing is very high. The aerosol particles are highly penetrating
because of their size distribution and are highly searching because of their concentration. The aerosol is
capable of passing through the smallest flow within the cartridge construction.

Bacterial Aerosol Challenge Results Summary

Total
% Detectable Bacterial
Cartridge Aerosol Challenge*2 Sterile
Serial No. Penetration*1 (cfu) Effluent L.R.V.

MER-AZ PASS 4.5 x 1010 YES 10.65

(A) (ZERO)
MER-AZ PASS 4.04 x 1010 YES 10.61
(B) (ZERO)
MER-AZ PASS 1.17 x 1011 YES 11.07
(C) (ZERO)
MER-AZ 0.0036 9.12 x 1010 NO 10.96
(1)
MER-AZ 0.026 8.16 x 1010 NO 10.91
(3)
MER-AZ 0.5 5.68 x 1010 NO 10.75
(4)
MER-AZ 0.042 1.43 x 1011 YES 11.16
(6)
MER-AZ 0.014 1.11 x 1011 YES 11.05
(7)
MER-AZ PASS 1.11 x 109 YES 9.05
(D) (ZERO)

*1 Quantity of detectable aerosol penetration using the VALAIRDATA Integrity Tester. The allowable limit set
by domnick hunter for a sterilising grade filter is <0.00005% penetration.
*2 Total bacterial challenge (in colony forming units) calculated from upstream challenge level.

Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges 4
 Discussion of Bacterial Challenge Results
The function of a sterilising grade of compressed air and gas filter is to completely remove all microbiological
particles yet allow the maximum flow of sterile gas. Most popular types and makes of air and gas filter rely
on the same mechanisms of filtration, for further detailed consideration of these, please see references 11,
12 and 13. BIO-X II has been optimised for voids volume and depth of active filtration medium (hence
increased flow capacity).

Certain sized particles are much more penetrating, or more difficult to filter, than others. The most difficult
size range to filter is 0.1 - 0.3m (ref. 12). The removal efficiency for this range of particle sizes is therefore
a powerful aid in the design, development and testing of high efficiency air filters such as BIO-X II. If the
filter removes these sizes of particles then it will have even greater efficiency with smaller and larger
particles. A challenge to the air filter of a very high concentration of 0.1 - 0.3m sized particles is a very
realistic and highly searching test to assure the operational efficiency hence integrity of the filter. This is the
basis of the internationally recognised aerosol challenge test procedure for air filters and is the basis for the
domnick hunter integrity test equipment: VALAIRDATA.

The choice of a single test organism, Brevundimonas diminuta was made because the vegetative cells of this
organism are in the most penetrating size range and sufficiently robust to withstand the rigours of the
domnick hunter bacterial aerosol challenge procedure. In order to demonstrate the minimum bacterial
retention capability of BIO-X II, a series of airborne challenges were made. It is useful to compare these
results with a typical worst-case challenge that the filter might receive during its service life.

The actual numbers of viable micro-organisms in a gas stream depend on many variables (e.g. site location,
local industrial and agricultural activity, the weather etc.). Estimates of numbers in atmospheric air vary
widely, with average counts from 99 to 850 cfu per m3 (colony forming units per cubic metre) having been
reported (reference 14). Half the particles carrying bacteria were reported to be larger than 8m which would
be easily removed by prefiltration. Average counts for the environment within a fermentation facility have
been reported as between 120 and 706 cfu per m3 (reference 15). A dramatically high count of approximately
40,000 cfu per m3 was reported (reference 8) during a sandstorm in the Nevada desert!

In reality, a very significant proportion of viable micro-organisms in atmospheric air will fail to reach the
sterilising filter because of inactivation during passage through the compressor system and removal by
pre-filtration. However, for the purposes of this exercise and in order to assure a worst-case analysis, the
following assumptions will be made.

- the filter is a BIO-X II MER-AZ, as used in the bacterial challenge test

- the flow rate is 12 Nm3 per hour.
- the operational service life is 350 continuous days per year
- the atmospheric count is 2,000 cfu per m3 for all 350 days
- all atmospheric micro-organisms reach the final filter in a viable state and have the potential to
contaminate the sterile process

The total challenge under these conditions per year =

2000 cfu/m3 micro-organism concentration

x 12 m3/h flow rate
x 24 h/day hours in the day
x350 day days in the year operation
Total 2 x 108 cfu estimated maximum annual challenge

By comparison, the highest challenge reported was: 1.43 x 1011 cfu for the test filter BIO-X II AND WAS
ABSOLUTELY RETENTIVE AT THIS CHALLENGE LEVEL.

The safety margin for BIO-X II of 715 times minimum retention capacity over the worst case annual challenge
is, in reality, increased by many orders of magnitude because of microbial inactivation, removal by
prefiltration and lower initial atmospheric concentrations.

5 Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges
 Conclusions
It is very important to use correct and meaningful types of test methods in order to determine true operational efficiencies
when validating and integrity testing filters for the sterilisation of compressed air and gases.

Such validating and integrity testing procedures must be carried out in conditions that closely represent actual filter
operation.

BIO-X II - has been rigorously tested under correct and meaningful conditions.

BIO-X II - was absolutely retentive when challenged with 1.43 x 1011 airborne
bacterial particles.

BIO-X II - showed zero detectable penetration when challenged with more than
1012 aerosol particles.

BIO-X II - is a sterilising grade filter for compressed air and gas applications.

Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges 6
 Performance Evaluation
REVERSE STEAM IN-SITU STERILISATION
This procedure was used to evaluate the elements as it is generally accepted to be the most arduous method.

OUTLINE PROCEDURE

CONTROL
CHART
BOX
RECORDER

TEMPERATURE
CONNECTIONS
PROBE
TO SOLENOID

ELEMENTS UNDER TEST

STEAM
4
STEAM 3
1 PRESS
REGULATOR 5

2
CONDENSATE
TRAP
CONDENSATE
TRAP

AIR

REGULATOR 6

FIGURE 1: Automated in-situ, reverse flow steam exposure test rig for the dynamic testing of BIO-X II filter cartridges

A typical temperature profile for the sterilisation cycle is shown in Fig. 3.

30 mins

142C

TEMP.
40C

1 hour
TIME

Fig.3.

The reverse steam cycle is comprised of the following steps.

1. 30 minute reverse steaming at 140 C. The flow of steam through the element was regulated by a 1mm
orifice. This ensures each test is carried out under the same conditions.
2. The steam terminated and the vessel and its contents are allowed to cool for a period of 10 minutes.
3. Air then enters the vessel in the forward direction at an inlet pressure of 30 psi. Air flows through the
element for a period of 20 minutes before returning to reverse steaming.

3 x MER12-SRH elements were steamed simultaneously in the housing and were periodically checked for
integrity throughout the test. A total of 6 elements were tested.

7 Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges
RESULTS
Element Element Number of Cycles at 140 C
Identity Type Aerosol % Penetration

Nom 1 MER12SRH 19 29 35 54 61 72 87 107

0 0 0 0 0 0 0 0
Nom 2 MER12SRH 19 29 358 54 61 70 87 107
0 0 0 0 0 0 0 0
Nom 3 MER12SRH 17 40 51 64 78 91 104
0 0 0 0 0 0 0
Nom 4 MER12SRH 19 41 52 65 79 92 104
0 0 0 0 0 0 0
Nom 5 MER12SRH 19 41 52 65 79 92 104
0 0 0 0 0 0 0
Nom 6 MER12SRH 21 44 63 70 81 96 104
0 0 0 0 0 0 0

CONCLUSION
The BIO-X product can withstand a minimum of 100 cycles at 142C in the reverse direction while
remaining integral.

DYNAMIC HEAT TRIALS

OUTLINE PROCEDURE
1. An MER4SRH element was placed in a ZVA-011 housing and air flowed through at 200 l/min in the
forward direction via a hot air gun. The temperature of the air at the inlet to the housing was 200C and at
the exit 150C.
2. The hot air was switched off and air from the ring main flowed in the reverse direction for a period of
10 minutes at 30 scfm (rated flow).
3. The direction of flow was then reversed for a period of 10 minutes at 30 scfm.

This was done to achieve maximum stress on the filter medium. The element was integrity tested by the
aerosol challenge method after each stage.

RESULTS
Element Element Air Flow Air Temp. No. of Cycles
Identity Type Rate C % Aerosol Penetration

SKB-A MER4SRH 200 l/min 200 - 150 8 14 18 21 29

0 0 0 0 0
35 43 52 56 80
0 0 0 0 0

CONCLUSION
The BIO-X product will remain integral for a minimum of 80 dynamic heat cycles.

STATIC HEAT TRIALS

An MER4SRH was placed in an oven at 200C and air flow tested periodically at ambient conditions. The
element was both forward and reverse flowed at rated conditions, being integrity tested after each stage.

RESULTS Element Element Air Temp. No. of hours @ 200C

Identity Type C % Aerosol Penetration

SKB-B MER4SRH 200 117 140 239 328 352 376

0 0 0 0 0 0

CONCLUSION
The BIO-X product will remain integral for a minimum 376 hours of static heat at 200C.

Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges 8
 Airborne Bacterial Challenge Procedure
APPARATUS

The layout of the apparatus used in the filter tests is shown in Figure 4 and was based on that described in reference 2.
The assembly consisted of a nebuliser for the generation of the bacterial aerosol, a sterile filtered compressed air supply,
the test filter housing and the upstream and downstream aerosol sampling devices - all-glass impingers and a slit
sampler respectively.

Connecting pipework for the transport of compressed air and aerosols to the test sections was 25mm O.D. 316 stainless
steel tubing and all valves were one inch stainless steel butterfly valves. Welded joints were full penetration, continuous
smooth, and crevice free and all bends were smoothly angled to avoid trapping any airborne bacteria.

Various inlet and outlet ports were included in the system for steam sterilisation and sampling. Additionally, facilities to
control other experimental variables such as temperature, pressure and air flow rate were also included.

1. Nebuliser 8. Steam inlet

2. Test filter housing 9. Exhaust gas sterilising filter
3. Impinger samplers 10. Sampling ports
4. Slit sampler 11. Drainage ports
5. Flowmeters 12. Flow control valve
6. Inlet air sterilising filters 13. Prefilter
7. Pressure meters

FIGURE 4: Challenge test apparatus used to validate BIO-X II filter cartridges for airborne bacterial challenge.

9 Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges
FLOWMETERS
Flowmeters arranged in series with the upstream challenge samplers used to determine the proportion of the total air flow
sampled in each case. Sterilising grade air filters were placed on all exhausts to prevent the release of bacterial aerosols
and the potential ingress of contaminants.

AEROSOL GENERATION
Bacterial aerosols are conveniently and efficiently produced by a Collison type nebuliser, originally designed at
Microbiological Research Establishment (MRE), Porton Down, England. A description of this nebuliser and its mode of
action have already been reported (reference 2).

The nebuliser is operated by a stream of filtered, sterile air which refluxes a suspension of the test micro-organisms,
producing an aerosol of the correct droplet size such that on average one droplet contains one cell. Oversized droplets
impinge on the side walls of the reservoir vessel.

It was found that nebuliser pressure should always be greater than the line pressure by 20-25 psi in order for an efficient
nebulising action to occur. These pressures were measured by in-line pressure meters and manually adjusted to the
required values by pressure regulators. The aerosol is injected into the main airflow and carried along the pipeline at a
pre-set flow rate, to create the desired challenge at the test filter.

Since monodispersed bacterial aerosols (i.e. single bacteria per aerosol droplet) are highly desirable, to ascertain actual
challenge rates and filter efficiencies the size distribution of aerosol droplets from the nebuliser were experimentally
determined using an aerosol particle size counter (reference 2). An average particle diameter of 0.4-0.5 micron was
measured, indicating that most of the aerosol droplets produced by this design of nebuliser were unlikely to carry more
than a single bacterium per droplet.

SAMPLING TECHNIQUES
One of the main requisites in air filter testing is the accurate determination of the number of bacteria in the aerosol
challenging the filter. It is possible to estimate the concentration released from the nebuliser as an aerosol by
performing bacterial counts in serial dilutions of the suspensions before and after nebulising and measuring the net loss
of liquid from the vessel. While such procedures do measure the net loss of organisms and liquid from the nebuliser, the
estimates tend to be inaccurate and cannot take into account losses which may occur by trapping in the lines and
equipment downstream from the nebuliser or any loss of viability of the organisms. Furthermore, the results will be
affected by any disproportionate losses of liquid due to evaporation.

A more accurate method which obviates these difficulties and one which is essential in the intricate assembly used in this
system, is to measure the actual airborne organism concentrations directly by sampling the aerosol. In practice, routine
aerosol sampling is carried out at one sampling port immediately upstream from the filter housing to measure the
organism in the challenge aerosol encountering the filter (viz. the actual bacterial challenge).

Control experiments also involved sampling at points downstream from the filter housing without a test filter installed, to
ensure that during test runs all organisms passing through any filter are detected and not lost by trapping or cell death in
this section of the pipeline.

Aerosol sampling was carried out by two different systems, namely double liquid impingers and a slit sampler. To be
effective in any test system such samplers must be highly efficient and shown to capture nearly 100% of airborne
particles. These types of samplers were chosen because of their demonstrated high efficiencies and suitabilities for
microbial concentrations involved. Very high organism concentrations, such as found in the upstream challenge, were
sampled by chilled liquid entrapment in all-glass impingers of the Porton design. Low organism concentrations in the
downstream air flow required whole flow sampling using a slit sampler.

During challenge monitoring trials, many of the usual aqueous impinger liquids proved inadequate because the operating
conditions caused secondary nebulisation within the sampler leading to serious losses of liquid and trapped organisms.
Alternative trapping liquids were tested in the impinger and the escape of organisms monitored. Liquids of high
viscosity, such as glycerol, were found to be sufficiently effective (reference 2) and were used routinely.

100% glycerol was demonstrated to be highly efficient in capturing organisms, especially when pre-cooled to -20C, as at
this temperature it was not itself nebulised; thus liquid losses were negligible. Nevertheless, although organism capture
was highly reproducible, it was apparent that organisms were still escaping from a single impinger sampling system.
Virtually 100% trapping was demonstrated by linking a second impinger, also containing pre-cooled glycerol, in series with
the first.

A large flow capacity slit sampler (Casella 2109/79 Mk5 bacterial slit sampler) fitted with a high volume sampling head
(700 l/min) was used to sample the whole volume of air downstream from the test filters in order to capture any
organisms passing through. It was difficult to precisely determine the capture efficiency of the slit sampler because the
design is optimised for the sampling of large volumes of air containing very small numbers of micro-organisms which are
concentrated onto a single agar plate. No organisms were ever detected in the exhaust gas from the slit sampler under
normal experimental conditions and the efficiency of entrapment was considerable, approaching 100%, especially as the
whole downstream flow was sampled during filter testing. A considerable design advantage over partial and switched flow
sampling systems.

Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges 10
 Bacterial Challenge Test Organism
STOCK CULTURES: CARE AND MAINTENANCE

These were carried out in general accordance with HIMA Report No. 78-4 (reference 5).
Reconstitute lyophilised preparations of Brevundimonas diminuta ATCC 19146, as per the directions and recommenda-
tions of the American Type Culture Collection. Check the purity of the reconstituted cultures.
Prepare stock cultures by inoculation of Tryptone Soya Agar (TSA) plates and incubate at 30C for 24 hours.

ORGANISM DESCRIPTION

Organism: Brevundimonas diminuta ATCC 19146

Microscopic Examination: Non-sporing, Gram negative, cocco-bacillus 0.3-0.4m x 0.8-1.0m with a single polar flagel-
lum occurring primarily as single cells and showing characteristic zig-zag motility (hanging drop method).
Colony Morphology: Colonies on TSA (aerobic, 30C) are pinpoint at 2 hours and are 1-2mm at 36-48 hours. These
colonies are slightly convex, shiny, entire and beige in colour.

CHARACTERISTICS ENABLING IDENTIFICATION

Brevundimonas diminuta ATCC 19146 shows limited biochemical activity and is unable to utilise most carbohydrates as
primary carbon sources. The organisms is an obligate aerobe and is cytochrome oxidase and catalase positive.

Summary of characteristics enabling identification of Brevundimonas diminuta ATCC 19146.

Test Results Test Results

Spore formation - DNAse -

Gram stain - Centrimide tolerance -
Cocco-bacillus + Cytochrome oxidase (indophenol) +
Motility + Indole -
Single polar flagellum + Dentification +
Obligate aerobe + Growth of glucose -
Voges/Proskauer - Lactose -
Methyl red - Ethanol +
Gelatinase - Xylose -
Catalase + MacConkey Agar +

+ = positive result for that test or possession of attribute

- = negative result for that test or lack of attribute

PREPARATION OF BACTERIAL CHALLENGE SUSPENSION

(Generally in accordance with reference 5).

Inoculate 20ml Tryptone Soya Broth. Use 0.1ml of TSB Culture with test from stock cultures to inoculate 200ml of Saline
Lactose Broth. Incubate at 30C for 24 hours.

Centrifuge whole culture for not more than 20 minutes (4000 rpm) at 2-4C.

Aseptically remove half the volume of supernatant and resuspend cell pellet in remaining volume.

Use concentrate cell suspension immediately or it can be stored for up to 8 hours at 2-4C.

11 Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges
 References
1. Operating instructions for Sterile Air Filters. domnick hunter Publication Reference 547/6/89 (P.110).

2. Croy, R.R.D. Fielding, R.M. Billiet, C.T. (1984) A bacterial challenge test system to determine the
effetiveness of filters for the cold sterilisation of compressed air. Process Biochemistry, Dec. 1984.
(Available from domnick hunter Process Division.)

3. VALAIRDATA

4. Health Industry Manufacturers Association (HIMA) Report No. 78-4, 12/1980. Medical Filter Sterilisation
Monograph: Guidelines for the Validation of Filters for Sterilising Liquids.

5. BIO-X II

6. HIGH FLOW HOUSINGS (1988). domnick hunter Publication Reference 524/7/90.

7. Bourdillon, R.B., Lidwell, O.M. and Thomas, J.C. (1941). J. Hyg. Camb., 41, 197.

8. British Standards Institution BS5726 (1979), Specification for microbiological safety cabinets.

9. Restall, S.W.F. (1978), Lab. Practice, July, P.557.

10.Blakie, E.W. (1987) Theory and practice of compressed air filtration and Sterilization in the Production of
Antibiotics.

11. Billiet, C.T., Stenhouse, J.I.T. (1985) Theoretical study of filter system used in the production of sterile
air. Filtration and Separation, Nov, Dec. 1985. (Available from domnick hunter Process Division.)

12.Sterilisation of compressed air and gases in the pharmaceutical and fermentation industries (1985).
domnick hunter Publication Reference 45/12/85.

13.Bovallius, A., Bucht, B., Roffey, R., Annas, P. (1978) Three year investigation of the natural airborne
bacterial flora at four localities in Sweden. Appl. Environ. Microbiol. 35 (5), 847-852.

14.Martinez, K.F./ (1986) In-depth survey report: Control technology assessment of enzyme fermentation
processes.... National Institute of Occupational Safety and Health Report Number CT-116-15b, December
1986, Cincinnati, Ohio.

15. Domnick, K.R. (1983) Compressed air sterilisation. Publication DMF 101 available through domnick hunter ltd.

Quality Assurance Document for BIO-X II Air Sterilisation Grade Filter Cartridges 12
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Telefax: +60 3 735 1699
domnick hunter (RL) Thailand Co. Limited
Room 12A, 12th Floor, SNC Tower,
domnick hunter (RL) 33 Soi South Nana,
Sukhumvit 4 Road,
Singapore Pte Limited Klong Toey Bangkok 10110, Thailand
APS Industrial Building, Tel: +66 2 267 9760
49 Jalan Pemimpin 05-11, Telefax: +66 2 267 9764
Singapore 577203
Tel: +65 250 4088
Telefax: +65 250 4223
domnick hunter inc.
5900 B Northwoods Parkway,
domnick hunter Iberica Charlotte, NC 28269, USA
Calle Jordi de Sant Jordi 19, Tel: +1 (704) 921 9303
08027 Barcelona, Spain Toll free: 1-800-345-8462
Tel: +34 (0)3 351 4807 Telefax: +1 (704) 921 1960
Telefax: +34 (0)3 351 7102