Validation Guide Sterisart NF

Size: px
Start display at page:

Download "Validation Guide Sterisart NF"

Transcription

1 Validation Guide Sterisart NF

2

3 Validation Guide Sterisart NF Version 1.0 Disposable Filtration Units for Sterility Testing ACD -GBD ACD -GBD ACD -GBD GBD

4

5 Table of Contents 1. Introduction Validation of Production Procedures and Test s The Sterility Test The Purpose of this Validation Guide cgmp Quality Assurance from Sartorius Quality Assurance Prevention of Contamination Complete Traceability Quality Management Systems Requirements to be Fulfilled by a System for Sterility Testing According to Pharmacopoeial s Specific Requirements for Avoidance of False Positives Specific Requirements for Avoidance of False Negatives Microbiological Tests Bacteria Challenge Tests Hydrophilic 0.45 µm Sartochem Membrane Filters Hydrophobic Venting Filter Membranes Venting Filter on the Dual-Needle Metal Spike (0.2 µm PTFE Membrane Filter) By-packed Needle with Venting Filter (0.2 µm PTFE Membrane Filter) Sterilization Validation Presterilization Bioburden Validation of Gamma Irradiation Examination of the Validation Dose mapping Validation Sterility Test with Soybean-Casein Digest Broth (TSB) and Thioglycolate Broth after Irradiation at a Reduced Dose of 5 kgy Growth Promotion Test Performance Test after Irradiation with 25 kgy Performance Test 50 kgy Validation of Ethylene Oxide Gas Sterilization Sterility Test with Bioindicators Sterility Test with Soybean-Casein Digest Broth (TSB) and Thioglycolate Broth Growth Promotion Test Degassing Curve Performance Test after Double Sterilization Physical Tests Integrity Test of the Membrane Filters Venting Filters: 0.2 µm PTFE Membrane Filter On Top of the Containers, in the Dual-Needle Metal Spikes of 16466, and and on the Venting Needles of and Container: 0.45 µm Sartochem Membrane Filters (In -> Out) µm PTFE Membrane Filter (Venting Filter on the Container) Water Penetration Test µm PTFE Membrane Filter of the Container Venting Filter Burst Pressure Burst Pressure of the Container Burst Pressure of the Container Venting Filter Pressure Hold Test of the Rubber Cap on the Container Venting Filter Flow Rates with Liquids Sartochem Membrane Filters with RO Water. Sample Taken Out of an Open Vessel Sartochem Membrane Filters with RO Water. Sample Taken Out of a Closed Vessel (1 Liter Bottle) Sartochem Membrane Filters with Glucose Solution 20% Sartochem Membrane Filters with Glucose Solution 40% Flow Rates for Air µm PTFE Membrane Filter of the Dual Needle Metal Spike Venting Filter Unit µm PTFE Membrane Filter of the Venting Needle Supplied with Sterisart types and µm PTFE Membrane Filter of the Venting Needle Spike Supplied with Sterisart types and µm PTFE Membrane Filter in the Container Venting Filter Wetting Time Sartochem Membrane Filters Leak Test of the System Tubing Systems of Sterisart Versions 16466, 16467, 16468, and (all with different adapters needles) Tubing System of Sterisart Version (Two Different Dual Needle Metal Spikes) Sterisart NF Containers and Connection to the Tubing System Container Venting Filter Housing Equal Conveyance of the Liquid into the Two Sterisart NF Containers Accuracy of the Graduation of the Containers Integrity of the Packaging Integrity of the Package Sealing Integrity of the Package Sealing of Sterisart Type Analytical Test Extractable Substances in the Filtrate According to USP and Ph Eu Determination of ph Value and Conductivity Non-Volatile Residue 51 5

6 5.1.3 Determination of Oxidizable Substances Determination of Chlorides Determination of Sulfates Determination of Ammonia Determination of Heavy Metals Desorption Test with Antibiotics and Preservatives Design of the Sterisart Containers Filtration of Red Ink Solution (100%) Adsorption and Desorption of Antibiotics and Preservatives and Growth Promotion Test 56 Growth Promotion Test The Packaging Material s Impermeability to Gas Chemical Tests Chemical Compatibility Visual Inspections Container Venting Filter Sterisart Devices Quality Assurance Incoming Inspection Qualification of the Sartochem Membrane Filter Material Qualification of the PTFE Membrane Filter Qualification of the Plastic Parts Material Qualification of the Tubes In-Process Control Sterisart NF Container Sterisart NF Tubing System and Spike µm PTFE Membrane Filter Venting Filter Final Control Sterisart NF System 66 6

7 1. Introduction 1.1 Validation of Production Procedures and Test s Pharmaceuticals 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 pharmaceutical manufacturer is obliged to supply proof of their safety and sterility according to Pharmacopoeia 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 Pharmaceutique (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 reproducibly and in the desired quality, in keeping with the established production and quality control procedures. 1.2 The Sterility Test The required method for sterility testing of finished products, for all products which can be filtered or made to be filterable, is the Membrane Filter : Pharmacopoeial articles are to be tested by the Membrane Filtration 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 growthinhibitory substances and subsequently 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 disinfectant taken through unsuitable device packaging during sterilization of the isolator. Since it is an absence presence test for microorganisms, 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 Guide Our 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, implementation 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. 7

8 1.4 cgmp Quality Assurance from Sartorius The 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 batchto-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 Assurance For 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. Documentation 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 conditions, 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 specifications. 1.6 Prevention of Contamination Sterisart NF devices are individually 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 contaminating 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 2 O 2 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 Traceability The 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. 8

9 1.8 Quality Management Systems DIN EN ISO 9001 Certificates Sartorius Stedim Biotech implemented Quality Management Systems to assure consistent high quality of Membrane Filters, Ultra Filters, Filter Cartridges and Disposables. 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 EEC) The complete Quality Systems Certificates are continuously updated and can be downloaded on our website: qm-certificates 9

10 1.9 Requirements to be Fulfilled by a System for Sterility Testing According to Pharmacopoeial s General Requirements User s requirement This necessitates Tests made The product must be sterile, and the sterilizing process must not affect the function a) Validated sterilization process b) No change in the product itself after sterilization Sterilization validation Sterility test (3.2, 3.3) Sterilization validation Performance test (3.2, 3.3) Reliable, significant quality assurance a) A non-destructive, routine test of system integrity must be correlated to the ability to retain microorganisms b) Effective Quality Control procedures must be determined and exercised Bacteria challenge tests and correlation to integrity test data (2.1, 4.1) and water penetration test (4.2) In-process control (8.2), final control (8.3) No deterioration during transport and storage 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 Integrity of the packaging (4.11) Shelf life (4.11) The system must allow the shortest possible test time Quickest possible filtration of sample Flow rates with liquids (4.5) Flow rates for air (4.6) Wetting time (4.7) Accuracy of graduation (4.10) The system must be practical a) Graduations to simplify filling, e.g. of culture media b) Equal splitting of samples c) Chemical compatibility with most test products Accuracy of graduations (4.10) Equal conveyance (4.9) Chemical compatibility (6.1) User environment safety must be given The container must be sufficiently pressure resistant Burst pressure (4.3), Pressure hold test on cap (4.4) 10

11 1.9.1 Specific Requirements for Avoidance of False Positives User s requirement This necessitates Tests made 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) Specific Requirements for Avoidance of False Negatives User s requirement This necessitates Tests made Microorganisms in the test sample must be reliably 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 Bacteria challenge test (2.1) Design of the Sterisart containers (5.2.1) No hindrance to the growth of any microorganisms retained a) No residues from the sterilization process b) No residues from other sterilization 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 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) 11

12 2. Microbiological Tests 2.1 Bacteria Challenge Tests Hydrophilic 0.45 µm Sartochem Membrane Filters Background USP 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 microorganisms 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 and DIN 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 10 7 Serratia marcescens per cm 2 filter area. Compliance with this Bacteria Challenge Test will conclusively demonstrate that microorganisms are certainly and reliably retained. Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD A suspension of $ 10 7 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 Brevundimonas 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 ). 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. 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) The Sartochem membrane filter has a retention of $ 10 7 Serratia marcescens organisms per cm 2 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. 12

13 2.1.2 Hydrophobic Venting Filter Membranes Background In accordance with the HIMA Doc. No. 3, Vol. 4, ASTM and DIN 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 10 7 Brevundimonas diminuta per cm 2 filter area. Meeting the requirements 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 Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD A suspension of $ 10 7 Brevundimonas 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 ). 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. 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) INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ The PTFE membrane venting filter on the top of the container has 100% retention of $ 10 7 Brevundimonas diminuta organisms per cm 2 of effective filtration area. The PTFE membrane venting filter meets the requirements of HIMA Doc. No. 3, Vol. 4, ASTM and DIN part 3. 13

14 2.1.3 Venting Filter on the Dual- Needle Metal Spike (0.2 µm PTFE Membrane Filter) Sterisart type: ACD -GBD, GBD, GBD A suspension of $ 10 7 Brevundimonas 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 ). 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. 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) INQ INQ INQ INQ INQ INQ INQ INQ In Sterisart NF systems sterilized by ethylene oxide gas, the PTFE membrane venting filter on the spike has a retention of $ 10 7 Brevundimonas diminuta organisms per cm 2 of effective filtration area. The PTFE membrane venting filter meets the requirements of HIMA Doc. No. 3, Vol. 4, ASTM and DIN part 3. 14

15 2.1.4 By-packed Needle with Venting Filter (0.2 µm PTFE Membrane Filter) Sterisart types: ACD -GBD, ACD -GBD Background A suspension of $ 10 7 Brevundimonas 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 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 ). 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. 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) INQ INQ INQ INQ INQ INQ INQ In Sterisart NF systems sterilized by ethylene oxide gas, the PTFE membrane venting filter on the venting needle has a retention of 10 7 Brevundimonas diminuta organisms per cm 2 of effective filtration area. The PTFE membrane venting filter meets the requirements of HIMA Doc. No. 3, Vol. 4, ASTM and DIN part 3. 15

16 3. Sterilization Validation Background A 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 microorganisms. 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 and ethylene oxide sterilization in compliance with DIN EN 550 and ISO Presterilization Bioburden Background The European Pharmacopoeia describes how the safety assurance level (SAL) of sterilization processes is calculated. The microbial load of a system is determined before sterilization to ensure that the burden is not too high for the sterilization process to be reliable. Sterisart types: ACD -GBD, ACD -GBD, GBD, GBD, GBD The 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 ) and incubated for 7 days at 30 C. The resulting colonies were counted. Type Lot no. No. of Average Average Average units bioburden bioburden bioburden tested outer surface inner surface total [cfu] [cfu] [cfu] The bioburden of the Sterisart systems proved are low enough to ensure that any standard sterilization procedure using gammairradiation or ethylene oxide gas will reduce the bioburden to zero. 16

17 3.2 Validation of Gamma Irradiation Sterisart types: GBD, GBD, GBD, GBD, GBD, GBD Examination of the Validation The validation of gamma-irradiation follows the VD Max -method in which the average bioburden of 3 lots (total of 12 Sterisart units) is determined. For each Sterisart lot further 26 units are gammairradiated with a minimum-dose of 5 kgy. Afterwards a sterility test with these Sterisart units is performed. Type Lot no. No. of Average Average Average units bioburden bioburden bioburden tested outer surface inner surface total [cfu] [cfu] [cfu] 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 sterile 26 sterile sterile 26 sterile sterile 26 sterile The 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. 17

18 3.2.2 Dose mapping Validation During dose mapping, the dosimeters 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, USA Dosimeter type: Nylon dosimeter FWT Photometer type: Aérial Spectrophotometer Photometer manufacturer: FWT Far West Technology, USA Calibrated with: NPL (National Physical Laboratory, England) Alanin Dosimeter 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 Dosimeter in the geometric middle: Height 15 cm 33.8 kgy 34.6 kgy 35.0 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 Dosimeter in the geometric middle: Height 161 cm 28.0 kgy 28.3 kgy 28.9 kgy Dosimeter in the geometric middle: Height 15 cm 25.5 kgy 26.3 kgy 27.3 kgy When the validation procedure is followed, Sterisart units are gamma-irradiated as required, with a minimum dose of 25 kgy reaching all areas. 18

19 3.2.3 Sterility Test with Soybean-Casein Digest Broth (TSB) and Thioglycolate Broth after Irradiation at a Reduced Dose of 5 kgy To ensure that the system remains sterile even at the minimum dose of 5 kgy, two GBD packages (types ) were irradiated accordingly. The result is transferable to types 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. Type Lot no. No. of Sterility test with Sterility test with systems TSB broth; thioglycolate broth; tested no. of containers no. of containers sterile 26 sterile sterile 26 sterile sterile 26 sterile The 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. 19

20 3.2.4 Growth Promotion Test The 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 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 Soybean-casein digest 22.5 ± 2.5 C 5 days Aerobic D Clostridium sporogenes ATCC 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 No. of containers with substantial growth. Type Lot no. A B C D E F Colony count No. of containers with no growth < 100 cfu < 100 cfu < 100 cfu < 100 cfu < 100 cfu < 100 cfu < 100 cfu 0 The bacterial growth tests show that the sterile materials and gamma-irradiation sterilization do not inhibit the growth of microorganisms. 20

21 3.2.5 Performance Test after Irradiation with 25 kgy Ready-manufactured, unsterilized Sterisart NF units and individually packaged hydrophobic venting filter units (lot no ) were gammairradiated 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 microbiological and physical performance of the systems was tested. The 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 passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed 0 failed passed 20 passed 20 passed 10 passed No tested 0 failed 0 failed 0 failed 0 failed passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed 0 failed passed 20 passed 20 passed 10 passed No tested 0 failed 0 failed 0 failed 0 failed passed 24 passed 24 passed 12 passed 3 passed 0 failed 0 failed 0 failed 0 failed 0 failed passed 40 passed 39 passed 20 passed 3 passed 0 failed 0 failed 0 failed 0 failed 0 failed passed 40 passed 40 passed 20 passed 3 passed 0 failed 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 passed 3 passed 0 failed 0 failed 21

22 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) passed 10 passed: 10 passed: 10 passed: Not irradiated 0 failed bar bar ml/min 0 failed 0 failed 0 failed passed 10 passed: 10 passed: 10 passed: Irradiated with 0 failed bar bar ml/min 25 kgy 0 failed 0 failed 0 failed A 25-kGy dose of gamma-irradiation does not influence the performance of Sterisart NF systems or the PTFE membrane filter in the venting filter. 22

23 3.2.6 Performance Test 50 kgy Ready-manufactured, unsterilized Sterisart NF units were gammairradiated with minimum doses of 50 kgy. After sterilization, the units were visually inspected for cracks or discoloration and the microbiological and physical performance of the systems was tested. The venting filters were irradiated separately. The 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 passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed 0 failed passed 20 passed 20 passed 10 passed 3 passed 0 failed 0 failed 0 failed 0 failed 0 failed passed 20 passed 20 passed 10 passed 3 passed 0 failed 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 passed 3 passed 0 failed 0 failed 23

24 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) passed 10 passed: 10 passed: 10 passed: Not irradiated 0 failed bar bar ml/min 0 failed 0 failed 0 failed passed 10 passed: 10 passed: 10 passed: Irradiated with 0 failed bar bar ml/min 50 kgy 0 failed 0 failed 0 failed Gamma-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. 24

25 3.3 Validation of Ethylene Oxide Gas Sterilization Sterisart types: ACD, ACD A 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 Sterility Test with Bioindicators The Sterisart NF units were prepared with Bacillus subtilis endospore strips with a concentration of 10 6 spores per strip. Some units were gassed with ethylene oxide for a minimum of 2 hours (concentration 820 mg/m 2 ). The remaining units were gassed with ethylene oxide for an average of 4 hours (concentration 820 mg/m 2 ). 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. After the ethylene oxide gas sterilization all bioindicators (Bacillus subtilis spore strips) were sterile. Number and distribution of bioindicators Cycle No. Number of bioindicators used half-cycle half-cycle half-cycle full-cycle half-cycle of bioindicator tests Type Lot Gassing time Result of bioindicator test hours all bioindicators sterile hours all bioindicators sterile hours all bioindicators sterile hours all bioindicators sterile 25

26 3.3.2 Sterility Test with Soybean- Casein Digest Broth (TSB) and Thioglycolate Broth A 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. 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 sterile 10 sterile Sterility Test 2. 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 sterile 10 sterile Sterility Test 3. 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 sterile 10 sterile Ethylene oxide sterilization with a concentration of 820 mg/m 2 ensures that all surfaces of the Sterisart NF system in contact with the sample are sterile. All half-cycles are controlled with bio-indicators. 26

27 3.3.3 Growth Promotion Test The 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/m 2. 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 microorganisms 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 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 Soybean-casein digest 22.5 ± 2.5 C 5 days Aerobic D Clostridium sporogenes ATCC 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 Number of containers with substantial growth Type Lot no. A B C D E F Colony count No. of containers with no growth < 100 cfu < 100 cfu < 100 cfu < 100 cfu < 100 cfu < 100 cfu 0 The bacterial growth tests demonstrate that effective ethylene oxide sterilisation neither promote nor inhibit the growth of microorganisms. 27

28 3.3.4 Degassing Curve Background This test is necessary to determine the quarantine time required before ethylene oxide sterilized units are transferred to stock. The 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. After 24 hours After 48 hours 7.03 mg ethylene oxide product 3.15 mg ethylene oxide product After 3 days, the maximum limit value had not been exceeded Performance Test after Double Sterilization The Sterisart NF units were gassed twice with ethylene oxide as described in 3.3. After double sterilization, the systems were visually inspected for cracks or discoloration and the microbiological and physical performance of the systems were tested. 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) passed 20 passed 20 passed 10 passed 0 failed 0 failed 0 failed passed 20 passed 20 passed 10 passed 0 failed 0 failed 0 failed No color changes or damage such as cracks were observed. Double sterilization with ethylene oxide using a two-time dose does not affect the performance of the Sterisart NF units. 28

29 4. Physical Tests 4.1 Integrity Test of the Membrane Filters Background The 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; and 3.3.2) Venting Filters: 0.2 µm PTFE Membrane Filter On Top of the Containers, in the Dual-Needle Metal Spikes of 16466, and and on the Venting Needles of and Sterisart types: ACD -GBD, ACD -ACD, ACD -GBD, GBD, GBD Remark Generally, venting filters from the same lot are used in particular Sterisart and lots. Purple 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. 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 INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ The venting filters of the spikes, the containers and the vending needles are integer when the PTFE membrane filter is sealed within the specified parameters. 29

30 4.1.2 Container: 0.45 µm Sartochem Membrane Filters (In -> Out) Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD A 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. Type Lot. no. No. of containers No. of containers No. of containers tested > 2.5 bar # 2.5 bar The container is intact when the Sartochem membrane filter is clamped at parameters within the specifications. 30

31 µm PTFE Membrane Filter (Venting Filter on the Container) Sterisart types: GBD, ACD, ACD -GBD, GBD, GBD The 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. 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 INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : If the bubble point is higher than 0.9 bar, the membrane retains > 1 ƒ 10 7 bacteria/cm 2 filter area. 31

32 4.2 Water Penetration Test Background Membrane 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 µm PTFE Membrane Filter of the Container Venting Filter Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD Deionized 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. 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 INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : At 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. 32

33 4.3 Burst Pressure Background Peristaltic 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. Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD Burst Pressure of the Container Positive 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. Type Lot. no. No. of containers Burst pressure Burst pressure tested $ 5.0 bar < 5.0 bar

34 4.3.2 Burst Pressure of the Container Venting Filter Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD Positive 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. 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 INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ INQ The minimum burst pressure is 6 bar. The recommended maximum operation pressure is set at 4 bar to allow for a safety margin. 34

35 4.4 Pressure Hold Test of the Rubber Cap on the Container Venting Filter Background A 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. Sterisart types: ACD, ACD The 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. Type Container venting filter lot no.: INQ; , assembled in Sterisart NF lot no.: Pressure No. of removed rubber caps No. of removed rubber caps [bar] that fitted tightly that fitted loose Type Venting filter lot no.: INQ; , assembled in Sterisart NF lot no.: Pressure No. of removed rubber caps No. of removed rubber caps [bar] that fitted tightly that fitted loose Loose 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 bar, preventing the bursting of the containers used. 35

36 4.5 Flow Rates with Liquids Background Sartochem is an extremely hydrophilic 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 Sartochem Membrane Filters with RO Water. Sample Taken Out of an Open Vessel. Sterisart types: ACD, ACD 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. Type Type Lot. no. No. of systems Flow rate in ml/min. tested at pump rate setting Type Type Lot. no. No. of systems Flow rate in ml/min. tested at pump rate setting Remark The inner diameter of the metal needle of 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. The average flow rate of RO water in the Sterisart systems is dependent on the pump speed. 36

37 4.5.2 Sartochem Membrane Filters with RO Water. Sample Taken Out of a Closed Vessel (1 Liter Bottle) Sterisart types: ACD, ACD To demonstrate that the venting of the closed bottle by adapter types and 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. Type Type Lot. no. No. of systems Flow rate in ml/min. tested at pump rate setting Type Type Lot. no. No. of systems Flow rate in ml/min. tested at pump rate setting Remark The inner diameter of the metal needle of 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. The average flow rate of RO water in the Sterisart systems is dependent on the pump speed Sterisart type: GBD First, 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 The dualneedle 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. Type Type Lot. no. No. of systems Flow rate in ml/min. tested at pump rate setting Remark Since the inner diameter of the syringe adapter is smaller than that of the dual needle metal spike (type 16466) throughput measurements at a pump rate setting 700 were not carried out. The average flow rate of RO water in the Sterisart systems is dependent on the pump speed. 37

38 Sterisart type: GBD First, 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. Type Type Lot no. No. of Filling time in seconds systems tested at pump rate setting Type Lot no. No. of Draining time in seconds systems tested at pump rate setting

39 Sterisart type: GBD First, 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. Type Type Lot no. No. of Filling time in seconds systems tested at pump rate setting Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting Type Lot no. No. of systems Draining time in seconds tested at pump rate setting With 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. 39

40 Sample Taking out of Collapsible Bags Sterisart type: ACD -GBD First, the double tubing was placed in the pump head of the Sterisart universal pump 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. Type Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting The average flow rate of RO water in the Sterisart systems is dependent on the pump speed. 40

41 4.5.3 Sartochem Membrane Filters with Glucose Solution 20% Sterisart types: ACD, ACD According to chapter 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). Type Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting Type Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting Viscous 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 Sartochem Membrane Filters with Glucose Solution 40% Sterisart types: ACD, ACD According to chapter 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). Type Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting Type Type Lot no. No. of systems Flow rate in ml/min tested at pump rate setting % glucose solution can be filtered up to a pump setting of 300; the flow rate is virtually the same for both types. 41

42 4.6 Flow Rates for Air µm PTFE Membrane Filter of the Dual Needle Metal Spike Venting Filter Unit Background Sterisart type: ACD The 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. Article no. Lot no. Built in Sterisart No. of venting Average flow rate lot no. filters tested in l/min INQ INQ INQ INQ INQ INQ µm PTFE Membrane Filter of the Venting Needle Supplied with Sterisart types and Background Sterisart types: ACD, ACD -GBD The venting needle of the types and were adapted on the measuring device. The air throughput was measured applying 0.1 bar to the venting filter (PTFE membrane). Article no. Lot no. Assembled in Lot. no. No. of venting Average flow Sterisart type filters tested rate in l/min INQ INQ INQ INQ INQ Since the venting needle supplied with type 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 µm PTFE Membrane Filter of the Venting Needle Spike Supplied with Sterisart types and The needles of the Sterisart types and were adapted on the measuring device. The air throughput was measured applying 0.1 bar to the venting filter (PTFE membrane). Article no. Lot no. Assembled in Lot. no. No. of venting Average flow Sterisart type filters tested rate in l/min INQ INQ INQ

43 µm PTFE Membrane Filter in the Container Venting Filter Background The 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: ACD, ACD, ACD -GBD, GBD, GBD Positive 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. 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 INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : INQ : The airflow rate of the PTFE membrane is sufficiently high enough to facilitate an expeditious release of pressure in the system. 43

44 4.7 Wetting Time Sartochem Membrane Filters Background Sartochem is an extremely hydrophilic 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 international pharmacopoeias require the immediate filtration of the sample after wetting the membrane. A 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. Type Lot. no. Membrane No. of membranes No. of membranes No. of membranes lot tested < 1 sec $ 1 sec The hydrophilic Sartochem membrane filter is wet within 1 second, meaning sample filtration is possible immediately. 44

45 4.8 Leak Test of the System Tubing Systems of Sterisart Versions 16466, 16467, 16468, and (all with different adapters needles) Background Peristaltic pumps are able to build up high pressures inside a system. The leak test guarantees the system remains leak-proof throughout the procedure. Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD The 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. Type Lot. no. No. of systems No. of systems No. of systems tested $ 1.5 bar < 1.5 bar The system is leak-proof when the tubing system is assembled according to the internal manufacture and test specifications. 45

46 4.8.2 Tubing System of Sterisart Version (Two Different Dual Needle Metal Spikes) Background Peristaltic pumps are able to build up high pressures inside a system. The leak test guarantees the system remains leak-proof throughout the procedure. Type Lot. no. No. of systems No. of systems No. of systems tested $ 4.0 bar < 4.0 bar Sterisart type: GBD The 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 Sterisart NF Containers and Connection to the Tubing System Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD The 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. Type Lot. no. No. of systems No. of systems No. of systems tested $ 4.0 bar < 4.0 bar The container is leak-proof when the system is assembled according the to internal manufacture and test specifications. 46

47 4.8.4 Container Venting Filter Housing Sterisart types: ACD, ACD, ACD -GBD, GBD, GBD The 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. 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 INQ INQ INQ INQ INQ INQ INQ The container venting filter is leak-proof when the venting filter is manufactured according to the internal manufacture and test specifications. 47

48 4.9 Equal Conveyance of the Liquid into the Two Sterisart NF Containers Background One 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. Sterisart types: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD RO water was pushed into the Sterisart NF units by a Sterisart pump (Type 16413) at maximum speed pump rate setting 700 for and pump rate setting 400 for (all outlets were closed). After 10 seconds for Type 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. Type Lot. no. No. of systems Average volume Average volume tested in container A in container B [g] [g] The volume of containers A and B did not differ by more than 10%. Equal 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 specifications. 48

49 4.10 Accuracy of the Graduation of the Containers Background The capacity of one Sterisart NF container is 120 ml with graduation marks at 50, 75, and 100 ml levels. According to international regulations the graduations should have an accuracy greater than 10%. Sterisart types: ACD, ACD RO water was filled into a Sterisart NF container up to the 50 ml graduation mark. The exact volume filled into the container was determined 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. Type lot no and lot no Test conditions t = 20 C, p= 1013 mbar, q (water)= g/ml Test run no. Sterisart NF lot no. Weight of the Calculated filling volume filling volume [g] [ml] 50 ml ml ml ml ml ml The 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. 49

50 4.11 Integrity of the Packaging Background The packaging of the Sterisart NF units must remain intact until usage, ensuring protection of the devices from any environmental contamination. The sealing of the 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: ACD -GBD, ACD -GBD, ACD -GBD, GBD, GBD Integrity of the Package Sealing The package containing Sterisart was placed with the Tyvek material on a water surface. After a contact time of one hour, the package was visually inspected to check if the Tyvek was still sealed perfectly. The Tyvek was removed and the remainder of the sealing matrix was inspected for continuity on the PETG plastic. Type Lot. no. No. of packages No. of packages No. of packages tested passing the test failing the test Integrity of the Package Sealing of Sterisart Type Type Lot. no. No. of packages No. of packages No. of packages tested passing the test failing the test

51 5. Analytical Test Remark Since 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 Background The 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 nonvolatile residues, oxidizable substances, ph and conductivity changes, heavy metal, ammonia, sulfate and chloride levels. Sterisart types: GBD, ACD, ACD s 50 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 Determination of ph Value and Conductivity A ph and conductivity meter was used to determine the ph and the conductivity of the extracts Non-Volatile Residue 200 ml of the filtrate was evaporated until dry and then dried further for one hour at 105 C. The residue was weighed Determination of Oxidizable Substances 10 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 Determination of Chlorides 1 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 Determination of Sulfates 0.1 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 Determination of Ammonia 1 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 when the test solution is not more intensely colored than the standard solution prepared at the same time Determination of Heavy Metals 200 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 when the test solution is not a darker color than the standard solution prepared at the same time. 51

52 Lot no. Conductivity Non-Volatile Residue Oxidizable Chloride Sulfate Ammonia Heavy [µs/cm] ph [g/200 ml] Substances Metals Blank negative negative negative negative negative (= 0.6 mg/200 ml = 0.3 mg/100 ml) Difference negative negative negative negative negative (= 0.2 mg/200 ml = 0.1 mg/100 ml) Difference 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: NVR < 0.003% (= 3 mg/100 ml) The 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. 52

53 5.2 Desorption Test with Antibiotics and Preservatives Background Microorganisms can propagate quickly when inhibitor substances are absent. Any residue of preservatives 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 inhibitory 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. 53

54 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) Scanning Electron Micrograph (SEM): Vertical Cut of the Sartochem Membrane Filter 54

55 Scanning Electron Micrographs (SEM): Surface of the Sartochem Membrane Filter 1. Sartochem Membrane Filter Hydrophilic zone Sartochem Membrane Filter Membrane with inner and outer clamped zones Sartochem Membrane Filter Membrane with inner and outer clamped zones ƒ 2000, SEM 3. Outer clamped zone 2. Inner clamped zone 1. Hydrophilic membrane filter approx. original size ƒ 195, SEM 2. Sartochem Membrane Filter inner clamped zone ƒ 1405, SEM Comparable structure of the surface of polypropylene foil plastic 55

56 5.2.2 Filtration of Red Ink Solution (100%) Background Red ink solution will stain every part of a matrix red where contact is made. Red 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 conditions. The clamped zones of both Sartochem membrane filters were inspected visually Adsorption and Desorption of Antibiotics and Preservatives and Growth Promotion Test : Adsorption and Desorption Test The 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 1 st, 2 nd, 10 th and 20 th 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 1 st, 101 st and 300 th ml. The 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. Sterisart with Sartochem Filter holder device with Sartochem The specialized Sterisart clamping technique prevents the diffusion of any sample components into the clamped zone. 56