Tài liệu Procedures & guidelines_guideline for microbiological evaluation of commercially sterile products

Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products FSQ-588002-0104 Table of contents 1. Preface............................................................................................... 1 1.1. Evaluation of customer requirements...........................................................1 1.2. Verification of commercial sterility ...............................................................1 1.3. Low acid vs. High acid products ...................................................................1 1.4. Procedural overview.......................................................................................2 2. Pre-trial preparations........................................................................ 3 2.1. Verification of plant pre-requisites................................................................3 2.2. Verification of Package Integrity ...................................................................3 3. Filling Test......................................................................................... 3 4. Sterility Test for low acid products (pH >4,6) ................................. 3 4.1. Test Protocol ...................................................................................................4 4.2. Test product and raw materials .....................................................................4 4.3. Trial Procedure................................................................................................4 4.3.1. 4.4. 4.4.1. 4.4.2. 4.4.3. 4.4.4. 4.5. 4.5.1. 4.5.2. 4.5.3. 4.5.4. 4.5.5. 4.5.6. 4.5.7. Suggested test routine:........................................................................................................................... 5 Sampling procedure .......................................................................................5 Sample size............................................................................................................................................ 5 Defect level to be verified ..................................................................................................................... 5 Choice of sampling plans....................................................................................................................... 6 Incubation.............................................................................................................................................. 6 Procedure for Laboratory Control .................................................................7 End product test procedures .................................................................................................................. 7 Evaluation methods vs. statistical result ................................................................................................ 7 Early detection of severe failure ............................................................................................................ 8 Detection of Thermophilic sporeformers (optional) .............................................................................. 8 If pH-Measurement procedure is used for evaluation............................................................................ 8 Interpretation of Results ........................................................................................................................ 9 What should be done if the defect limit is exceeded?............................................................................ 9 4.6. Flow Chart for Microbiological Test Procedure (low acid pH >4,6) ..........10 4.7. Report / certificate / agreement ...................................................................11 5. Sterility Test for high acid products (pH <4,6) .............................. 12 5.1. Test protocol .................................................................................................12 5.2. Test product and raw material .....................................................................12 5.3. Trial procedure..............................................................................................12 5.4. Sampling procedure .....................................................................................12 5.5. Procedure for Laboratory Control ...............................................................13 5.5.1. 5.5.2. 5.5.3. 5.5.4. Incubation............................................................................................................................................ 13 End product test procedures ................................................................................................................ 13 Interpretation of results........................................................................................................................ 14 What should be done if the defect limit is exceeded?.......................................................................... 14 FSQ – 588002-0104 i Table of contents 5.6. Flow Chart for Microbiological Test Procedure (high acid pH <4,6) ........14 5.7. Report / certificate / agreement ...................................................................15 6. Commercial Sterility Level Verification (Optional) ....................... 16 6.1. Sterility Verification ......................................................................................16 6.2. Refinement of Production Routines ............................................................17 6.3. Efficiency Verification Period ......................................................................17 Appendix 1: Statistical Evaluation ........................................................ 18 Appendix 2: Sterile Test Protocols........................................................ 19 FSQ – 588002-0104 ii Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 1. Preface This document is intended to support the future commissioning of aseptic equipment delivered by a company in the Tetra Pak Group of Companies to a company, which is the recipient of the delivered equipment. The scope of the document is concerned with the verification of commercial sterility of the packaged product. Commercial and financial matters are not part of the scope of this document and should be covered in a separate contract. The result of a verification of commercial sterility of the packaged product does not necessarily reflect the long-term performance of the line, which is the cumulative result of many variable factors. The document is supplied as a guideline only, not as a guarantee in itself. It establishes a common ground on which the parties named above can use as a starting point in the individual case. It presumes that all relevant equipment operating manuals, recommended maintenance schedules, as well as generally accepted good manufacturing practices etc are followed. 1.1. Evaluation of customer requirements The general guidelines contained in this document must be supplemented with additional specifications for each individual installation. It is the responsibility of the local Tetra Pak Market Company to fully evaluate the customer requirements, including appropriate legal requirements, for supply and performance of equipment and services. This will form the basis for determining the scope of supply. The following items have to be specified by the project partners during the project realisation phase: § § Sterility Test: Number of samples, sterility level for line release for commercial production (normally 1 : 1’000). Minimal requirement for laboratory equipment and staff to perform above tests. 1.2. Verification of commercial sterility This guideline is designed to verify the commercial sterility of the complete production line, based upon the evaluation of the end product. It is intended to demonstrate readiness for commencement of commercial production. The procedure outlined in this guideline is intended for application in the following situations: • • • • The construction of a completely new plant The installation of a new line in an existing plant The installation of a new module in an existing plant where the aseptic integrity of the existing product transfer is compromised in order to make the installation. The introduction of new products. 1.3. Low acid vs. High acid products This guideline distinguishes between procedures for low-acid products (pH >4,6), described in chapter 4 and high acid products (pH <4,6), which are handled in chapter 5. FSQ – 588002-0104 1 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 1.4. Procedural overview Note: Commissioning is the demonstration that the equipment, production line or plant performs as specified and mutually agreed upon by the customer and Tetra Pak. Commissioning should be carried out after equipment installation and prior to the verification of commercial sterility. The following flow chart serves as a guideline for the project after equipment installation: Commissioning/Pre-trial preparations • Finalisation of software program of the controller • Calibration of instruments and control loops • Mechanical test of the complete line with water including production, CIP and SIP (Sterilisation in place) runs. • Training of operators, maintenance and QA/QC staff • Verification of package integrity, raw material quality, utilities, thermal process and environmental hygiene Low acid products ~15 days High acid products: ~20-28 days depending on the test method. Time estimate: Filling Test With skim milk or product Sterility Test 1 With skim milk or product Sterility Test 2 With skim milk or product Sterility Test 3 With skim milk or product Incubation Period (ending with Evaluation of Commercial Sterility) According to appendix 1 Line Release of Routine Commercial Production Based on sterility requirements agreed between the project partners Optional (according to local Market company/customer contract) Sterility Verification Period Production period with increase sampling (5 – 20 days). Refinement of Production Routines Completion of Training for operators, maintenance and QA/QC staff. Efficiency Verification Period Measuring of the target line efficiency over a period of 1 to 3 weeks (acc. To PM) Final Line Approval Final hand-over of Line to the customer, after commencement of routine commercial production FSQ – 588002-0104 2 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 2. Pre-trial preparations 2.1. Verification of plant pre-requisites Verification of the listed plant pre-requisites and quality of packed product is recommended when: • A completely new plant is built • New equipment is installed in an existing plant • Key components are changed • Programme changes are made • It is legally required The following pre-requisites should be verified according to the applicable Good Manufacturing Practice for liquid food production/manufacturers specifications: • raw material quality • thermal process • CIP critical control points • utilities • environmental hygiene • Training level of operator, maintenance and QA/QC staff 2.2. Verification of Package Integrity Package integrity should be verified during commissioning/water pre-trials and during the following Filling Test. It is recommended to simulate a production run, including the whole production line, on water. A minimum of 30 minutes uninterrupted production of finished, good quality packages should be obtained. i.e. short stops and mechanical faults should not occur in any part of the production line. If problems occur during the water test, appropriate corrective action must be carried out before a new test is performed. It is unacceptable to start sterile tests without prior corrective action. The procedure for testing package integrity supplied by the relevant Tetra Pak Business Unit should be followed. 3. Filling Test The installation should be run on water without faults and whenever possible the plant should have run on product, followed by CIP, before sterility tests start. The purpose of the filling test is 3 fold: • Finalisation of software program of the controller • Calibration of instruments and control loops • Mechanical test of the complete line with product 4. Sterility Test for low acid products (pH >4,6) Sterility tests are necessary for new or considerably modified UHT plants or heat exchanger, respectively, components of the aseptic transfer (including the aseptic tank and valve cluster) or fillers in order to validate the sterile performance of the entire line. FSQ – 588002-0104 3 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products A sterility test is only useful, if the installation is completed from both, a mechanical and software point of view and any problems rectified during the prior filling tests outlined above. During the test run it is recommended to build in events, which could occur during routine production runs. These events could include short stops, splicing of packaging material and strip or other events appropriate for the respective Tetra Pak machine system. Packages or carton trays need to be numbered and the events have to be documented with the respective package or carton number to control such events. 4.1. Test Protocol Test protocols shall be well documented for record purposes. The following areas should be covered. • • • • • Raw material quality, e.g. raw milk or juice Processing Filling Package Integrity Microbiological evaluation of finished test product 4.2. Test product and raw materials For low-acid products (> pH 4,6) it is preferred to use plain white milk, e.g. skim milk. If possible, use the product intended for commercial production to get the most representative test results. The packaging material used should be suitable for the product application, e.g. TBA/m material when filling milk. The material intended for commercial production should be used (not test material delivered with the machine). Longitudinal strip used should be suitable for the product application and packaging material used. Hydrogen Peroxide according to Tetra Pak specification for the specific filling machine type should be used. 4.3. Trial Procedure It is recommended that 3 separate test runs be carried out. This will ensure that average conditions during normal production are simulated. These conditions should include stops, CIP and intermediate storage of product in an aseptic tank (if applicable). The total number of packages taken for evaluation should correspond to the recommended number in the sampling plan used (see ‘Choice of sampling plans’ page 6). According to statistical protocol, the results from all 3 tests must be accumulated and evaluated as a single test production. FSQ – 588002-0104 4 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 4.3.1. Suggested test routine: Day 1 One test run, including UHT processing and aseptic filling Finish with a full CIP Day 2 – Two test runs including UHT processing and aseptic filling (separated by intermediate cleaning of Process and Filling to verify the performance of this function) Note: If an aseptic tank is part of the installation to be verified, it is recommended that the product from test 3 be run into the aseptic tank and stored at ambient temperature and filled on day 3. 4.4. Sampling procedure 4.4.1. Sample size It is the customer’s responsibility to define the acceptable quality level to be tested. The number of packages to be tested depends upon the desired quality level and will be based on Poisson distribution. The results of the samples taken in each run should be added together for purposes of statistical evaluation. 4.4.2. Defect level to be verified A sampling guideline based upon a commonly used defect level of 1/1000 is given below as one example. A confidence level of 95% is used according to common world standard. It requires a minimum sample size of 3000 packages to test a defect rate of 1/1000. If a more stringent defect level is required, the table below is supplied as a further sampling guide: Defect rate percentage Defect rate to be tested Minimum sample size with 95% confidence 1,0 0,1 0,01 0,001 0,0001 1:100 1:1000 1:10 000 1:100 000 1:1 000 000 300 3000 30 000 300 000 3 000 000 FSQ – 588002-0104 5 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 4.4.3. Choice of sampling plans The table below will assist in the choice of sample size and the evaluation of the test runs. The sample size, in the column second from the left, represents the total number of samples in 3 separate test runs. The maximum number of defects allowed (third column from left) corresponds to a defect rate limit of 1:1000 (0,1%) with a confidence level of 95%. It is recommended to choose sampling plan 4 below (7800). According to the table, this plan will allow a maximum of 3 defects in 7720 packages still fulfilling the requirement of a failure less than 1:1000. • Plan # Sample size 1 2 3 4 5 3000 4730 6270 7720 9151 Maximum Detection defects allowed probability with a defect rate of 1/1000 0 1 2 3 4 95% 95% 95% 95% 95% Besides the determination of defect rates legal requirements must be fulfilled including pathogens (e.g. EU, PMO or National legislation directives). Note: If the total allowed number of defects occurs in one run or are distributed over the 3 runs, the result is still considered to be acceptable. 4.4.4. Incubation Low-acid (pH >4,6) Incubate all packages from each run (packs/trays to be coded) for 7 days at 30°C, according to the flow diagram on page 10. Follow the recommended temperatures. After a period of 3 days incubation the first streak and pH measurement and/or ATP measurement could be done from approximately 300 additional samples, to act as a screening check for severe failure. The result of this screening test cannot be added to the final test result for statistical purposes. These samples have to be taken as additional samples and should not be taken from the sample plan 4 (7720 packages) above mentioned. Note: If the filling temperature is below incubation temperature care must be taken to ensure that the stacking pattern adopted allows for all packages to increase in temperature rapidly and evenly. FSQ – 588002-0104 6 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 4.5. Procedure for Laboratory Control 4.5.1. End product test procedures Verification of commercial sterility i.e. testing of end product quality shall be carried out on every test package incubated at 30°C for 7 days. Example: Sample plan 4 - total number of packs to be tested is 7720. Maximum allowed defects 3 (see page 6). After incubation, a minimum of 2574 packs from each test run are tested according to the following procedure: The recommended procedure method is by streak plating* the contents of each aseptically sampled package, using a calibrated 10µl loop (maximum 4 streaks per plate) on PC-Agar plates. The plates shall be read after 72 hours incubation at 30°C. It is recommended that a suitable procedure for media control (agar sterility) be done parallel to the sterility testing. The method of package opening should be such that the seals of the packages are left intact, (if packs are required for future integrity testing). Care must be taken to sterilise the external surfaces of the packages sampled for microbiological purposes with alcohol and/or flaming prior to aseptic opening with sterilised scissors or blade. *Streak plating. Refers to the technique of applying liquid product to the surface of an agar plate with a 10µl calibrated loop. The method of application is a straight-line inoculation, beginning approx. 5 mm from the edge of the plate and ending about 5 mm from the opposite edge of the plate. Note: Although packs from each run are incubated and tested separately, the results will be accumulated for statistical purposes only if they are tested in the same way. 4.5.2. Evaluation methods vs. statistical result According to statistical rules and logic, only one evaluation method (e.g. streak plating) should be used. Mixing different methods (pH, sensory, streak plate, ATP etc) introduces the risk of evaluating different contaminating sources/ types of bacteria. The same rule applies for the use of different incubation times and temperatures for the test. If a reliable statistical result is to be achieved, all samples should be incubated for the same time / temperature and evaluated by one method only. (Streak plating is the most sensitive method of detecting bacteria) FSQ – 588002-0104 7 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 4.5.3. Early detection of severe failure Additional samples could be taken during the tests for early detection of severe failure. 100 packages at each test run (~300 packs in total). All packs are incubated at 30°C for 3 days. As a part of early detection of severe failure, daily control of blown samples should be included (check all packages incubated). 4.5.4. Detection of Thermophilic sporeformers (optional) Additional samples could be taken during the tests to detect Thermophilic sporeformers. 100 packages at each test run (~300 packs in total). This is particularly important to verify a new processing profile. All packs are incubated at 55°C for 5 days. The product shall be streaked, in the way described previously, on PCA (if national legislation does not require any different) and incubated at 55°C for 5 days, after which they are read. An alternative recommendation would be to screen the raw material for the presence of these organisms prior to commencement of the test. If pH-Measurement procedure is used for evaluation This method is less sensitive than streak plating. Some bacteria might not be detected by this method. For each batch of samples the average pH-value (= arithmetic mean of 10 randomly taken samples) has to be determined prior to measurements. If a pH-deviation of > 0.1 from the average value is observed in a sample, proceed as follows: • The sample has to be checked immediately in view of possible type of micro-organisms in order to confirm that the deviation is of microbiological and not chemical origin • The pH-electrode has to be washed before the next measurement • A recalibration of the electrode has to be made regularly (e.g. every 30 minutes); especially if drift of the measured pH-values or a slow-down of the reaction time during the test occurs. 4.5.5. Note: • The tolerable deviation from average pH-value is max. 0.1 units. Average pH depends on the product vs. process application and might vary in different plants. • All sensory and/or pH failures are to be verified by microbiological testing. • All packages failing sensory, pH and/or microbiological testing are to be saved for package integrity testing. FSQ – 588002-0104 8 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 4.5.6. Interpretation of Results The results of all test runs are to be added together before consulting the statistical table (See Appendix 1). If the total allowed defects occur in one run or are distributed over all 3 runs, the result is still considered to be acceptable. For sterility verification during commercial production all results obtained from microbiological testing performed on incubated packs should be accumulated for statistical purposes, i.e. samples from 1 months production could be accumulated and evaluated in the same way as in the sterile test. (See also page 16; section 6) 4.5.7. What should be done if the defect limit is exceeded? If the defect limit is exceeded, systematic troubleshooting to find the root cause must be undertaken. Appropriate corrective action must be carried out before a new test series is performed. It is unacceptable to repeat tests without prior corrective action. To commercially release a production line for normal production without corrective actions is regarded as “ commercial sterility not achieved”. FSQ – 588002-0104 9 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 4.6. Flow Chart for Microbiological Test Procedure (low acid pH >4,6) 1. Samples from 3 test runs (e.g. 7720 packs) 2. Early detection of severe failure (additional ~300 samples) Incubate for 7 days @ 30°C Incubate for 3 days @ 30°C Recommended to streak all samples and incubate @ 30ºC for 72 hours Evaluate by rapid methods (i.e.sensoric, pH, blown, ATP, streak) 3. Detection of Thermophilic sporeformers (Optional) Incubate for 5 days @ 55°C Recommended to streak all samples and incubate in a sealed container @ 55ºC for 5 days. Note: Number sampled packages and streak plates respectively. Rinse and save the packages for further investigation. Any package with microbiological, visual, sensory or pH deviation must be checked for package integrity. FSQ – 588002-0104 10 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products Interpretation of test results (low acid products, pH <4,6) The sample is considered to be commercially sterile if: • A streak on an agar plate shows < 10 colonies AND • The package shows no visual defects (blown, leaks etc.) AND • The product shows no signs of deterioration (clotting coagulation, smell odour etc.) AND • No pH deviation of the product was observed The sample is considered not to be commercially sterile if: • A streak on an agar plate shows > 10 colonies (the streak is usually full if the pack is unsterile) • The streak is sterile but the pH deviation of the product exceeds 0.1 units from the average value (in these cases the product has to be examined microscopically). OR • The package shows visual defects (blown, leaks, etc) AND/OR • The product shows signs of deterioration (clotting, coagulation, smell, odour etc.) AND/OR • The pH deviation of the product exceeds 0.1 units from the average value 4.7. Report / certificate / agreement It is required to document that actual test fulfilled the test criteria. The plant/ equipment should be taken over by the customer, and is ready for commercial production. Suitable document to be supplied by the local Tetra Pak Market Company. FSQ – 588002-0104 11 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 5. Sterility Test for high acid products (pH <4,6) Sterility tests are necessary for new or considerably modified heat exchanger or pasteurizer, respectively, components of the aseptic transfer (including the aseptic tank and valve cluster, if applicable) or fillers in order to validate the sterile performance of the entire line. A sterility test is only useful, if the installation is completed from both, a mechanical and software point of view and any problems rectified during the prior filling tests outlined above. During the test run it is recommended to build in events, which could occur during routine production runs. These events could include intermediate stops, splicing of packaging material and strip. Packages or carton trays needs to be numbered and the events have to be documented with the respective package or carton number to control such events. 5.1. Test protocol Follow instructions for Test protocol on page 4. 5.2. Test product and raw material For high-acid products (less or equal to pH 4,6) it is preferred to use a clear juice, i.e. apple juice, to be able to detect turbidity/cloudiness. The packaging material used should be suitable for the product application, e.g. TBA/j material when filling juice. The material intended for commercial production should be used (not test material delivered with the machine). Longitudinal strip used should be suitable for the product application and packaging material used. Hydrogen Peroxide according to Tetra Pak specification for the specific filling machine type should be used. 5.3. Trial procedure Follow instructions for Trial procedure on page 4. 5.4. Sampling procedure Follow instructions for Sampling procedure on page 5 FSQ – 588002-0104 12 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 5.5. Procedure for Laboratory Control 5.5.1. Incubation Incubate all packages from each run (packs/trays to be coded) for 15 days in 20-25 oC, Screening for severe failure of the test could be made during the incubation period by means of visual inspection of blowing packages (gas formation). Blown packages found at screening should be added to the final test result for statistical purposes. Any blown package must be checked for pack integrity. Note: There is no known legislation governing incubation of high acid products. If the filling temperature is below 20°C care must be taken to ensure that the stacking pattern adopted allows for all packages to increase in temperature rapidly and evenly. 5.5.2. End product test procedures Verification of commercial sterility shall be carried out on every package incubated. The recommended procedure method is by streak plating* the contents of each aseptically sampled package, using a calibrated 10µl loop (maximum 4 streaks per plate) on a suitable media for high-acid products (see below). The method of package opening should be such that the seals of the packages are left intact, (if packs are required for future integrity testing). Care must be taken to sterilise the external surfaces of the packages sampled for microbiological purposes with alcohol and/or flaming prior to aseptic opening with sterilised scissors or blade. The following methods are recommended • Visual (blown packages) • Turbidity (by visual check on clear juice only) • Sensory • Microbiological testing by - streak plating using a 10µl calibrated loop - surface plate count using a 1ml or 0.1 ml inoculums • Samples to be plated on - Orange serum agar or equivalent e.g. malt extract agar, Saboraud dextrose agar for total spoilage organisms - ACM for acetic acid bacteria - MRS for lactic acid bacteria • Direct phase contrast microscopy *Streak plating. Refers to the technique of applying liquid product to the surface of an agar plate with a 10µl calibrated loop. The method of application is a straight-line inoculation, beginning approx. 5 mm from the edge of the plate and ending about 5 mm from the opposite edge of the plate. FSQ – 588002-0104 13 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 5.5.3. Interpretation of results The results of all test runs are to be added together before consulting the statistical table (See Appendix 1). If the total allowed defects occur in one run or are distributed over all runs, the result is still considered to be acceptable. It is not significant if the defects occur in one run. For sterility verification during commercial production all results obtained from microbiological testing performed on incubated packs should be accumulated for statistical purposes. 5.5.4. What should be done if the defect limit is exceeded? If the defect limit is exceeded, systematic troubleshooting to find the root cause must be undertaken. Appropriate corrective action must be carried out before a new test series is performed. It is unacceptable to repeat tests without prior corrective action. To commercially release a production line for normal production without corrective actions is regarded as “ commercial sterility not achieved”. Note: • All sensory failures are to be verified by microbiological testing. • All packages failing sensory and/or microbiological testing are to be saved for package integrity testing. 5.6. Flow Chart for Microbiological Test Procedure (high acid pH <4,6) Samples from 3 test runs (e.g. 7720) Incubate all packages for 15 days @ 20-25ºC Daily inspection for blown packages (Early detection of severe failure) Recommended to streak all packs Alternatively, streak plate 10% and inspect product and package for moulds, plus blown packs Read result Note: Number sampled packages and streak plates respectively. Rinse and save the packages for further investigation. Any package with microbiological, visual, sensory or pH deviation must be checked for package integrity. FSQ – 588002-0104 14 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products Interpretation of test results The sample is considered to be commercially sterile if: • A streak on an agar plate shows < 10 colonies AND The package shows no visual defects (blown. leaks etc.) AND • The product shows no signs of deterioration (turbidity/cloudiness, moulds, smell odour etc.) The sample is considered not to be commercially sterile if: • A streak on an agar plate shows > 10 colonies (The streak is usually full if the pack is unsterile) OR The package shows visual defects (blown, leaks, etc) AND/OR • The product shows signs of deterioration (turbidity/cloudiness, moulds, smell, odour etc.) 5.7. Report / certificate / agreement It is required to document that actual test fulfilled the test criteria. The plant/ equipment should be taken over by the customer, and is ready for commercial production. Suitable document to be supplied by the local Tetra Pak Market Company. FSQ – 588002-0104 15 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 6. Commercial Sterility Level Verification (Optional) 6.1. Sterility Verification The purpose of this period is to verify higher level of commercial sterility over an extended period of time with the added advantage of a much larger statistical sample base. All samples incubated and tested during this period, should be accumulated and the results evaluated against the pre-agreed* target defect rate with 95% confidence. The sterility level is expressed as the total number of defective packages (microbiological/integrity infection) within the total of incubated and tested packs. The following table gives an upper limit for the defect rates on 95 % confidence level with a given sample size and a given number of defects. Example 1: 700 samples have been tested and one defect has been found, the conclusion is that the defect rate is better than 0.68%. Maximum defect rates, % Example 2: In order to verify 1:10’000 (0.01%) sterility, a minimum of 30’000 samples have to be tested. However, this sample size would allow for zero defects (0.01%) or 1 defect (0.016%). If more than one defective pack should be allowed, then a greater sample size should be utilised which would allow for 1-4 defects (see bold figures in the table below). Sample size Number of defects 0 1 2 3 4 5 0.03 0.047 0.063 0.077 0.091 0.105 10 000 0.01 0.016 0.021 0.026 0.03 0.035 30 000 0.008 0.012 0.016 0.02 0.023 0.026 40 000 0.006 0.01 0.013 0.016 0.018 0.021 50 000 *The Defect rate tested and related sample size to be the subject of agreement by the Customer/Tetra Pak project team. FSQ – 588002-0104 16 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 6.2. Refinement of Production Routines The purpose of this period (which runs concurrently with the extended verification of commercial sterility outlined above) is to allow the operational staff to gain the necessary skill level required to operate the equipment in an optimum way in order to achieve the targeted line efficiency. The duration of this period is variable between according to the current skill level of the operational staff but may not exceed 6 months. Example 1: In a new plant with inexperienced staff who have no previous operating experience, this period could be as long as four months, but may not exceed six months. During this period, it is the responsibility of the joint Tetra Pak/Customer project team to ensure that the necessary training in operating, maintenance and quality control methodology takes place. Example 2: Where equipment is installed in a fully operational plant with experienced operational staff, this period could be shortened considerably. The duration of this period should be at the discretion of the joint Tetra Pak/Customer project team but may not exceed 6 months. 6.3. Efficiency Verification Period The efficiency verification period should commence when, at the discretion of the joint Tetra Pak/Customer project team, the operating staff have the necessary skill levels to achieve the targeted line efficiency and all equipment works satisfactorily. During an extended period of between 5 - 20 working days, the filling operation is closely monitored. The line efficiency is monitored during this time and checked against the mutually agreed target. The length of the monitoring period should be decided upon by the joint Tetra Pak/Customer project team according to the nature and size of the project, the amount and type of equipment installed, and the experience level of the operating staff. During this period, all events during production should be carefully recorded and adequate corrective actions initiated. With the collection of the appropriate data the line performance can be determined. In the latest generation of TBA filling machines, this PLMS facility is standard. FSQ – 588002-0104 17