validation guide sterisart® nf1. introduction 7 1.1 validation of production procedures and test...

Validation GuideSterisart® NF

Validation GuideSterisart® NFVersion 1.0

Disposable Filtration Units for Sterility Testing

16466-ACD|-GBD 16467-ACD|-GBD 16468-ACD|-GBD

16469|16470|16475-GBD

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1. Introduction 71.1 Validation of Production

Procedures and Test Methods 71.2 The Sterility Test 71.3 The Purpose of this Validation Guide 71.4 cGMP Quality Assurance

from Sartorius 81.5 Quality Assurance 81.6 Prevention of Contamination 81.7 Complete Traceability 81.8 Quality Management Systems 91.9 Requirements to be Fulfilled

by a System for Sterility Testing According to Pharmacopoeial Methods 10

1.9.1 Specific Requirements for Avoidance of False Positives 11

1.9.2 Specific Requirements for Avoidance of False Negatives 11

2. Microbiological Tests 122.1 Bacteria Challenge Tests 122.1.1 Hydrophilic 0.45 µm Sartochem®

Membrane Filters 122.1.2 Hydrophobic Venting Filter

Membranes 132.1.3 Venting Filter on the Dual-Needle

Metal Spike (0.2 µm PTFE Membrane Filter) 14

2.1.4 By-packed Needle with Venting Filter (0.2 µm PTFE Membrane Filter) 15

3. Sterilization Validation 163.1 Presterilization Bioburden 163.2 Validation of Gamma Irradiation 173.2.1 Examination of the Validation 173.2.2 Dose mapping Validation 183.2.3 Sterility Test with Soybean-Casein

Digest Broth (TSB) and Thioglycolate Broth after Irradiation at a Reduced Dose of 5 kGy 19

3.2.4 Growth Promotion Test 203.2.5 Performance Test after

Irradiation with 25 kGy 213.2.6 Performance Test 50 kGy 233.3 Validation of Ethylene Oxide

Gas Sterilization 253.3.1 Sterility Test with Bioindicators 253.3.2 Sterility Test with Soybean-Casein

Digest Broth (TSB) and Thioglycolate Broth 26

3.3.3 Growth Promotion Test 273.3.4 Degassing Curve 283.3.5 Performance Test after Double

Sterilization 28

4. Physical Tests 294.1 Integrity Test of the

Membrane Filters 294.1.1 Venting Filters: 0.2 µm PTFE

Membrane Filter On Top of the Containers, in the Dual-Needle Metal Spikes of 16466, 16469 and 16475 and on the Venting Needles of 16467 and 16468 29

4.1.2 Container: 0.45 µm Sartochem® Membrane Filters (In -> Out) 30

4.1.3 0.2 µm PTFE Membrane Filter (Venting Filter on the Container) 31

4.2 Water Penetration Test 324.2.1 0.2 µm PTFE Membrane Filter of

the Container Venting Filter 324.3 Burst Pressure 334.3.1 Burst Pressure of the Container 334.3.2 Burst Pressure of the Container

Venting Filter 344.4 Pressure Hold Test of the Rubber

Cap on the Container Venting Filter 35

4.5 Flow Rates with Liquids 364.5.1 Sartochem® Membrane Filters

with RO Water. Sample Taken Out of an Open Vessel 36

4.5.2 Sartochem® Membrane Filters with RO Water. Sample Taken Out of a Closed Vessel (1 Liter Bottle) 37

4.5.3 Sartochem® Membrane Filters with Glucose Solution 20% 41

4.5.4 Sartochem® Membrane Filters with Glucose Solution 40% 41

4.6 Flow Rates for Air 424.6.1 0.2 µm PTFE Membrane Filter of

the Dual Needle Metal Spike Venting Filter Unit 42

4.6.2 0.2 µm PTFE Membrane Filter of the Venting Needle Supplied with Sterisart® types 16467 and 16468 42

4.6.3 0.2 µm PTFE Membrane Filter of the Venting Needle Spike Supplied with Sterisart® types 16469 and 16475 42

4.6.4 0.2 µm PTFE Membrane Filter in the Container Venting Filter 43

4.7 Wetting Time Sartochem® Membrane Filters 44

4.8 Leak Test of the System 454.8.1 Tubing Systems of Sterisart®

Versions 16466, 16467, 16468, 16469 and 16475 (all with different adapters|needles) 45

4.8.2 Tubing System of Sterisart® Version 16470 (Two Different Dual Needle Metal Spikes) 46

4.8.3 Sterisart® NF Containers and Connection to the Tubing System 46

4.8.4 Container Venting Filter Housing 474.9 Equal Conveyance of the Liquid into

the Two Sterisart® NF Containers 484.10 Accuracy of the Graduation of

the Containers 494.11 Integrity of the Packaging 504.11.1 Integrity of the Package Sealing 504.11.2 Integrity of the Package Sealing

of Sterisart® Type 16470 50

5. Analytical Test 515.1 Extractable Substances in the

Filtrate According to USP and Ph Eu 51

5.1.1 Determination of pH Value and Conductivity 51

5.1.2 Non-Volatile Residue 51

Table of Contents

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5.1.3 Determination of Oxidizable Substances 51

5.1.4 Determination of Chlorides 515.1.5 Determination of Sulfates 515.1.6 Determination of Ammonia 515.1.7 Determination of Heavy Metals 515.2 Desorption Test with Antibiotics

and Preservatives 535.2.1 Design of the Sterisart® Containers 545.2.2 Filtration of Red Ink Solution

(100%) 565.2.3 Adsorption and Desorption of

Antibiotics and Preservatives and Growth Promotion Test 56

Growth Promotion Test 595.3 The Packaging Material‘s

Impermeability to Gas 60

6. Chemical Tests 616.1 Chemical Compatibility 61

7. Visual Inspections 637.1 Container Venting Filter 637.2 Sterisart® Devices 64

8. Quality Assurance 658.1 Incoming Inspection 658.1.1 Qualification of the Sartochem®

Membrane Filter Material 658.1.2 Qualification of the PTFE

Membrane Filter 658.1.3 Qualification of the Plastic

Parts Material 658.1.4 Qualification of the Tubes 658.2 In-Process Control 668.2.1 Sterisart® NF Container 668.2.2 Sterisart® NF Tubing System

and Spike 668.2.3 0.2 µm PTFE Membrane Filter

Venting Filter 668.3 Final Control 668.3.1 Sterisart® NF System 66

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1.2 The Sterility TestThe required method for sterility testing of finished products, for all products which can be filtered or made to be filterable, is the Membrane Filter Method: “Pharmacopoeial articles are to be tested by the Membrane Filtration Method under Test for Sterility of the Product to be Examined where the nature of the Product permits (Excerpt from the USP 28, <71> Sterility Tests). Specific details on this method are given in the various Pharmacopoeias. It entails the filtration of mixed or successive samples from a product lot through membrane filters, washing the membranes to remove growth-inhibitory substances and subse-quently incubating the membranes in contact with nutrient media. The product lot is considered to be sterile when no turbidity is formed in the nutrient media, provided that it is certain that the absence of turbidity (of microbial growth) is not caused by insufficient washing of a product containing growth-inhibitory substances, by bacterial passage through a membrane filter of inadequate pore size, or by residues of disin-fectant taken through unsuitable device packaging during steril-ization of the isolator. Since it is an absence|presence test for micro-organisms, and the possibility of re-testing is very limited, it is vital that no secondary contamination be allowed to enter the samples, wash liquid, nutrient media or test system during the sterility test procedure. Such aseptic testing requires the use of an appropriately dedicated system (e.g. Sterisart® NF) in an appropriate environment (e.g. an isolator). The Sterisart® NF System consists of a disposable device, complete with a needle for liquid uptake, tubing for splitting and transferring liquid, and containers with membrane filters for filtration and incubation. The Sterisart® Universal-Pump, a peristaltic pump, moves the liquid from needle to test containers.

1.3 The Purpose of this Validation GuideOur intention, as manufacturer of the Sterisart® NF disposable devices, is to help you as much as possible in the preparation for the validation of your sterility testing procedure. This “Validation Guide“ supplies you with a variety of information on the testing methods that are used by Sartorius QA|QC in the production of Sterisart® NF devices, from raw materials to finished products, and the specifications that must be met before they are released for sale. The purpose of this material is to give you justified confidence in using the products described and to provide you with the documented basis for the planning, implemen-tation and documentation that goes along with the validation of your specific sterility testing procedure, which will correspond with the pharmaceutical products tested and the exact procedure used. Should you have questions on this, or require assistance regarding the validation of your sterility testing procedure or on microbiological monitoring of the air in a test environment, please do not hesitate to contact our Validation Service Department.

1. Introduction

1.1 Validation of Production Procedures and Test MethodsPharmaceuticals such as parenterals, ophthalmics, veterinary medicines and other products that come into contact with the blood stream, or otherwise enter the body below the skin surface, must be sterile to avoid health risks via microbial contamination. The pharmaceu-tical manufacturer is obliged to supply proof of their safety and sterility according to Pharmaco-poeia guidelines. An indispensable part of this proof is the validation of procedures used. This is a logical supplement and significant part of cGMP regulations, which have been in force for many years.

Guidelines for validation are given in the US Code of Federal Regulations Title 21 and in various pharmacopoeias such as the USP or PhEu. In addition, guidelines have been established jointly by the Committee for Laboratories and Official Drug Product Inspection Services as well as the Department of Industrial Pharmacists of the Federation Internationale Pharma-ceutique (F.I.P.): „Validation, as used in these guidelines, comprises the systematic testing of essential production steps and equipment in the R & D and production departments, including testing and inspection of pharmaceutical products with the goal of ensuring that finished products can be manufactured reliably and repro-ducibly and in the desired quality, in keeping with the established production and quality control procedures“.

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1.4 cGMP Quality Assurance from SartoriusThe consistent high quality of Sartorius membrane filters and devices containing membrane filters is assured by careful selection of raw materials, well planned and validated production technologies and an exceptionally efficient Quality Assurance Department, all of which results in excellent batch-to-batch reproducibility. The test procedures used are based both on external standard methods, such as the USP, Ph Eu, JP and ASTM, and on in-house methods which are the result of Sartorius’ extensive experience in filtration technology over the past 70 years.

1.5 Quality AssuranceFor quality assurance, all materials are selected carefully in accordance with current regulations, such as the FDA CFRs, cGMPs in-house guidelines and the specifications of our Research and Development Department including the terms of delivery and acceptance of our Purchasing Department. Documen-tation begins with the inspection of incoming raw materials including in-process materials, molded parts and sealing materials, etc. for manufacture. Adherence to cGMP requirements (clean room condi-tions, gowning and employee hygiene, etc.), which are monitored by documented in-process controls, ensures optimal quality control in standard operating procedures for production. Finished Sartorius Sterisart® NF systems undergo strict final product quality control. This involves individual tests carried out on a representative number of samples. A lot is not released until all in-process and final quality control data are available and confirmed to be within specifica-tions.

1.6 Prevention of ContaminationSterisart® NF devices are individ-ually sealed in gas permeable protective plastic boxes in a controlled production area. Following this step, the Sterisart® NF alpha types are packed with 10 systems in a box and are treated using a validated ethylene oxide sterilization. The Sterisart® NF gamma types are sealed in an additional gas-impermeable plastic bag prior to packing them with 10 systems in a box, and sterilized by a validated gamma irradiation process. This double-packaging rules out the possibility of contami-nating the Sterisart® NF system with a disinfectant that is used in isolators for the sterility test, such as Vapor Phase Hydrogen Peroxide (VHP): Following disinfection of the equipment’s surface, it must be shown that no H

2O2 residues remain in the system as these could inhibit the growth of microorganisms, and could cause false-negative results. The most reliable way to ensure that VHP does not remain in the system is to use specialized packaging that VHP cannot penetrate. Sterisart® NF gamma has therefore been specifically developed with VHP impermeable packaging for use in isolators.

1.7 Complete TraceabilityThe type, lot number, sterlization and expiration date are printed on the label of the protective plastic box and on the label of the box where the 10 Sterisart® NF units are packed. Additionally type, lot number and individual container number are printed on each Sterisart® container. The traceable lot number allows convenient retrieval of all data compiled on the materials used, production steps and QC tests.

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1.8 Quality Management Systems

DIN EN ISO 9001 CertificatesSartorius Stedim Biotech imple-mented Quality Management Systems to assure consistent high quality of Membrane Filters, Ultra Filters, Filter Cartridges and Dispos-ables.

Exemplary Quality Systems Certificates:

Global Quality Systems Certificates| Quality Certificates (ISO 9001:2000)

Global Quality Systems Certificates| Quality Certificates for Medical Devices (ISO 13485:2003 and directive 93|42|EEC)

The complete Quality Systems Certificates are continuously updated and can be downloaded on our website:

www.sartorius-stedim.com/ qm-certificates

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1.9 Requirements to be Fulfilled by a System for Sterility Testing According to Pharmacopoeial Methods

User’s requirement This necessitates Tests made

General Requirements

The product must be sterile, and the sterilizing process must not affect the function

Reliable, significant quality assurance

No deterioration during transport and storage

The system must allow the shortest possible test time

The system must be practical

User|environment safety must be given

a) Validated sterilization process

b) No change in the product itself after sterilization

a) A non-destructive, routine test of system integrity must be correlated to the ability to retain microorganisms

b) Effective Quality Control proce-dures must be determined and exercised

a) Integrity of the packaging must be maintained up to time of use

b) No aging which could affect the function should occur, at least up to the given expiry date

Quickest possible filtration of sample

a) Graduations to simplify filling, e.g. of culture media

b) Equal splitting of samples

c) Chemical compatibility with most test products

The container must be sufficiently pressure resistant

Sterilization validation | Sterility test (3.2, 3.3)

Sterilization validation | Performance test (3.2, 3.3)

Bacteria challenge tests and corre-lation to integrity test data (2.1, 4.1) and water penetration test (4.2)

In-process control (8.2), final control (8.3)

Integrity of the packaging (4.11)

Shelf life (4.11)

Flow rates with liquids (4.5)Flow rates for air (4.6)Wetting time (4.7)Accuracy of graduation (4.10)

Accuracy of graduations (4.10)

Equal conveyance (4.9)

Chemical compatibility (6.1)

Burst pressure (4.3), Pressure hold test on cap (4.4)

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1.9.1 Specific Requirements for Avoidance of False Positives


Avoidance of secondary contamination during the test

a) Outside air must only be capable of entering the system through filters certain to sterilize it

b) Maintenance of the closed system while operating

Bacteria challenge test (2.1)

Leak test (4.8)Burst pressure (4.3)

1.9.2 Specific Requirements for Avoidance of False Negatives


Microorganisms in the test sample must be reliably retained

No hindrance to the growth of any microorganisms retained

a) The membranes must be validated for the retention of microorganisms

b) Tight sealing of the Sartochem® membrane by a specially developed membrane clamp technique

a) No residues from the sterilization process

b) No residues from other steril-ization processes such as used in isolators

c) No interfering extractables from the system

d) Minimal adsorption of any growth inhibitors in the sample, and easy removal of the traces adsorbed prior to incubation with culture media

e) Bio-inert materials

Bacteria challenge test (2.1)

Design of the Sterisart® containers (5.2.1)

Sterilization validation (3.2, 3.3)

Gas-impermeability of the packaging (5.3)

Extractables (5.1)

Desorption test (5.2)

Growth Promotion test (3.2.4, 3.3.3)

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2. Microbiological Tests

2.1 Bacteria Challenge Tests

2.1.1 Hydrophilic 0.45 µm Sartochem® Membrane Filters

BackgroundUSP and Ph Eu call for the use of “membrane filters having a nominal pore size not greater than 0.45 µm whose effectiveness to retain micro-organisms has been established”. Serratia marcescens is used as a challenge microorganism analogous to Brevundimonas diminuta used for challenge tests on 0.2 µm pore sized membranes.

Therefore according to the HIMA Doc. No. 3, Vol. 4, ASTM 838-83 and DIN 58355 part 3, for 0.2 µm “sterilizing-grade” membrane filters, the 0.45 µm Sartochem® membrane used in Sterisart® NF is to be tested in an analogue procedure. This is to show if a sterile effluent is produced when challenged with a minimum concentration of 107 Serratia marcescens per cm2 filter area. Compliance with this Bacteria Challenge Test will conclusively demonstrate that microorganisms are certainly and reliably retained.

Sterisart® types: 16466-ACD|-GBD, 16467-ACD|-GBD, 16468-ACD|-GBD, 16469-GBD, 16475-GBD

ConclusionThe Sartochem® membrane filter has a retention of $ 107 Serratia marcescens organisms per cm2 of effective filtration area. Therefore the pore size of the Sartochem® membrane is 0.45 µm. A 0.45 µm membrane complies with official requirements of the international pharmacopoeia for sterility testing membrane filters.

Results

Type Lot no. No. of No. of No. of Sartochem® sterile filtrates filtrates showing tested (acc. to both S. marcescens methods) (cfu counted on agar)

16466 010015 20 20 0

16466 020014 20 20 0

16466 020015 20 20 0

16466 020016 20 20 0

16466 020020 20 20 0

16466 020021 20 20 0

16467 020012 20 20 0

16467 020017 20 20 0

16468 040006 20 20 0

16469 040038 20 20 0

16469 040039 20 20 0

16475 040021 20 20 0

16475 040042 20 20 0

MethodA suspension of $ 107 Serratia marcescens per ml of liquid nutrient media acc. to HIMA, Doc. 3 (since the test specified in HIMA Doc. 3 is used for evaluation of 0.2 µm pore sized filters the original test uses Brevun-dimonas diminuta instead of the larger microorganism Serratia marcescens, but of the same concentration as used in this analogous test!), was filtered through the Sartochem® membrane in the sterile Sterisart® NF container at 3 bar. Filtrate from each container was collected in a sterile vessel. Half of the filtrate was filtered through a 0.45 µm cellulose nitrate gridded membrane, which was then placed onto nutrient agar (article no. 14144). The collected filtrates and membranes on nutrient agar were incubated at 30°C for 5 days. All red Serratia marcescens colonies on the gridded membranes and red turbid filtrates were counted.

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2.1.2 Hydrophobic Venting Filter Membranes

BackgroundIn accordance with the HIMA Doc. No. 3, Vol. 4, ASTM 838-83 and DIN 58355 part 3, the 0.2 µm membrane filters used in the Sterisart® NF systems should produce a sterile effluent when challenged with a minimum concentration of 107 Brevundimonas diminuta per cm2 filter area. Meeting the require-ments of these tests ensures safe and reliable sterile filtration of air passing through the vent filters of the dual-needle metal spike, the vent filter of the venting spike and on the top of the Sterisart® NF containers.

0.2 µm PTFE Membrane Venting Filter on the Container


MethodA suspension of $ 107 Brevun-dimonas diminuta per ml liquid nutrient media acc. to HIMA, Doc. 3, was filtered through a sterilized, ethanol pre-wet container venting filter at 1.0 bar. Filtrate from each venting filter was collected in a sterile vessel. Half of the filtrate was filtered through a 0.45 µm cellulose nitrate gridded membrane, which was then placed onto nutrient agar (article no. 14144). The collected filtrates and membranes on nutrient agar were incubated at 30°C for 5 days. All cream Brevundimonas diminuta colonies on the gridded membranes and cream turbid filtrates were counted.

ConclusionThe PTFE membrane venting filter on the top of the container has 100% retention of $ 107 Brevun-dimonas diminuta organisms per cm2 of effective filtration area. The PTFE membrane venting filter meets the requirements of HIMA Doc. No. 3, Vol. 4, ASTM 838-83 and DIN 58355 part 3.

Results

Article no. Lot no. Assembled No. of No. of No. of filtrates in Sterisart® venting sterile showing B. diminuta lot no. filters filtrates (cfu counted tested on agar)

17526-INQ 010294 010015 50 50 0

17526-INQ 020253 020014|12|15 50 50 0

17526-INQ 020825 020020 50 50 0

17526-INQ 020581 020017 50 50 0

17526-INQ 030429 030008|9|10 50 50 0

17526-INQ 030456 030011|15|16|20 50 50 0

17526-INQ 030621 030017|18|19 50 50 0

17526-INQ 031013 040006 50 50 0

17526-INQ 040016 040014 50 50 0

17526-INQ 040056 040021 50 50 0

17526-INQ 040135 040037 50 50 0

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2.1.3 Venting Filter on the Dual-Needle Metal Spike (0.2 µm PTFE Membrane Filter)

Sterisart® type: 16466-ACD|-GBD, 16469-GBD, 16475-GBD

MethodA suspension of $ 107 Brevun-dimonas diminuta per ml liquid nutrient media acc. to HIMA, Doc. 3, was filtered through a sterilized, ethanol pre-wet venting filter on the spike at 0.5 and 1 bar. Filtrate from each container was collected in a sterile vessel. Half of the filtrate was filtered through a 0.45 µm cellulose nitrate gridded membrane, which was then placed onto nutrient agar (article no. 14144). The collected filtrates and membranes on nutrient agar were incubated at 30°C for 5 days. All cream Brevundimonas diminuta colonies on the gridded membranes and cream turbid filtrates were counted.

ConclusionIn Sterisart® NF systems sterilized by ethylene oxide gas, the PTFE membrane venting filter on the spike has a retention of $ 107 Brevundimonas diminuta organisms per cm2 of effective filtration area. The PTFE membrane venting filter meets the requirements of HIMA Doc. No. 3, Vol. 4, ASTM 838-83 and DIN 58355 part 3.

Results


17526-INQ 010294 010015 50 50 0

17526-INQ 020253 020014|15 50 50 0

17526-INQ 020825 020020 50 50 0

17526-INQ 030429 030009|10 50 50 0

17526-INQ 030456 030015|16|20 50 50 0

17526-INQ 030621 030019 50 50 0

17526-INQ 040056 040021 50 50 0

17526-INQ 040135 040037 50 50 0

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2.1.4 By-packed Needle with Venting Filter (0.2 µm PTFE Membrane Filter)

Sterisart® types: 16467-ACD|-GBD, 16468-ACD|-GBD

BackgroundA suspension of $ 107 Brevun-dimonas diminuta per ml liquid nutrient media acc. to HIMA, Doc. 3, was filtered through the sterilized, ethanol pre-wet venting filter of the venting needle of 16467 at 0.5 and 1 bar. Filtrate from each container was collected in a sterile vessel. Half of the filtrate was filtered through a 0.45 µm cellulose nitrate gridded membrane, which was then placed onto nutrient agar (article no. 14144). The collected filtrates and membranes on nutrient agar were incubated at 30°C for 5 days. All cream Brevundimonas diminuta colonies on the gridded membranes and cream turbid filtrates were counted.

ConclusionIn Sterisart® NF systems sterilized by ethylene oxide gas, the PTFE membrane venting filter on the venting needle has a retention of ≥ 107 Brevundimonas diminuta organisms per cm2 of effective filtration area. The PTFE membrane venting filter meets the require-ments of HIMA Doc. No. 3, Vol. 4, ASTM 838-83 and DIN 58355 part 3.

Results


17526-INQ 020253 020012 50 50 0

17526-INQ 020581 020017 50 50 0

17526-INQ 030429 030008 50 50 0

17526-INQ 030456 030011 50 50 0

17526-INQ 030621 030017|18 50 50 0

17526-INQ 031013 040006 50 50 0

17526-INQ 040016 040014 50 50 0

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BackgroundA series of tests is needed to confirm that a sterilization procedure has been carried out successfully. Sterility testing of a system is used to prove that the entire contents of the inner packaging are sterile. The growth promotion test ensures that no residue from the sterilization process remains in the packaging that could promote or inhibit the subsequent growth of microor-ganisms. The performance test ensures that the sterilization procedure does not damage any part of the product or lead to system malfunctions.

Sartorius uses two different methods for sterilization: The gamma-irradiation method is employed for all Sterisart® NF gamma versions (GBD) and the ethylene oxide gas method for all Sterisart® NF alpha versions (ACD). Gamma-irradiation of products is carried out as required by DIN EN 552 and ISO 11137 and ethylene oxide sterilization in compliance with DIN EN 550 and ISO 11135.

3.1 Presterilization Bioburden

BackgroundThe European Pharmacopoeia describes how the safety assurance level (SAL) of sterilization processes is calculated. The microbial load of a system is determined before steril-ization to ensure that the burden is not too high for the sterilization process to be reliable.

Sterisart® types: 16466-ACD|-GBD, 16467-ACD|-GBD, 16469-GBD, 16470-GBD, 16475-GBD

MethodThe bioburden was determined of all inner and outer Sterisart® surfaces by filling, shaking and rinsing the units in succession. The rinsing solution collected was filtered through a 0.45 µm gridded membrane filter made of cellulose nitrate. The filter was transferred to nutrient agar (article no. 14144) and incubated for 7 days at 30°C. The resulting colonies were counted.

ConclusionThe bioburden of the Sterisart® systems proved are low enough to ensure that any standard steril-ization procedure using gamma-irradiation or ethylene oxide gas will reduce the bioburden to zero.

Results

Type Lot no. No. of Average Average Average units bioburden bioburden bioburden tested outer surface inner surface total [cfu] [cfu] [cfu]

16466 040011 10 114.1 5.2 122.7

16467 040019 10 50.6 2.4 103.6

16468 040014 10 43.1 1.6 49.7

16470 040026 4 19.5 2.75 22.25

16475 040041 10 90.5 3.4 93.9

3. Sterilization Validation

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3.2 Validation of Gamma Irradiation

Sterisart® types:16466-GBD, 16467-GBD, 16468-GBD, 16469-GBD, 16470-GBD, 16475-GBD

3.2.1 Examination of the Validation

MethodThe validation of gamma-irradiation follows the VDMax-method in which the average bioburden of 3 lots (total of 12 Sterisart® units) is determined. For each Sterisart® lot further 26 units are gamma-irradiated with a minimum-dose of 5 kGy. Afterwards a sterility test with these Sterisart® units is performed.

ConclusionThe results demonstrate that even a minimum dose of 5 kGy sterilizes all systems tested. Therefore it can be assumed that a sterilization with 25 kGy always produces a sterile product. A constant level of 25 kGy irradiation is proved by the dose mapping procedure.

Results

Type Lot no. No. of Average Average Average units bioburden bioburden bioburden tested outer surface inner surface total [cfu] [cfu] [cfu]

16466 040011 10 114.1 5.2 122.7

16468 040014 10 43.1 1.6 49.7

16475 040041 10 90.5 3.4 93.9

Sterility test after gamma-irradiation with 5 kGy:

Type Lot no. No. of Sterility test with Sterility test with systems TSB broth; thioglycolate broth; tested no. of containers no. of containers

16466 040022 26 26 sterile 26 sterile

16467 040020 26 26 sterile 26 sterile

16475 040021 26 26 sterile 26 sterile

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3.2.2 Dose mapping Validation

MethodDuring dose mapping, the dosim-eters are placed on the packed palette at various depths on two parallel levels according to a predefined raster pattern, once on the surface and once in the middle of the palette. The density and the specific weight of the load was determined to establish a dose mapping guide value for the sterilizer.

After sterilization, all dosimeters were subjected to photometric analysis.

Dosimeter manufacturer: FWT Far West Technology, USADosimeter type: Nylon dosimeter FWT 60-00

Photometer type: Aérial SpectrophotometerPhotometer manufacturer: FWT Far West Technology, USA

Calibrated with: NPL (National Physical Laboratory, England) – Alanin Dosimeter

ConclusionWhen the validation procedure is followed, Sterisart® units are gamma-irradiated as required, with a minimum dose of 25 kGy reaching all areas.

Level I

Width 5 cm 26.5 cm 53 cm

Dosimeter on the surface: Height 55 cm 37.5 kGy 37.8 kGy 37.5 kGy

Dosimeter in the geometric middle: Height 161 cm 34.1 kGy 37.1 kGy 36.4 kGy


Level II

Width 5 cm 26.5 cm 53 cm

Dosimeter on the surface: Height 55 cm 30.7 kGy 31.1 kGy 31.3 kGy



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ConclusionThe results demonstrate that even a minimum dose sterilizes all systems tested.The gamma-irradiation procedure with 25 kGy ensures that all surfaces of the Sterisart® NF systems in contact with the sample are sterile.

3.2.3 Sterility Test with Soybean-Casein Digest Broth (TSB) and Thiogly-colate Broth after Irradiation at a Reduced Dose of 5 kGy

MethodTo ensure that the system remains sterile even at the minimum dose of 5 kGy, two GBD packages (types 16466|67|70|75) were irradiated accordingly. The result is trans-ferable to types 16468|69. A sterility test is performed with the Sterisart® NF systems using a Sterisart® pump (type 16413). A container from each system was filled with 50 ml of TSB broth (tryptone soya broth, Oxoide code no. B00509M) and 100 ml of thioglycolate medium (Oxoide code no. B00510M). All containers were incubated together with a bottle of unused TSB broth and thioglycolate medium (sterility control with unused media) for 14 days at 30°C.

Results

Type Lot no. No. of Sterility test with Sterility test with systems TSB broth; thioglycolate broth; tested no. of containers no. of containers

16466 040022 26 26 sterile 26 sterile

16467 040020 26 26 sterile 26 sterile

16475 040021 26 26 sterile 26 sterile

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3.2.4 Growth Promotion Test

MethodThe growth test was carried out in accordance with USP and Ph Eu versions, valid at the time the tests had been carried out.The sterile Sterisart® NF gamma units (sterilized with a minimum dose of 25 kGy) were filled with fluid nutrient broth. Each container was inoculated with not more than 100 microorganisms and incubated together with the positive control under defined conditions. The growth of microorganisms was monitored daily to determine whether the growth of the samples was comparable to the positive control.

Microorganism Nutrient Incubation Incubation Incubation strain media temperature time condition

A Aspergillus niger ATCC 16404 Soybean-casein digest 22.5 ± 2.5°C 5 days Aerobic

B Bacillus subtilis ATCC 6633 Soybean-casein digest 22.5 ± 2.5°C 3 days Aerobic

C Candida albicans ATCC 10231 Soybean-casein digest 22.5 ± 2.5°C 5 days Aerobic

D Clostridium sporogenes ATCC 11437 Thioglycolate 32.5 ± 2.5°C 3 days Anaerobic

E Pseudomonas aeruginosa ATCC 9027 Thioglycolate 32.5 ± 2.5°C 3 days Aerobic

F Staphylococcus aureus ATCC 6538P Thioglycolate 32.5 ± 2.5°C 3 days Aerobic

Conclusion The bacterial growth tests show that the sterile materials and gamma-irradiation sterilization do not inhibit the growth of micro-organisms.

Type Lot no. A B C D E F Colony count No. of containers with no growth

16466 040022 1 1 1 1 1 1 < 100 cfu 0

16467 040020 1 1 1 1 1 1 < 100 cfu 0

16469 040038 1 1 1 1 1 1 < 100 cfu 0

16469 040039 1 1 1 1 1 1 < 100 cfu 0

16470 040033 1 1 1 1 1 1 < 100 cfu 0

16470 040034 1 1 1 1 1 1 < 100 cfu 0

16475 040021 1 1 1 1 1 1 < 100 cfu 0

ResultsNo. of containers with substantial growth.

| 21

3.2.5 Performance Test after Irradiation with 25 kGy

MethodReady-manufactured, unsterilized Sterisart® NF units and individually packaged hydrophobic venting filter units (lot no. 030429) were gamma-irradiated at doses not less than 25 kGy and not more than 50 kGy. After sterilization, the units were visually inspected for cracks or discoloration and the microbio-logical and physical performance of the systems was tested.

ResultsThe color of the Sterisart® NF containers and the Y-connector changed to light brown. Most of this discoloration disappeared after 2-3 weeks. Damage such as cracks was not observed.

Type Lot no. Sartochem® Sartochem® Burst pressure Leak Hardness BCT integrity test container test of the tubing (Section 2.1.1) (Section 4.1.2) (Section 4.3.1) (Section 4.8) 1 h at pump rate setting 700

16466 030009 20 passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed 0 failed

16466 030015 10 passed 20 passed 20 passed 10 passed No tested 0 failed 0 failed 0 failed 0 failed


16467 030018 10 passed 20 passed 20 passed 10 passed No tested 0 failed 0 failed 0 failed 0 failed




Type Lot no. Leak Hardness test of the tubing (Section 4.8) 1 h at pump rate setting 700

16470 040033 20 passed 3 passed 0 failed 0 failed

22 |

ConclusionA 25-kGy dose of gamma-irradiation does not influence the performance of Sterisart® NF systems or the PTFE membrane filter in the venting filter.

Lot no. Venting filter Venting filter Venting filter Venting filter BCT Bubble Point air flow rate ethanol flow rate (Section 2.1.2) (Section 4.1.3) (Section 4.6.1)

030429 50 passed 10 passed: 10 passed: 10 passed: Not irradiated 0 failed 1.2–1.3 bar 1.3–1.8 bar 54–87 ml/min 0 failed 0 failed 0 failed

030429 50 passed 10 passed: 10 passed: 10 passed: Irradiated with 0 failed 1.15–1.3 bar 1.3–1.5 bar 45–67 ml/min 25 kGy 0 failed 0 failed 0 failed

| 23

3.2.6 Performance Test 50 kGy

MethodReady-manufactured, unsterilized Sterisart® NF units were gamma- irradiated with minimum doses of 50 kGy. After sterilization, the units were visually inspected for cracks or discoloration and the microbio-logical and physical performance of the systems was tested. The venting filters were irradiated separately.

ResultsThe color of the Sterisart® NF containers and the adapter to the Y-connector changed to brown. The tubing and the Y-connector turned light brown. Most of these discolorations disappeared after 2-3 weeks. Damage such as cracks was not observed.

Type Lot no. Sartochem® Sartochem® Burst pressure Leak Hardness BCT integrity test container test of the tubing (Section 2.1.1) (Section 4.1.2) (Section 4.3.1) (Section 4.8) 1 h at pump rate setting 700

16466 030009 20 passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed

16467 030008 20 passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed




Type Lot no. Leak Hardness test of the tubing (Section 4.8) 1 h at pump rate setting 700

16470 040033 10 passed 3 passed 0 failed 0 failed

24 |

ConclusionGamma-irradiation at a dose of 50 kGy does not influence the performance of the Sterisart® NF systems or of the PTFE membrane filter in the venting filter units.

Lot no. Venting filter Venting filter Venting filter Ethanol flow rate BCT Bubble Point air flow rate (Section 2.1.2) (Section 4.1.3) (Section 4.6.1)

030429 50 passed 10 passed: 10 passed: 10 passed: Not irradiated 0 failed 1.2–1.3 bar 1.3–1.8 bar 54–87 ml/min 0 failed 0 failed 0 failed

030429 50 passed 10 passed: 10 passed: 10 passed: Irradiated with 0 failed 1.2–1.25 bar 1.3–1.6 bar 55–70 ml/min 50 kGy 0 failed 0 failed 0 failed

| 25

3.3 Validation of Ethylene Oxide Gas Sterilization

Sterisart® types: 16466-ACD, 16467-ACD

MethodA total of 6 sterility tests were performed for the validation of the ethylene oxide gas sterilization. 3 half-cycles packed with the maximum load, 1 half-cycle packed with the minimum load (1 palette) and 3 full cycles packed with the maximum load were carried out. The first cycle was double sterilized. At least 120 bioindicators (Bacillus subtilis spore strips) were used for the half-cycles. Sterility tests were run for Sterisart® as well as for Minisart type 0.2 µm. Physical experiments were carried out for all products sterilized with ethylene oxide gas.

3.3.1 Sterility Test with Bioindicators

MethodThe Sterisart® NF units were prepared with Bacillus subtilis endospore strips with a concen-tration of 106 spores per strip. Some units were gassed with ethylene oxide for a minimum of 2 hours (concentration 820 mg/m2). The remaining units were gassed with ethylene oxide for an average of 4 hours (concentration 820 mg/m2). The gassing was repeated three times. After sterilization, the spore strips were removed under aseptic conditions and poured onto liquid soybean casein digest medium and incubated for 7 days at 30°C. Sterility was demonstrated if no microbial growth was observed.

ResultsAfter the ethylene oxide gas steril-ization all bioindicators (Bacillus subtilis spore strips) were sterile.

Number and distribution of bio indicators

Cycle No. Number of bioindicators used

half-cycle 11040411 120

half-cycle 25040411 130

half-cycle 29040411 260

full-cycle 11050411 20

half-cycle 19050412 34

Results of bioindicator tests

Type Lot Gassing time Result of bioindicator test

16466 040032 4 hours all bioindicators sterile




26 |

3.3.2 Sterility Test with Soybean-Casein Digest Broth (TSB) and Thioglycolate Broth

MethodA sterility test was performed on the Sterisart® NF systems using a Sterisart® pump (type 16413). A container from each system was filled with 50 ml of TSB broth (tryptone soya broth, Oxoide code no. B00509M) and 100 ml of thioglycolate medium (Oxoide code no. B00510M). All containers were incubated together with a bottle of unused TSB broth and thioglycolate medium (sterility control with unused media) for 14 days at 30°C.

Results

Sterility Test|1. Half-Cycle

Type Lot no. No. of Sterility test with TSB broth; Sterility test with thioglycolate systems tested no. of containers broth; no. of containers

16467 040019 10 10 sterile 10 sterile



16467 040019 10 10 sterile 10 sterile



16467 040019 10 10 sterile 10 sterile

Conclusion Ethylene oxide sterilization with a concentration of 820 mg/m2 ensures that all surfaces of the Sterisart® NF system in contact with the sample are sterile. All half-cycles are controlled with bio-indicators.

| 27

3.3.3 Growth Promotion Test

MethodThe growth test was carried out in accordance with USP and Ph Eu versions, valid at the time the tests had been carried out.

The Sterisart® NF units were sterilized by ethylene oxide at a concentration of 700 mg/m2. Half of the Sterisart® NF units were filled with fluid soybean-casein digest medium as defined in the European Pharmacopoeia. Each container was inoculated with not more than 100 microorganisms and then incubated alongside the positive control at 22.5 ± 2.5°C for a maximum of 3 days under predefined conditions. The growth of microorganisms was monitored daily to determine whether the growth in the samples was comparable to the positive control.

The remaining Sterisart® NF units were filled with fluid thioglycolate medium as defined in the European Pharmacopoeia. Each container was inoculated with not more than 100 microorganisms and then incubated alongside the positive control at 32.5 ± 2.5°C for a maximum of 3 days under defined conditions. The growth of micro-organisms was monitored daily to determine whether the growth in the samples was comparable to the positive control.

Microorganism Nutrient Incubation Incubation Incubation strain media temperature time condition

A Aspergillus niger ATCC 16404 Soybean-casein digest 22.5 ± 2.5°C 5 days Aerobic

B Bacillus subtilis ATCC 6633 Soybean-casein digest 22.5 ± 2.5°C 3 days Aerobic

C Candida albicans ATCC 10231 Soybean-casein digest 22.5 ± 2.5°C 5 days Aerobic

D Clostridium sporogenes ATCC 11437 Thioglycolate 32.5 ± 2.5°C 3 days Anaerobic

E Pseudomonas aeruginosa ATCC 9027 Thioglycolate 32.5 ± 2.5°C 3 days Aerobic

F Staphylococcus aureus ATCC 6538P Thioglycolate 32.5 ± 2.5°C 3 days Aerobic

Conclusion The bacterial growth tests demon-strate that effective ethylene oxide sterilisation neither promote nor inhibit the growth of micro-organisms.

Type Lot no. A B C D E F Colony count No. of containers with no growth

16466 040025 1 1 1 1 1 1 < 100 cfu 0

16466 040032 1 1 1 1 1 1 < 100 cfu 0

16466 040036 1 1 1 1 1 1 < 100 cfu 0

16467 040019 1 1 1 1 1 1 < 100 cfu 0

16467 040024 1 1 1 1 1 1 < 100 cfu 0

16467 040035 1 1 1 1 1 1 < 100 cfu 0

ResultsNumber of containers with substantial growth

28 |

3.3.4 Degassing Curve

BackgroundThis test is necessary to determine the quarantine time required before ethylene oxide sterilized units are transferred to stock.

MethodThe Sterisart® NF units were sterilized with ethylene oxide as described in 3.3. After sterilization, the Sterisart® NF units were filled with 450 ml of deionized water at 37°C and left to stand for 24 hours. The ethylene oxide residue in the water was determined at regular intervals.

3.3.5 Performance Test after Double Sterilization

MethodThe Sterisart® NF units were gassed twice with ethylene oxide as described in 3.3. After double steril-ization, the systems were visually inspected for cracks or discolor-ation and the microbiological and physical performance of the systems were tested.

After 24 hours After 48 hours

7.03 mg ethylene oxide|product 3.15 mg ethylene oxide|product

ConclusionAfter 3 days, the maximum limit value had not been exceeded.

Results

Type Lot no. Sartochem® Sartochem® Burst pressure Leak BCT integrity test container test (Section 2.1.1) (Section 4.1.2) (Section 4.3.1) (Section 4.8)

16466 020020 20 passed 20 passed 20 passed 10 passed 0 failed 0 failed 0 failed

16467 030004 20 passed 20 passed 20 passed 10 passed 0 failed 0 failed 0 failed

No color changes or damage such as cracks were observed.

ConclusionDouble sterilization with ethylene oxide using a two-time dose does not affect the performance of the Sterisart® NF units.

| 29

ConclusionThe venting filters of the spikes, the containers and the vending needles are integer when the PTFE membrane filter is sealed within the specified parameters.

4. Physical Tests

4.1 Integrity Test of the Membrane Filters

BackgroundThe pressure hold test is carried out to ensure that the membrane seal is secure and not damaged. The membrane is wet out (PTFE with ethanol and Sartochem® with RO water) and the membrane must hold a specified positive pressure. See also chapter gamma|ETO sterilization sterility testing (3.2.3; 3.3.1 and 3.3.2).

4.1.1 Venting Filters: 0.2 µm PTFE Membrane Filter On Top of the Containers, in the Dual-Needle Metal Spikes of 16466, 16469 and 16475 and on the Venting Needles of 16467 and 16468

Sterisart® types: 16466-ACD|-GBD, 16467-ACD|-ACD, 16468-ACD|-GBD, 16469-GBD, 16475-GBD

Remark Generally, venting filters from the same lot are used in particular Sterisart® 16466|16467|16468| 16469 and 16475 lots.

MethodPurple stain solution (crystal-violet) is applied at a pressure of 1.5 bar on the upstream side of the PTFE membrane filter (inlet side of venting filter). The PTFE membrane filter is intact when no colored water drops are observed from the outlet of the venting filter after 1 minute.

Results

Article no. Lot no. Assembled in Sterisart® No. of No. of No. of venting venting venting filters filters tested filters Type Lot. no. tested > 1.5 bar # 1.5 bar

17526-INQ 010294 16466 010015 205 205 0

17526-INQ 010653 16466 020005|8|9 240 240 0

17526-INQ 020253 16467 020012 240 240 0

17526-INQ 020253 16466 020014|15 240 240 0

17526-INQ 020581 16467 020017 240 240 0

17526-INQ 020491 16466 020016 130 130 0

17526-INQ 020825 16466 020020 140 140 0

17526-INQ 020929 16466 020021 240 240 0

17526-INQ 031013 16468 040006 335 335 0

17526-INQ 040016 16468 040014 335 335 0

17526-INQ 040056 16475 040021 340 340 0

17526-INQ 040135 16469 040037 340 340 0

17526-INQ 040174 16475 040042 330 330 0

30 |

4.1.2 Container: 0.45 µm Sartochem® Membrane Filters (In -> Out)


MethodA positive pressure of 2.5 bar was applied to the water-wet Sartochem® membrane filter from the top of the Sterisart® NF container. The venting filter of the container was closed. The Sartochem® membrane filter is intact when no air bubbles rise from the outlet of the container after 1 min.

Results

Type Lot. no. No. of containers No. of containers No. of containers tested > 2.5 bar # 2.5 bar

16466 010015 20 20 0

16466 020005 20 20 0

16466 020014 20 20 0

16466 020015 20 20 0

16466 020016 20 20 0

16466 020020 20 20 0

16466 020021 20 20 0

16467 020012 20 20 0

16467 020017 20 20 0

16468 040006 20 20 0

16469 040037 40 40 0

16469 040038 40 40 0

16475 040021 40 40 0

16475 040042 40 40 0

ConclusionThe container is intact when the Sartochem® membrane filter is clamped at parameters within the specifications.

| 31

4.1.3 0.2 µm PTFE Membrane Filter (Venting Filter on the Container)

Sterisart® types: 16466-GBD, 16467-ACD, 16468-ACD|-GBD, 16469-GBD, 16475-GBD

MethodThe PTFE membrane was wet with isopropyl alcohol and positive pressure was applied to the membrane from the inlet of the venting filter housing. The venting filter was placed in a vessel containing water and the bubble point was taken at the pressure at which a continuous stream of bubbles emerged from the outlet.

ConclusionIf the bubble point is higher than 0.9 bar, the membrane retains > 1 ƒ 107 bacteria/cm2 filter area.

Results

Article no. Lot no. Assembled in Sterisart® No. of venting No. of BP No. of BP Sterile filtration filters tested $ 0.9 bar < 0.9 bar result Type Lot. no. sterile|unsterile

17526-INQ 010294 16466 010015 10 10: 1.3–1.5 0 50|0

17526-INQ 020253 16467 020012 10 10: 1.3–1.5 0 50|0

17526-INQ 020253 16466 020014|15 10 10: 1.3–1.5 0 50|0

17526-INQ 020581 16467 020017 10 10: 1.3–1.5 0 50|0

17526-INQ 020825 16466 020020 10 10: 1.3–1.5 0 50|0

17526-INQ 030429 16467 030008 10 10: 1.2–1.5 0 50|0

17526-INQ 030429 16466 030009 10 10: 1.2–1.5 0 50|0

17526-INQ 030456 16466 030015|16 10 10: 1.4–1.5 0 50|0

17526-INQ 030621 16467 030017|18 10 10: 1.4–1.5 0 50|0

17526-INQ 031013 16468 040006 10 10: 1.3–1.4 0 50|0

17526-INQ 040016 16468 040014 10 10: 1.3–1.4 0 50|0

17526-INQ 040056 16475 040021 10 10: 1.3–1.4 0 50|0

17526-INQ 040135 16469 040037 10 10: 0.9–1.5 0 50|0

17526-INQ 040174 16469 040042 10 10: 1.2–1.4 0 50|0

32 |

4.2 Water Penetration Test

BackgroundMembrane filters for venting are made of hydrophobic materials such as PTFE to avoid membrane wetting by the humidity of filtered gas. A high water penetration point assures that under high operating pressure liquid will not pass through the membrane. Note: The hydrophobic filters in the spikes are not tested, because they are only exposed to negative pressure and the pressure difference cannot reach the water penetration point.

4.2.1 0.2 µm PTFE Membrane Filter of the Container Venting Filter


MethodDeionized water is forced with slowly rising pressure onto the PTFE membrane in the venting filter. The water penetration point (WPP) is identified as a continuous stream of water drops at the outlet.

ConclusionAt operating pressures lower than 3.0 bar, no liquid enters the pore structure, subsequently wetting the PTFE membrane filter. This ensures pressure release before media filling and air passage during aerobic incubation.

Results

Article no. Lot no. Assembled in Sterisart® No. of No. of No. of venting WPP WPP filters $ 3.0 bar < 3.0 bar Type Lot. no. tested

17526-INQ 010294 16466 010015 10 10: 4.6–4.9 0

17526-INQ 010653 16466 020005|8|9 10 10: 4.5–4.8 0

17526-INQ 020253 16467 020012 10 10: 4.0–4.9 0

17526-INQ 020253 16466 020014|15 10 10: 4.0–4.9 0

17526-INQ 020581 16467 020017 10 10: 4.2–4.6 0

17526-INQ 020491 16466 020016 10 10: 4.5–4.9 0

17526-INQ 020825 16466 020020 10 10: 3.9–4.5 0

17526-INQ 020929 16466 020021 10 10: 3.4–5.8 0

17526-INQ 030429 16466 030009 10 10: 4.0–4.5 0

17526-INQ 030456 16466 030015|16 10 10: 4.5–4.8 0

17526-INQ 030621 16467 030017|18 10 10: 4.6–4.8 0

17526-INQ 031013 16468 040006 10 10: 4.3–4.7 0

17526-INQ 040016 16468 040014 10 10: 4.2–5.2 0

17526-INQ 040056 16475 040021 10 10: 4.1–4.6 0

17526-INQ 040135 16469 040037 10 10: 4.1–4.8 0

17526-INQ 040174 16469 040042 10 10: 4.2–4.5 0

| 33

4.3 Burst Pressure

BackgroundPeristaltic pumps are capable of building up high pressure within a system. A high burst pressure assures that the system maintains its integrity and does not leak during usage.


4.3.1 Burst Pressure of the Container

MethodPositive pressure was applied from the top of the Sterisart® NF container and gradually increased. The venting filter and the outlet of the container were closed. The pressure was increased to the burst pressure of the container, or to a maximum of 5 bar.

Results

Type Lot. no. No. of containers Burst pressure Burst pressure tested $ 5.0 bar < 5.0 bar

16466 010015 20 20 0

16466 020005 20 20 0

16466 020014 18 18 0

16466 020015 19 19 0

16466 020016 20 20 0

16466 020020 20 20 0

16466 020021 19 19 0

16466 030009 20 20 0

16466 030015 19 19 0

16466 030016 20 20 0

16467 020012 20 20 0

16467 020017 20 20 0

16467 030008 20 20 0

16467 030017 20 20 0

16467 030018 20 20 0

16468 040006 20 20 0

16468 040007 20 20 0

16469 040037 19 19 0

16469 040038 20 20 0

16475 040021 20 20 0

16475 040042 20 20 0

34 |

Results

Article no. Lot no. Assembled in Sterisart® No. of venting No. of No. of filters tested burst pressure burst pressure Type Lot. no. $ 6.0 bar < 6.0 bar

17526-INQ 010294 16466 010015 125 125 0

17526-INQ 010653 16466 020005|8|9 240 240 0

17526-INQ 020253 16467 020012 140 140 0

17526-INQ 020253 16466 020014|15 140 140 0

17526-INQ 020581 16467 020017 140 140 0

17526-INQ 020491 16466 020016 110 110 0

17526-INQ 020825 16466 020020 140 140 0

17526-INQ 020929 16466 020021 240 240 0

17526-INQ 030429 16466 030009 140 140 0

17526-INQ 030456 16466 030015|16 140 140 0

17526-INQ 030621 16467 030017|18 190 190 0

17526-INQ 031013 16468 040006 335 335 0

17526-INQ 040016 16468 040014 335 335 0

17526-INQ 040056 16475 040021 340 340 0

17526-INQ 040135 16469 040037 340 340 0

17526-INQ 040174 16469 040042 330 330 0

4.3.2 Burst Pressure of the Container Venting Filter


MethodPositive pressure was applied to the inlet of the venting filter and gradually increased. The outlet of the housing was closed. The pressure was increased to the burst pressure of the venting filter housing, or to a maximum of 6 bar.

ConclusionThe minimum burst pressure is 6 bar. The recommended maximum operation pressure is set at 4 bar to allow for a safety margin.

| 35

4.4 Pressure Hold Test of the Rubber Cap on the Container Venting Filter

BackgroundA peristaltic pump is able to build up high pressure in a system. The pressure to avoid bursting of the Sterisart® NF system should blow off the rubber cap.


MethodThe container was closed at the exit with plugs and the hose was cut near the entrance of the container. The rubber cap was inserted onto the connection of the venting filter. The pressure source was connected to the hose connector of the container and then the pressure was gradually increased by 0.2 bar/5.0 sec. up to the point where the rubber cap blew off.

ConclusionLoose fitted rubber caps will be blown away at a pressure of < 3.0 bar. Tightly fitted rubber caps should blow away between a pressure of 3.3–4.1 bar, preventing the bursting of the containers used.

Results Type 16466

Container venting filter lot no.: 17526-INQ; 020929, assembled in Sterisart® NF lot no.: 020021

Pressure No. of removed rubber caps No. of removed rubber caps [bar] that fitted tightly that fitted loose

2.3–2.4 - -

2.5–2.6 - 5

2.7–2.8 - 3

2.9–3.0 - 2

3.1–3.2 - -

3.3–3.4 3 -

3.5–3.6 4 -

3.7–3.9 2 -

4.0–4.1 1 -

Results Type 16467

Venting filter lot no.: 17526-INQ; 020253, assembled in Sterisart® NF lot no.: 020012

Pressure No. of removed rubber caps No. of removed rubber caps [bar] that fitted tightly that fitted loose

2.3–2.4 - -

2.5–2.6 - 4

2.7–2.8 - 3

2.9–3.0 - 3

3.1–3.2 - -

3.3–3.4 2 -

3.5–3.6 3 -

3.7–3.9 4 -

4.0–4.1 1 -

36 |

4.5 Flow Rates with Liquids

BackgroundSartochem® is an extremely hydro-philic membrane. No chemical procedure is required for conversion (as is required for membranes which are not naturally hydrophilic e.g., PVDF), due to raw material itself being hydrophilic. This is an important factor for the flow rate of liquids.

4.5.1 Sartochem® Membrane Filters with RO Water. Sample Taken Out of an Open Vessel.


MethodA peristaltic pump at pump rate settings of 200 (only 16467), 400 and 700 (only 16466) transports RO water through the system. Every minute the filtrate was measured by weight.

Results Type 16466

Type Lot. no. No. of systems Flow rate in ml/min. tested at pump rate setting 400 700

16466 020016 5 475.6 856.3

16466 020021 5 478.2 860.8

Results Type 16467


16467 020012 5 232.1 406.3

16467 020017 5 231.2 404.3

RemarkThe inner diameter of the metal needle of 16467 is smaller than that of the adapter 16466, thus limiting the throughput at pump rate settings > 400 rpm. This means that there is no parallel increase of throughput at increasing pump rate settings > 400.

ConclusionThe average flow rate of RO water in the Sterisart® systems is dependent on the pump speed.

| 37

4.5.2 Sartochem® Membrane Filters with RO Water. Sample Taken Out of a Closed Vessel (1 Liter Bottle)

4.5.2.1 Sterisart® types: 16466-ACD, 16467-ACD

MethodTo demonstrate that the venting of the closed bottle by adapter types 16466 and 16467 is sufficient and no negative influence of the throughput performance occurs, a sample from a closed bottle with RO water was tested. In the case of type 16467, an additional venting needle was pierced into the closure of the bottle.

A peristaltic pump at pump rate settings of 200 (only 16467), 400 and 700 (only 16466) transports RO water through the system. Every minute the filtrate was measured by weight.

Results Type 16466


16466 020016 5 473.6 838.2

16466 020021 5 476.2 839.1

Results Type 16467


16467 020012 5 228.4 396.8

16467 020017 5 226.4 392.7

RemarkThe inner diameter of the metal needle of 16467 is smaller than that of the adapter 16466, thus limiting the throughput at pump rate settings > 400 rpm. This means that there is no parallel increase of throughput at increasing pump rate settings > 400.


4.5.2.2 Sterisart® type: 16469-GBD

MethodFirst, the connector of the syringe adapter was closed with a plug. Then the double tubing was placed in the pump head of the Sterisart® universal pump 16413. The dual-needle metal spike was inserted into the septum of a 1-liter bottle that was filled with RO water. The throughput was tested at various settings of the Sterisart® pump. To measure the exact throughput volume, the water pumped was not collected until a constant water level was attained inside the container (constant pressure level in the container, constant throughput of water). The throughput volume of water was measured for 1 minute.

Results Type 16469


16469 040038 3 238.8 424.2

16469 040039 3 226.6 382.4

RemarkSince the inner diameter of the syringe adapter is smaller than that of the dual needle metal spike (type 16466) throughput measure-ments at a pump rate setting 700 were not carried out.


38 |


MethodFirst, the part of the tubing not identified by plastic rings was placed in the pump head of the Sterisart® pump. The dual-needle metal spike (yellow color-coded) was inserted into the septum of a 1-liter bottle, filled with RO water. The second dual-needle metal spike (not color coded) was inserted into the septum of an empty 100-ml bottle. The time needed for filling and draining the 100-ml bottle was measured at various pump rate settings.

Results Type 16470

Type Lot no. No. of Filling time in seconds systems tested at pump rate setting 200.0 400.0 700.0

16470 040033 3 51.3 26.3 16.3

16470 040034 3 51.3 26.3 16.7

Type Lot no. No. of Draining time in seconds systems tested at pump rate setting 200 400 700

16470 040033 3 58 34 24.7

16470 040034 3 59 34 24.7

| 39


MethodFirst, the double tubing is placed in the pump head of the Sterisart® pump. The short dual-needle metal spike (without yellow color-coded venting filter) was inserted into the septum of a 100-ml bottle. The longer dual-needle metal spike (with yellow color-coded venting filter) was inserted into the septum of a 1-liter bottle that was filled with RO water.

First, the time it took to fill the 100-ml bottle was measured.For this purpose, the 1-liter bottle was hung and the 100-ml bottle was positioned upright. To measure the throughput of the total system, the pump was left running so that the RO water was pumped through both Sterisart® containers. To determine the exact throughput volume, the water pumped was not collected until a constant water level in the container was attained (constant pressure inside the container, constant throughput of water). The throughput volume of water was measured for 1 minute. Finally, the time it took to drain the 100-ml bottle was measured. For this purpose, the 1-liter bottle was hung and the 100-ml bottle positioned upright.

All three values were measured at various pump rate settings.

Results Type 16475

Type Lot no. No. of Filling time in seconds systems tested at pump rate setting 200.0 400.0 700.0

16475 040021 3 49.3 37.7 33.3

16475 040041 3 48 38.3 33.7

Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting 200 400 700

16475 040021 3 200.7 229.7 237.2

16475 040041 3 192.1 221.6 239.7

Type Lot no. No. of systems Draining time in seconds tested at pump rate setting 200 400 700

16475 040021 3 26.7 14.3 9.3

16475 040041 3 27.7 14.3 10.3

ConclusionWith increasing pump rate settings the filling and draining times of the 100-ml bottle are decreased. The throughput volume increases correspondingly. The filling times are longer than the time to drain because a vacuum has to be greated before in the 100-ml bottle. During the draining process the liquid is pumped directly.

40 |

4.5.2.5 Sample Taking out of Collapsible Bags

Sterisart® type: 16468-ACD|-GBD

Method First, the double tubing was placed in the pump head of the Sterisart® universal pump 16413. Then the yellow plug was removed from the detachable double female luer lock connector. The double female luer lock connector of the tubing system was attached to the connector of the collapsible infusion bag that was filled with 1 liter of water. The throughput at various pump rate settings was measured. To measure the exact throughput volume, the water pumped was not collected until a contant water level was attained inside the container (constant pressure level in the container, constant throughput of water). The throughput volume of the water filtered was measured for 1 minute.

Results Type 16468

Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting 200 400 700

16468 040014 3 230.8 457.5 868.7


| 41

4.5.3 Sartochem® Membrane Filters with Glucose Solution 20%


MethodAccording to chapter 4.5.1 sampling from open vessels.The maximum pump speed was selected in such a way that a tight fitting of the rubber caps was still guaranteed (approx. 3.0 bar).

Results Type 16466

Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting 200 400

16466 020016 5 228.1 460.5

16466 020021 5 229.7 462.4

Results Type 16467


16467 020012 5 226.9 379.0

16467 020017 5 226.1 377.6

ConclusionViscous solutions, such as glucose solution (20%) can be filtered up to a pump speed (pump rate setting) 400. The throughput performance of both adapter types is nearly the same.

4.5.4 Sartochem® Membrane Filters with Glucose Solution 40%

Sterisart® types:16466-ACD, 16466-ACD

MethodAccording to chapter 4.5.1 sample taking from open vessels.The maximum pump speed was selected in such a way that a tight fitting of the rubber caps was still guaranteed (approx. 3.0 bar).

Results Type 16466


16466 020016 5 234.7 367.4

16466 020021 5 237.3 372.8

Results Type 16467


16467 020012 5 241.1 350.6

16467 020017 5 246.2 352.8

Conclusion40% glucose solution can be filtered up to a pump setting of 300; the flow rate is virtually the same for both types.

42 |

4.6 Flow Rates for Air 4.6.1 0.2 µm PTFE Membrane Filter

of the Dual Needle Metal Spike Venting Filter Unit

Background

Sterisart® type:16466-ACD

MethodThe dual needle metal spike was adapted on the measuring device. The air throughput was measured applying 0.1 bar to the venting filter (PTFE membrane) in the spike.

Results

Article no. Lot no. Built in Sterisart® No. of venting Average flow rate lot no. filters tested in l/min

17526-INQ 010294 010015 10 0.859

17526-INQ 010653 020005 10 1.020

17526-INQ 020253 020014 10 0.933

17526-INQ 020491 020016 10 0.958

17526-INQ 020825 020020 10 1.049

17526-INQ 020929 020021 10 0.954

ConclusionSince the venting needle supplied with type 16467 has a greater inner diameter than the venting filter in the dual needle metal spike (16466), the air resistance is lower and consequently the air flow rate is higher than given in 4.6.1.

4.6.2 0.2 µm PTFE Membrane Filter of the Venting Needle Supplied with Sterisart® types 16467 and 16468

Background

Sterisart® types: 16467-ACD, 16468-ACD|-GBD

MethodThe venting needle of the types 16467 and 16468 were adapted on the measuring device. The air throughput was measured applying 0.1 bar to the venting filter (PTFE membrane).

Results

Article no. Lot no. Assembled in Lot. no. No. of venting Average flow Sterisart® type filters tested rate in l/min

17526-INQ 010653 16467 010009 10 1.339

17526-INQ 020253 16467 020012 10 1.271

17526-INQ 020581 16467 020017 10 1.422

17526-INQ 031013 16468 040006 10 1.712

17526-INQ 040016 16468 040014 10 1.733

4.6.3 0.2 µm PTFE Membrane Filter of the Venting Needle Spike Supplied with Sterisart® types 16469 and 16475

MethodThe needles of the Sterisart® types 16469 and 16475 were adapted on the measuring device. The air throughput was measured applying 0.1 bar to the venting filter (PTFE membrane).

Results

Article no. Lot no. Assembled in Lot. no. No. of venting Average flow Sterisart® type filters tested rate in l/min

17526-INQ 040135 16469 040037 10 1.667

17526-INQ 040056 16475 040021 10 1.707

17526-INQ 040174 16475 040042 10 1.724

| 43

Results

Article no. Lot no. No. of venting No. of flow rate No. of flow rate filters tested $ 1.1 ml/min < 1.1 ml/min

17526-INQ 010294 10 10: 1.21–1.55 0

17526-INQ 010653 10 10: 1.33–1.83 0

17526-INQ 020253 10 10: 1.10–1.80 0

17526-INQ 020581 10 10: 1.40–1.70 0

17526-INQ 020491 10 10: 1.41–1.72 0

17526-INQ 020825 10 10: 1.33–1.94 0

17526-INQ 020929 10 10: 1.27–1.75 0

17526-INQ 031013 10 10: 1.64–1.88 0

17526-INQ 040016 10 10: 1.50–1.91 0

17526-INQ 040056 10 10: 1.67–1.88 0

17526-INQ 040135 10 10: 1.55–1.96 0

17526-INQ 040174 10 10: 1.70–1.94 0

4.6.4 0.2 µm PTFE Membrane Filter in the Container Venting Filter

BackgroundThe operator removes the rubber caps from the Sterisart® NF containers before filling with nutrient media. The positive pressure inside the system is released through the venting filter. During incubation, gas exchange also takes place through the venting filter, therefore a high airflow rate for the venting filter is necessary.

Sterisart® types:16466-ACD, 16467-ACD, 16468-ACD|-GBD, 16469-GBD, 16475-GBD

MethodPositive pressure, 0.1 bar is applied to the PTFE membrane filter from the inlet of the container venting filter. The flow rate is measured by a flow meter.

ConclusionThe airflow rate of the PTFE membrane is sufficiently high enough to facilitate an expeditious release of pressure in the system.

44 |

4.7 Wetting Time Sartochem® Membrane Filters

BackgroundSartochem® is an extremely hydro-philic membrane. No chemical procedure is required for conversion (as is required for membranes which are not naturally hydrophilic e.g., PVDF), due to raw material itself being hydrophilic. The interna-tional pharmacopoeias require the immediate filtration of the sample after wetting the membrane.

MethodA cut disc Sartochem® membrane filter is placed on the surface of RO water. The water is drawn by the sponge effect of the membrane structure inside the pores and the membrane becomes transparent.

Results

Type Lot. no. Membrane No. of membranes No. of membranes No. of membranes lot tested < 1 sec $ 1 sec

16466 020021 0002773 10 10 0

16467 020012 0002773 10 10 0

16468 040014 0002773 10 10 0

16469 040037 0002773 10 10 0

16475 040021 0002773 10 10 0

ConclusionThe hydrophilic Sartochem® membrane filter is wet within 1 second, meaning sample filtration is possible immediately.

| 45

4.8 Leak Test of the System

4.8.1 Tubing Systems of Sterisart® Versions 16466, 16467, 16468, 16469 and 16475 (all with different adapters|needles)

BackgroundPeristaltic pumps are able to build up high pressures inside a system. The leak test guarantees the system remains leak-proof throughout the procedure.


MethodThe tubing system was completely submerged in water and from the side of the dual needle metal spike (16466) or the 6-cm metal needle adapter (16467), positive pressure (1.5 bar) was applied to the tubing system. Air bubbles in the water after 60 seconds identified leakage from the tubing.

ConclusionThe system is leak-proof when the tubing system is assembled according to the internal manufacture and test specifications.

Results

Type Lot. no. No. of systems No. of systems≥ No. of systems tested $ 1.5 bar < 1.5 bar

16466 010015 10 10 0

16466 020005 10 10 0

16466 020014 10 10 0

16466 020015 10 10 0

16466 020016 10 10 0

16466 020020 10 10 0

16466 020021 10 10 0

16466 030009 10 10 0

16466 030015 10 10 0

16466 030016 10 10 0

16467 020009 10 10 0

16467 020012 10 10 0

16467 020017 10 10 0

16467 030008 10 10 0

16467 030017 10 10 0

16467 030018 10 10 0

16468 040006 10 10 0

16468 040007 10 10 0

16469 040037 10 10 0

16469 040038 10 10 0

16475 040021 10 10 0

16475 040042 10 10 0

46 |

4.8.2 Tubing System of Sterisart® Version 16470 (Two Different Dual Needle Metal Spikes)

BackgroundPeristaltic pumps are able to build up high pressures inside a system. The leak test guarantees the system remains leak-proof throughout the procedure.

Sterisart® type: 16470-GBD

MethodThe tubing system was completely submerged in water and from the side of the dual needle metal spike positive pressure (4.0 bar) was applied to the tubing system. Air bubbles in the water after 60 seconds would identify leakage from the tubing.

Results


16470 040033 10 10 0

16470 040034 10 10 0

4.8.3 Sterisart® NF Containers and Connection to the Tubing System


MethodThe container venting filter and the outlet of the container were closed and the Sterisart® NF container and half of the tubing were completely submerged in water. Positive pressure (4.0 bar) was applied from the tubing. Leaking parts were identified by air bubbles in the water after 60 seconds.

Results


16466 010015 20 20 0

16466 020005 20 20 0

16466 020015 20 20 0

16466 020016 20 20 0

16466 020020 20 20 0

16466 020021 20 20 0

16466 030009 20 20 0

16466 030015 20 20 0

16466 030016 20 20 0

16467 020009 20 20 0

16467 020012 20 20 0

16467 020017 20 20 0

16467 030008 20 20 0

16467 030017 20 20 0

16467 030018 20 20 0

16468 040006 20 20 0

16468 040007 20 20 0

16469 040037 20 20 0

16469 040038 20 20 0

16475 040021 20 20 0

16475 040042 20 20 0

ConclusionThe container is leak-proof when the system is assembled according the to internal manufacture and test specifications.

| 47

4.8.4 Container Venting Filter Housing

Sterisart® types: 16466-ACD, 16467-ACD, 16468-ACD|-GBD, 16469-GBD, 16475-GBD

MethodThe container venting filter housing was completely submerged in water with the outlet closed. Positive pressure (4.0 bar) was applied via the inlet of the venting filter and after 30 seconds air bubbles in the water identified leaking parts.

ConclusionThe container venting filter is leak-proof when the venting filter is manufactured according to the internal manufacture and test specifications.

Results

Article no. Lot No. Assembled in Sterisart® No. of venting No. of leak-proof No. of leak-proof Type Lot. no. filters tested $ 4.0 bar < 4.0 bar

17526-INQ 010294 16466 010015 105 105 0

17526-INQ 010653 16466 02005|8 220 220 0

17526-INQ 020253 16466 020014|15 120 120 0

17526-INQ 010491 16466 020016 110 110 0

17526-INQ 010581 16467 020017 120 120 0

17526-INQ 010825 16466 020020 120 120 0

17526-INQ 010929 16467 020021 240 240 0

48 |

4.9 Equal Conveyance of the Liquid into the Two Sterisart® NF Containers

BackgroundOne of the features of the Sterisart® NF units is the division of the liquid into equal volumes during the filtration step. Following filtration, the containers are filled with different nutrient media.


MethodRO water was pushed into the Sterisart® NF units by a Sterisart® pump (Type 16413) at maximum speed – pump rate setting 700 for 16466 and pump rate setting 400 for 16467 (all outlets were closed). After 10 seconds for Type 16466 and 20 seconds for Type 16467, the pump stopped and the double tube was cut off. The filled volume of each container was determined by weight.

Results

Type Lot. no. No. of systems Average volume≥ Average volume tested in container A in container B [g] [g]

16466 030009 10 70.4 71.1

16466 030015 10 70.3 70.1

16466 030016 10 69.7 69.6

16467 020012 10 68.9 68.5

16467 020017 10 69.6 68.5

16467 030008 10 69.2 68.8

16467 030017 10 67.2 67.0

16467 030018 10 66.7 69.5

16468 040006 10 65.0 64.3

16468 040007 10 66.4 65.8

16469 040037 10 129.4 129.2

16469 040038 10 76.7 77.2

16475 040021 10 68.5 69.0

16475 040042 10 72.1 71.8

The volume of containers A and B did not differ by more than 10%.

ConclusionEqual volumes of liquid in the system are distributed into both containers when the Sterisart® NF system is manufactured according to the internal manufacture and test specifi-cations.

| 49

4.10 Accuracy of the Graduation of the Containers

BackgroundThe capacity of one Sterisart® NF container is 120 ml with graduation marks at 50, 75, and 100 ml levels. According to international regula-tions the graduations should have an accuracy greater than 10%.


MethodRO water was filled into a Sterisart® NF container up to the 50 ml graduation mark. The exact volume filled into the container was deter-mined by weight. The exact filled volume was calculated by dividing the weight by the density of the RO water. The procedure was repeated at the 75 ml and 100 ml graduation marks.

ResultsType 16466 lot no. 020021 and 16467 lot no. 020012

Test conditions t = 20°C, p= 1013 mbar, q (water)= 0.998230 g/ml

ConclusionThe accuracy of the graduation marks on the containers is greater than ± 10% when the Sterisart® NF system is manufactured according to the internal manufacture and test specifications.

Test run no. Sterisart® NF lot no. Weight of the Calculated filling volume filling volume [g] [ml]

50 ml 020021 49.90 50.01

50 ml 020012 50.02 50.11

75 ml 020021 75.10 75.23

75 ml 020012 75.02 75.15

100 ml 020021 100.02 100.20

100 ml 020012 99.94 100.12

50 |

4.11 IntegrityofthePackaging

BackgroundThe packaging of the Sterisart® NF units must remain intact until usage, ensuring protection of the devices from any environmental contamination. The sealing of the DuPontTM Tyvek® material must remain tight during storage at high or low humidity, so that the package cannot “open” during its shelf life. The corners and edges of the Sterisart® package are most at risk.

Sterisart®types:16466-ACD|-GBD, 16467-ACD|-GBD, 16468-ACD|-GBD, 16469-GBD, 16475-GBD

4.11.1 IntegrityofthePackageSealing

MethodThe package containing Sterisart® was placed with the DuPontTM Tyvek® material on a water surface. After a contact time of one hour, the package was visually inspected to check if the DuPontTM Tyvek® was still sealed perfectly. The DuPontTM Tyvek® was removed and the remainder of the sealing matrix was inspected for continuity on the PETG plastic.

Results

Type Lot.no. No.ofpackages No.ofpackages No.ofpackages tested passingthetest failingthetest

16466 040032 10 10 0

16466 040036 10 10 0

16466 040043 10 10 0

16467 040024 10 10 0

16467 040030 10 10 0

16467 040035 10 10 0

16468 040006 20 20 0

16468 040007 10 10 0

16469 040037 10 10 0

16469 040038 10 10 0

16475 040021 10 10 0

16475 040042 10 10 0

Results

Type Lot.no. No.ofpackages No.ofpackages No.ofpackages tested passingthetest failingthetest

16470 040033 10 10 0

16470 040034 10 10 0

4.11.2 IntegrityofthePackageSealingofSterisart®Type16470

DuPontTM and Tyvek® are trademarks or registered trademarks of E.I. du Pont de Nemours and Company.

| 51

RemarkSince all system parts of Sterisart® (with the exception of the spikes|-needles) remained unchanged, tests carried out with the forerunner model, Sterisart® 2000, will be referred to here.

These results can be taken over 100% to the newly designed Sterisart® NF types.

Further, more test series, for example regarding antibiotic compatibility etc., are planned with Sterisart® NF in the near future and will be described in separate documents.

5.1 Extractable Substances in the Filtrate According to USP and Ph Eu

BackgroundThe determination of extractable substances establishes the minimum required rinse volume where zero substances should be extracted by the sample when the Sterisart® system is in use (e.g., in cases where the filtrate is used further). The USP 23 and Ph Eu 3 requirements for “Sterile Water for Injection” are used as the test limit. Samples of filtrate are analyzed for non-volatile residues, oxidizable substances, pH and conductivity changes, heavy metal, ammonia, sulfate and chloride levels.

Sterisart® types: 16464-GBD, 16464-ACD, 16463-ACD

Methods50 ml of water for injection was circulated by a Sterisart® pump through the Sterisart® containers for 30 minutes. After 24 hours of contact time in the containers, water was pumped through the system again for 30 minutes.

5.1.1 Determination of pH Value and ConductivityA pH and conductivity meter was used to determine the pH and the conductivity of the extracts.

5.1.2 Non-Volatile Residue200 ml of the filtrate was evapo-rated until dry and then dried further for one hour at 105°C. The residue was weighed.

5.1.3 Determination of Oxidizable Substances10 ml of 2 N sulfuric acid and 0.4 ml 0.1 N permanganate solution was added to 50 ml filtrate and heated for 10 minutes at 100°C. If the solution retained its color, the sample passed the requirements of USP 23 and Ph Eu Suppl. 1.7.99.

5.1.4 Determination of Chlorides1 ml of nitric acid and 0.2 ml of silver nitrate solution was added to 10 ml filtrate. If the solution showed no change in appearance for at least 15 minutes, the sample passed the requirements of USP 23 and Ph Eu Suppl. 1.7.99.

5.1.5 Determination of Sulfates0.1 ml of hydrochloric acid and 0.1 ml of barium chloride solution was added to 10 ml of the filtrate. If the solution showed no change in appearance for at least one hour, the sample passed the requirements of USP 23 and Ph Eu Suppl. 1.7.99.

5.1.6 Determination of Ammonia1 ml of alkaline potassium tetraiodomercurate solution was added to 20 ml of the filtrate. After 5 minutes the solution was examined down the vertical axis of the tube. A standard is prepared by adding 1 ml of alkaline potassium tetraiodomercurate solution to a mixture of 4 ml of ammonium standard solution (1 ppm) and 16 ml of ammonium-free water (0.2 ppm). The test is negative according to USP 23 and Ph Eu Suppl. 1.7.99. when the test solution is not more intensely colored than the standard solution prepared at the same time.

5.1.7 Determination of Heavy Metals200 ml of the filtrate was heated in a glass evaporating dish until the volume was reduced to 20 ml. 12 ml of the concentrated solution complied with the test limit for heavy metals (0.1 ppm). The standard was prepared by using 10 ml of the lead standard solution (1 ppm Pb). The test is negative according to USP 23 and Ph Eu Suppl. 1.7.99 when the test solution is not a darker color than the standard solution prepared at the same time.

5. Analytical Test

52 |

Results

Lot no. Conductivity Non-Volatile Residue Oxidizable Chloride Sulfate Ammonia Heavy [µS/cm] pH [g/200 ml] Substances Metals

Blank 0.654 5.62

990002 1.193 5.62 0.0006 negative negative negative negative negative (= 0.6 mg/200 ml = 0.3 mg/100 ml)

Difference 0.539 0.0

990002 1.215 5.64 0.0002 negative negative negative negative negative (= 0.2 mg/200 ml = 0.1 mg/100 ml)

Difference 0.654 0.02

USP 23 < 2.6 µS 5.6 sample volume ≥ negative negative negative negative negative requirement $ 100 ml: NVR < 0.002% (= 2 mg/100 ml)

Ph Eu >10 ml: sample volume negative negative negative negative negative Suppl. 5 µS >10 ml: 1.7.99 NVR < 0.003% (= 3 mg/100 ml)

ConclusionThe non-volatile residue, the content of oxidizable substances, heavy metals, chloride, sulfate and ammonia, and the change of pH value and conductivity are below the requirements set by the USP 26 limits for “Sterile Water for Injection”.

| 53

5.2 Desorption Test with Antibiotics and Preservatives

BackgroundMicroorganisms can propagate quickly when inhibitor substances are absent. Any residue of preser-vatives or antibiotics, which may be contained in pharmaceutical samples, must be removed via rinsing before adding nutrient media into the system. Therefore, all parts that come in contact with the sample must be thoroughly rinsed to remove all residues. The sealing of the hydrophilic membrane filter is a potential location where in hibitory substances can diffuse under the plastic during the filtration, making removal by rinsing difficult.

The Sartochem® membrane filter is clamped in such a manner that the pore structure in the clamped zone is compressed to a foil structure. Diffusion of the sample into the clamped zone is therefore impossible.

54 |

5.2.1.1 Scanning Electron Micrograph (SEM):Vertical Cut of the Sartochem® Membrane Filter

5.2.1 Design of the Sterisart® Containers

Normal membrane filter pore structure

The pores and the matrix are tightly clamped to a foil (e.g., plastic)

| 55

5.2.1.2 Scanning Electron Micrographs (SEM): Surface of the Sartochem® Membrane Filter

1. Sartochem® Membrane Filter Hydrophilic zone

2. Sartochem® Membrane Filter inner clamped zone

Sartochem® Membrane FilterMembrane with inner andouter clamped zones

Sartochem® Membrane FilterMembrane with inner andouter clamped zones

approx. original size ƒ 195, SEM

ƒ 2000, SEM

ƒ 1405, SEM

2. Inner clamped zone

1. Hydrophilic membrane filter

3. Outer clamped zone

Comparable structure of the surface of polypropylene foil plastic

56 |

5.2.2 Filtration of Red Ink Solution (100%)

BackgroundRed ink solution will stain every part of a matrix red where contact is made.

MethodRed ink solution was filtered through the Sterisart® container at 3.0 bar positive pressure until the filter was blocked. The plastic parts of the container were removed by cutting. Simultaneously, a Sartochem® disc membrane filter was clamped in a re-useable filter holder and red ink solution was filtered through the membrane filter under the same condi-tions. The clamped zones of both Sartochem® membrane filters were inspected visually.

ResultsThe red ink solution only stains the effective filtration area of the Sartochem® membrane filter in the Sterisart® container. The boundary between the free filter area and the clamped zone is clear and distinct, showing no diffusion of red ink into the clamped zone. If the Sartochem® membrane filter is clamped in a re-useable filter holder, red ink can diffuse to some extent into the clamped zone.

5.2.3 Adsorption and Desorption of Antibiotics and Preservatives and Growth Promotion Test

Method: Adsorption and Desorption TestThe Sterisart® units were wetted by sodium chloride solution (0.9 %). Next, 100 ml of an inhibitory substance was filtered through the Sterisart® unit and filtrate fractions were collected. The constituents of the substance in the filtrate fractions were determined in the 1st, 2nd, 10th and 20th ml by Reverse Phase HPLC. Afterwards, the system was flushed with 3 ƒ 100 ml sterile isotonic sodium chloride solution and the substance in the filtrates was determined at the 1st, 101st and 300th ml.

ConclusionThe specialized Sterisart® clamping technique prevents the diffusion of any sample components into the clamped zone.

Sterisart® with Sartochem® Filter holder device with Sartochem®

| 57

Results: Adsorption and Desorption

Unspecific Adsorption of Benzyl alcohol (1.59%)

Filtrate1 ml 2 ml 10 ml 20 ml

0

5

10

15

20

Benzyl alcohol [mg/ml]

0.423

8.581 1.312 0.646

unspecific Adsorption

Benzyl alcoholin the filtrate

dilluted by a residue ofthe previous wetting

The higher the volume of preser-vative solution applied to the membrane, the lower the non-specific adsorption. Saturation of the membrane with preservative is reached after non-competitive, non-specific adsorption.

The low concentration of preser-vative in the first ml of the filtrate may be interpreted as high adsorption in the system, but it results from the residue of the wetting solution in the Sterisart® system.

Unspecific Adsorption of para-Hydroxy Benzoic acid ethyl ester (2.06%)

Filtrate1 ml 2 ml 10 ml 20 ml

0

5

10

15

20

25

PHB [mg/ml]

2.22

2.711 0 0

unspecific Adsorption

PHB-ester the filtrate

dilluted by a residue ofthe previous wetting

58 |

Removal of p-Hydroxy Benzoic acid ethyl ester by Sodium Chloride solution (0.9%)

Filtrate1 ml 101 ml 300 ml

0

5

10

15

PBH alcohol [mg/ml]

11

6.699

non-adsorbed residueof PBH in the system

PBH in the filtrate0.016

Sodium chloride solution rinses inhibitory substances out of the Sterisart® systems.

The high concentration of inhibitors in the first ml of the filtrate may be interpreted as a previous high adsorption in the system, but it results from the residue of the inhibitory solution in the Sterisart® system.

Removal of Benzyl alcohol by Sodium Chloride solution (0.9%)

Filtrate1 ml 101 ml 300 ml

0

5

10

15

Benzyl alcohol [mg/ml]

10.585

0.086

non-adsorbed residueof Benzyl alcohol inthe system

Benzyl alcoholin the filtrate

0.011

| 59

Method Growth Promotion TestFollowing the above rinsing procedure, the containers of the Sterisart® system were filled with fluid thioglycolate medium according to the European Pharma-copoeia. The containers were inoculated with 1-100 Micrococcus luteus (ATCC 9341) microorganisms and were incubated in parallel with the positive control at 32.5 ± 2.5 °C under anaerobic conditions, for a maximum of 3 days. The growth of microorganisms was checked daily to determine whether the growth of the samples was comparable to the positive control.

ConclusionBacterial growth tests have shown that the tested antibiotics and preservatives do not remain in the Sterisart® system, where they could enhance or inhibit the growth of microorganisms.

ResultsGrowth Promotion Test

Lot no. Inhibitory No. of microbes Growth substance M. luteus promotion [cfu]

970001 Benzyl alcohol equal to (1.59% in water|ethanol) 15 positive control

970001 p-Hydroxy-benzoic-acid-ethyl-ester equal to (2.06% in water|ethanol) 15 positive control

60 |

5.3 ThePackagingMaterial‘sImpermeabilitytoGas

BackgroundIn the current international pharmacopoeia, the environment for performing a sterility test is defined as clean rooms class A or an isolator. An isolator has proven to be the economical choice for most laboratories in order to comply with the pharmacopoeia. All equipment that is used in isolators for a sterility test is gassed with disinfectants such as vapor phase hydrogen peroxide (VHP) to prevent any secondary contami-nation. Following disinfection of the equipment’s surface, it must be shown that no H2O2 residues remain in the system to inhibit the growth of microorganisms, causing false-negative results. The most reliable way to ensure that VHP does not remain in the system is to use specialized packaging that VHP cannot penetrate. Sterisart® gamma has been specifically developed with VHP impermeable packaging for use in isolators.

Sterisart®types:Valid for all –GBD versions

MethodFour Sterisart® plastic boxes were filled with RO water and sealed with DuPontTM Tyvek® foil. The boxes were sealed in a gas-imper-meable plastic bag and gamma irradiated by 25 kGy. Nine spore strips (Bacillus stearothermophilus ATCC 12980 1.7 ƒ 106) were distributed in an isolator at the most inaccessible gas points. The four Sterisart® boxes were treated inside the isolator with VHP for 45 minutes (= B. stearo thermophilus 106 lethal doses of 96 g in H2O2 (35%)). After the H2O2 sterilization, Merckoquant No. 1.10011.00001 peroxide test determined the concentration of H2O2 inside the packaging. The spore strips were removed under aseptic conditions and placed in nutrient broth. The test vessels were incubated for a maximum of 7 days at 30 °C. Sterility is given when no microbial growth was observed.

ResultsThe results below show the peroxide concentration inside the plastic box and the wrapping plastic bag.

ConclusionVapor phase H2O2 in lethal doses for 106 Bacillus stearothermophilus does not penetrate the outer plastic bag packaging of Sterisart® gamma.

The blank (not VHP exposed) contains 2 ppm peroxide due to an ultraviolet step after the reverse osmosis treatment during the water treatment.

Boxno. Peroxideconcentration Peroxideconcentration insidetheplasticbox outsidetheplasticbox, butinsidetheplasticbag

1 2 ppm 2 ppm

2 2 ppm 2 ppm

3 2 ppm 2 ppm

4 2 ppm 2 ppm

Blank 2 ppm 2 ppm

Bioindicator Growthafter3days Growthafter4daysno. incubation incubation

1 zero growth zero growth









Positive control A Turbidity observed Turbidity observed

Positive control B Turbidity observed Turbidity observed

Negative control zero growth zero growth

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6.1 Chemical Compatibility

BackgroundThe choice of composition materials for a system is always a compromise between functionality and chemical compatibility. The Sterisart® system has been constructed to have a wide range of chemical resis-tance by utilizing fewer different materials.

Sterisart® types: 16464-GBD, 16464-ACD, 16463-ACD, 16466-ACD|-GBD, 16467-ACD|-GBD, 16468-ACD|-GBD, 16469-GBD, 16470-GBD, 16475-GBD

MethodVarying liquid chemical substances were conveyed through the Sterisart® units by a Sterisart® pump. After 24 hours contact time at 25°C, the chemical substance was rinsed out with water.

The system was checked with respect to:

Visual inspection (cracks or color changes)

Leak test on the tubing system at 1.5 bar (methods as above in section 4.8.1)

Integrity test of the Sartochem® membrane filter at 2.5 bar (methods as above in section 4.1.2)

Leak test of the container at 4.0 bar (methods as above in section 4.8.3)

6. Chemical Tests

Results

Chemical Visual Leak test on Pressure Leak test on substance inspection the tubing system hold test the container

Emulsifier: Isopropyl myristate (IPM) o. k. no leakage o. k. no leakage Polysorbate 80 o. k. no leakage o. k. no leakage

Preservatives o. k. no leakage o. k. no leakage

Antibiotics o. k. no leakage o. k. no leakage

Sesame oil* o. k. no leakage o. k. no leakage

Arachide oil* o. k. no leakage o. k. no leakage

Sodium phenolate 30% o. k. no leakage o. k. no leakage

Glycerol 50% o. k. no leakage o. k. no leakage

Lecitine 0.3%* o. k. not tested o. k. not tested

* = difficult to filter. The pump speed has to be adapted. In the case of viscous oils mixing with IPM helps filtration.

62 |

Additional literature Sartochem® PVC Styrene derivatives statements: membrane tubing spike container system Y-connector housing of vent filter

Alcohols: Ethanol R R R Methanol R R N

Aromatic hydrocarbons: Benzene R N N Toluene R N N

Aliphatic hydrocarbons: Hexane R R R

Halogenated hydrocarbons: Tetrachlormethane R N N

Acids: Salicylic acid R R R Ascorbic acid R R R Hydrochloric acid N R LR

Alkalis: Sodium hydroxide LR R LR Potassium carbonate R R R

Aqueous solutions: 0.1% casein peptone sol. R R R Thioglycollate broth R R R

Ethers R R LR

Esters R N LR

Oil: Silicon oil R R R

Saline: Phosphate buffer R R R Sodium chloride phys. R R R

ConclusionGeneral chemical compatibility is given (literature statements included):

Chemical Sartochem® PVC Styrene derivatives substance membrane tubing spike container system Y-connector housing of vent filter

Weak acids R R LR

Alcohols R R R

Weak alkalis LR R LR

Aqueous solutions R R R

Ethers R R LR

Emulsifiers R R R

Esters R N LR

Aromatic hydrocarbons R N N

Aliphatic hydrocarbons R R R

Halogenated hydrocarbons R N N

Oils R R R

Saline R R R

Legend:R = resistantLR = low resistantN = non resistant

NoteCompatibility is influenced by various factors, such as temper-ature, concentration, contact time etc. If necessary, test the chemical resistance using the solution in your validation of the sterility test procedure.

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7. Visual Inspections

BackgroundSterisart® units are manufactured under clean room conditions. The plastic parts are treated with care so there are no scratches, which might obscure the final obser-vation of turbidity. There should be no structural damage, which could cause false-negative or false-positive results.

Sterisart® types: 16466-ACD|-GBD, 16467-ACD|-GBD, 16468-ACD|-GBD, 16489-GBD, 16470-GBD, 16475-GBD

MethodThe Sterisart® units and their components were inspected visually for:

Injection molded particles > 0.1 mm

Loose particles or fibers > 0.1 mm on the unit or membrane

Molding faults

Residues of the production process

Incomplete parts

Scratches, cracks or other damage to the container

7.1 Container Venting Filter

Results

Article no. Lot no. Assembled in Sterisart® No. of venting Molding Residues Cracks, lot. no. filters tested faults damages

17526-INQ 020253 020014|15 100 0 0 0

17526-INQ 020825 020020 100 0 0 0

17526-INQ 030429 030009|10 100 0 0 0

17526-INQ 030456 030015|16|20 100 0 0 0

17526-INQ 030621 030019 100 0 0 0

17526-INQ 031013 040006 100 0 0 0

17526-INQ 040016 040016 100 0 0 0

17526-INQ 040056 040021 100 0 0 0

17526-INQ 040135 040037 100 0 0 0

17526-INQ 040174 040042 100 0 0 0

64 |

7.2 Sterisart® Devices

ConclusionWhen Sterisart® is manufactured under defined parameters the percentage of rejected devices is less than 0.1%.

Results

Type Lot no. No. of systems Injected Loose Molding Residues Incomplete Scratches Cracks, tested particles particles faults damages

16466 020016 10 < 2 0 0 0 0 0 0

16466 030006 10 < 2 0 0 0 0 0 0

16466 030010 10 < 2 0 0 0 0 0 0

16466 030015 10 < 2 0 0 0 0 0 0

16466 030019 10 < 2 0 0 0 0 0 0

16467 020012 10 < 2 0 0 0 0 0 0

16467 030004 10 < 2 0 0 0 0 0 0

16467 030007 10 < 2 0 0 0 0 0 0

16467 030008 10 < 2 0 0 0 0 0 0

16467 030011 10 < 2 0 0 0 0 0 0

16467 030017 10 < 2 0 0 0 0 0 0

16468 040007 10 < 2 0 0 0 0 0 0

16468 040014 12 < 2 0 0 0 0 0 0

16468 040015 12 < 2 0 0 0 0 0 0

16469 040037 10 < 2 0 0 0 0 0 0

16469 040038 10 < 2 0 0 0 0 0 0

16470 040033 10 < 2 0 0 0 0 0 0

16470 040034 10 < 2 0 0 0 0 0 0

16475 040021 10 < 2 0 0 0 0 0 0

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8. Quality Assurance

Routine testing methods of the Sterisart® NF

8.1 Incoming InspectionEach lot is tested by the following destructive test methods:

8.1.1 Qualification of the Sartochem® Membrane Filter Material

8.1.2 Qualification of the PTFE Membrane Filter

Test Method described in section

Bacteria challenge test with Serratia marcescens internal SOP

Bubble point with deionized water internal SOP

Flow rate with deionized water internal SOP

Wetting time with deionized water internal SOP

Thickness of the membrane internal SOP

Completness of Saponification internal SOP


Bacteria challenge test with Brevundimonas diminuta 2.1.2

Bubble point with isopropyl-alcohol 4.1.3

Flow rate with isopropyl-alcohol internal SOP

Flow rate with air 4.6

8.1.3 Qualification of the Plastic Parts Material


Visual inspection 7

8.1.4 Qualification of the Tubes


Measurements of the dimensions internal SOP

Measurement of the inner diameter internal SOP

Shore hardness internal SOP

66 |

8.2 In-Process Control

8.2.1 Sterisart® NF Container


Visual inspection 0

Integrity test of the Sartochem® membrane filter 4.1.2

Burst pressure of the container 4.3.1

8.2.2 Sterisart® NF Tubing System and Spike


Leak test of the system 4.8

Equal conveyance of liquid internal SOP

Penetration test internal SOP

8.2.3 0.2 µm PTFE Membrane Filter Venting Filter


Bacteria challenge test of the PTFE membrane filter with Brevundimonas diminuta 2.1.2

Integrity test of the PTFE membrane filters 4.1

Bubble point of the PTFE membrane filter 4.1.3

Burst pressure of the container vent housing 4.3.2

Water penetration point of the PTFE membrane filter 4.8

Flow rate with air 4.6

Flow rate with ethanol internal SOP

Leak test of the container vent housing 4.8.4

Visual inspection 7

8.3 Final Control

8.3.1 Sterisart® NF System


Visual inspection 7

Integrity of the package sealing 4.11

Bacteria challenge test of the Sartochem® membrane filter with Serratia marcescens 2.1.1

Sterility test 3.3.1, 3.3.2

Growth promotion test 3.3.3

Integrity test of the Sartochem® membrane filter 4.1.2

Burst pressure of the container 4.3.1

Leak test 4.8

Equal conveyance of liquids 4.9

Firm connection of the needles internal SOP

Sales and Service ContactsFor further contacts, visit www.sartorius-stedim.com

EuropeGermanySartorius Stedim Biotech GmbHAugust-Spindler-Strasse 1137079 Goettingen

Phone +49.551.308.0Fax +49.551.308.3289

www.sartorius-stedim.com

Sartorius Stedim Systems GmbHSchwarzenberger Weg 73–7934212 Melsungen

Phone +49.5661.71.3400Fax +49.5661.71.3702

www.sartorius-stedim.com

FranceSartorius Stedim Biotech S.A.Z.I. des PaludsAvenue de Jouques – BP 105113781 Aubagne Cedex

Phone +33.442.845600Fax +33.442.845619

Sartorius Stedim France4, rue Emile Baudot91127 Palaiseau Cedex

Phone +33.1.6919.2100Fax +33.1.6920.0922

AustriaSartorius Stedim Austria GmbHFranzosengraben 12A-1030 Vienna

Phone +43.1.7965763.18Fax +43.1.796576344

BelgiumSartorius Stedim Belgium N.V.Leuvensesteenweg, 248/B1800 Vilvoorde

Phone +32.2.756.06.80Fax +32.2.756.06.81

DenmarkSartorius Stedim Nordic A/SHoerskaetten 6D, 1.DK-2630 Taastrup

Phone +45.7023.4400Fax +45.4630.4030

ItalySartorius Stedim Italy S.p.A.Via dell’Antella, 76/A50012 Antella-Bagno a Ripoli (FI)

Phone +39.055.63.40.41Fax +39.055.63.40.526

NetherlandsSartorius Stedim Netherlands B.V.Edisonbaan 243439 MN Nieuwegein

Phone +31.30.6025080Fax +31.30.6025099

SpainSartorius Stedim Spain SAC/Isabel Colbrand 10–12, Planta 4, Oficina 121Polígono Industrial de Fuencarral28050 Madrid

Phone +34.91.3586102Fax +34.91.3588804

SwitzerlandSartorius Stedim Switzerland GmbHLerzenstrasse 218953 Dietikon

Phone +41.44.741.05.00Fax +41.44.741.05.09

U.K.Sartorius Stedim UK LimitedLongmead Business ParkBlenheim Road, EpsomSurrey KT19 9 QQ

Phone +44.1372.737159Fax +44.1372.726171

AmericaUSASartorius Stedim North America Inc.131 Heartland Blvd.Edgewood, New York 11717

Toll-Free +1.800.368.7178Fax +1.631.254.4253

Sartorius Stedim SUS Inc.1910 Mark CourtConcord, CA 94520

Phone +1.925.689.6650Toll-Free +1.800.914.6644Fax +1.925.689.6988

Sartorius Stedim Systems Inc. 201 South Ingram Mill RoadSpringfield, MO 65802

Phone +1.417.873.9636Fax +1.417.873.9275

Asia|PacificIndiaSartorius Stedim India Pvt. Ltd.10, 6th Main, 3rd Phase PeenyaKIADB Industrial AreaBangalore – 560 058

Phone +91.80.2839.1963|0461Fax +91.80.2839.8262

JapanSartorius Stedim Japan K.K.KY Building, 8–11 Kita Shinagawa 1-chomeShinagawa-kuTokyo 140-0001

Phone +81.3.3740.5407Fax +81.3.3740.5406

MalaysiaSartorius Stedim Malaysia Sdn. Bhd.Lot L3-E-3B, Enterprise 4Technology Park MalaysiaBukit Jalil57000 Kuala Lumpur

Phone +60.3.8996.0622Fax +60.3.8996.0755

SingaporeSartorius Stedim Singapore Pte. Ltd.10, Science Park Road, The Alpha#02-25, Singapore Science Park 2Singapore 117684

Phone +65.6872.3966Fax +65.6778.2494

AustraliaSartorius Stedim Australia Pty. Ltd.Unit 17/104 Ferntree Gully RoadWaverley Business ParkEast Oakleigh, Victoria 3166

Phone +61.3.9590.8800Fax +61.3.9590.8828

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validation guide sterisart® nf1. introduction 7 1.1 validation of production procedures and test...

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