ISO 11137 Microbiological Validation

Introduction
"Sterile" is an absolute term, but the assurance that any given item is sterile is a probability function. The Sterility Assurance Level (SAL) of a product is defined as the probability of any given unit being non-sterile after exposure to a validated sterilization process. For a product to be CE marked and labelled sterile, it needs to conform to the European Standard EN556. The SAL defined by EN556 is 10 -6 , that is one surviving microorganism per one million products.

The Standard that defines methods of verifying a given SAL for irradiated products is ISO11137 ( Sterilization of Healthcare Products - Requirements for Validation and Routine Control - Radiation Sterilization 1995 [Annex B]).

Validation Methodology and Dose Audits
ISO11137 outlines the two methodologies for dose setting to achieve a given SAL, described as Method 1 and Method 2. A brief description of the theories and protocols regarding these methods and supporting routine dose audits are outlined below. These services are available through Isotron Laboratories.

Method 1
Method 1 is used to determine the radiation sterilization dose necessary to achieve a chosen sterility assurance level (SAL). A standard table of microbiological resistances is used in this approach.

Initial Phase - Bioburden Evaluation
A minimum of ten samples from three separate batches/lots is required for bioburden analysis. For the process to be valid, products submitted for ISO11137 validation should not demonstrate bacteriostatic or bactericidal properties. Bioburden analysis should be to a validated protocol. If no validation protocol is available, Isotron Laboratories will assist in developing one suitable for that specific product. The average bioburden for the product is determined from each lot/batch, the overall average bioburden is then calculated using the 30 samples from the three batches. The bioburden count used for the validation is the overall average unless the average of one of the batches is two or more times greater than the overall average, in which case the highest batch average bioburden is used.

Phase Two - Sub process verification dose experiment
Once the bioburden estimate has been determined the sub process verification dose (to achieve a SAL of 10 -2 ) level can be determined using the reference table in the ISO11137 Standard. A SAL of 10 -2 is used because the results of sterility tests carried out on 100 samples irradiated at this dose level can, by extrapolation, be used to confirm the dose required for a SAL level of 10 -6 . The bioburden value in the reference table equal to or greater than the actual bioburden is used. 100 test samples from a single batch are irradiated at the calculated sub process verification dose. This delivered dose will have a maximum variance of +/-10%. The samples are then transferred into the isolator unit in preparation for sterility testing. Each of the 100 test samples is sterility tested independently. These samples are then incubated at 30°C +/- 2°C for 14 days and the number of positives determined. Periodic checks are made to determine the number of positive tests over the 14-day period.

Statistical verification
Statistical verification of the test is accepted if there are no more than two positive sterility tests from the 100 test samples (i.e. Validation is satisfactory). The required minimum processing dose can then be obtained, referencing the standard distribution of resistances table in the ISO11137 standard.

Any failures require further investigation, an augmented sterilization dose may be required.

Method 2
Method 2 is used to determine the radiation sterilization dose necessary to achieve a chosen Sterility Assurance Level. Regarding the resistance to radiation of microorganisms as they occur on the product. The process is complex and a number of calculations are used to determine this dose. Only a very brief outline of the process is given below. Contact Isotron for further details of the process.

Initial phase
A minimum of 280 samples from three separate batches (840 in total) must be available for analysis.

Phase Two
20 product items are irradiated at each dose level from each of three batches. A series of not less than nine dose levels and increasing by 2kGy increments (e.g. 2, 4, 6, 8,........, 18kGy) are used, and each of the delivered doses monitored. All the irradiated product items are then individually sterility tested.

Phase Three
Using the sterility test results from above, the calculation outlined in the Standard is used to determine an initial estimate of the dose required to achieve a SAL of 10 -2 . The sterility test results also determine which batch is used for further sterility testing. 100 product items from this batch are irradiated at the calculated verification dose and individually sterility tested.

Phase four
The results of all the sterility tests are used to calculate the required minimum sterilization dose.

Routine Dose Audits
To conform to the Standard, the performance of the sterilization process must be monitored periodically. The Standard requires dose audits to be performed quarterly to confirm that the current bioburden profile is equivalent to the one at the initial dose setting exercise.