Procedures guidelines guideline for microbiological evaluation of commercially sterile products

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 commercia

Procedures & Guidelines

Guideline for Microbiological Evaluation of

Commercially Sterile Products

FSQ-588002-0104

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 Suggested test routine: 5

4.4 Sampling procedure 5

4.4.1 Sample size 5

4.4.2 Defect level to be verified 5

4.4.3 Choice of sampling plans 6

4.4.4 Incubation 6

4.5 Procedure for Laboratory Control 7

4.5.1 End product test procedures 7

4.5.2 Evaluation methods vs statistical result 7

4.5.3 Early detection of severe failure 8

4.5.4 Detection of Thermophilic sporeformers (optional) 8

4.5.5 If pH-Measurement procedure is used for evaluation 8

4.5.6 Interpretation of Results 9

4.5.7 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 Incubation 13

5.5.2 End product test procedures 13

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

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

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:

Efficiency Verification Period

Measuring of the target line efficiency over a period of 1 to 3 weeks (acc To PM)

Refinement of Production Routines

Completion of Training for operators,

maintenance and QA/QC staff

Optional (according to local Market company/customer contract)

Incubation Period (ending with Evaluation of Commercial Sterility)

With skim milk or product

Line Release of Routine Commercial Production

Based on sterility requirements agreed between the project partners

Time estimate: Low acid products

• 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

Filling Test

With skim milk or product

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

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

• 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

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

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

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 Maximum

defects allowed

Detection probability with a defect rate

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

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)

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

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

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

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”

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 Evaluate by rapid methods

and incubate @ 30ºC for 72 hours (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.