Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products FSQ-588002-0104 Table of contents Preface 1.1 Evaluation of customer requirements 1.2 Verification of commercial sterility .1 1.3 Low acid vs High acid products 1.4 Procedural overview .2 Pre-trial preparations 2.1 Verification of plant pre-requisites 2.2 Verification of Package Integrity 3 Filling Test Sterility Test for low acid products (pH >4,6) 4.1 Test Protocol 4.2 Test product and raw materials .4 4.3 Trial Procedure 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: Sampling procedure .5 Sample size Defect level to be verified Choice of sampling plans Incubation Procedure for Laboratory Control End product test procedures Evaluation methods vs statistical result Early detection of severe failure Detection of Thermophilic sporeformers (optional) If pH-Measurement procedure is used for evaluation Interpretation of Results What should be done if the defect limit is exceeded? 4.6 Flow Chart for Microbiological Test Procedure (low acid pH >4,6) 10 4.7 Report / certificate / agreement 11 Sterility Test for high acid products (pH 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 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 tests must be accumulated and evaluated as a single test production FSQ – 588002-0104 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 4.3.1 Suggested test routine: Day One test run, including UHT processing and aseptic filling Finish with a full CIP Day – 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 be run into the aseptic tank and stored at ambient temperature and filled on day 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 30 000 300 000 000 000 FSQ – 588002-0104 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 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 below (7800) According to the table, this plan will allow a maximum of defects in 7720 packages still fulfilling the requirement of a failure less than 1:1000 • Plan # Sample size 3000 4730 6270 7720 9151 Maximum Detection defects allowed probability with a defect rate of 1/1000 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 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 days at 30°C, according to the flow diagram on page 10 Follow the recommended temperatures After a period of 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 (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 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 days Example: Sample plan - total number of packs to be tested is 7720 Maximum allowed defects (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 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 mm from the edge of the plate and ending about 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 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 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 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 Procedures & Guidelines Guideline for Microbiological Evaluation of Commercially Sterile Products 4.6 Flow Chart for Microbiological Test Procedure (low acid pH >4,6) Samples from test runs (e.g 7720 packs) Early detection of severe failure (additional ~300 samples) Incubate for days @ 30°C Incubate for 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) Detection of Thermophilic sporeformers (Optional) Incubate for days @ 55°C Recommended to streak all samples and incubate in a sealed container @ 55ºC for 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 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 Sterility Test for high acid products (pH [...].. .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... 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