Sterilisation Validation

Like any process, the sterilisation process also requires validation. Sterilisation Validation follows the same steps (IQ, OQ and PQ) as process validation, but the content of these steps differ, also per sterilisation process. Below some possible steps are described. Sterilisation validation is done according to ISO 11135 (EO) or 11137 (radiation), and 11737 (microbiological methods).

Packaging configuration

The box or pallet’s density influences the sterilisation process’s effectiveness. The protocol must define the number of products per box or pallet. When several product dimensions are sterilised in one run, this must be described and a worst-case scenario made.

Also, the packaging configuration has influence and needs to be defined. The number of products per blister or pouch, the number of these in a carton box, the number of carton boxes per transporter box, and the number of transporting boxes per pallet must be defined. Also, the way the boxes are stacked on the pallet is part of the configuration.

Test validations

Several tests are part of the sterilisation validation. The two most important ones are the bioburden test and the sterility test, but also tests such as residual ethylene oxide determination are part of the validation. These tests must be validated to value the outcome of the test results.

The Bioburden validation can be done with the Bioburden Recovery Efficiency (RE). It is intended to assess the efficiency of the extraction technique to remove viable microorganisms from a product. This efficiency provides a numerical value called the Correction Factor, which is applied to the product bioburden.

The Sterilisation test must also be validated with the growth promotion test. A known amount of microbiological contamination is placed on a product, which needs to be recovered during testing.

Validation Steps Irradiation

To determine the parameters for the sterilisation process, the baseline of the bioburden values for the product needs to be determined. Usually, this is done by testing ten products.

We use the ISO11137-2 norm and the bioburden results to define the verification dose. Samples (e.g., 10) are sterilised at this verification dose and tested on sterility. When all products are sterile, the test is considered a pass.

Validation Steps EO

Also, for Ethylene Oxide (EO) sterilisation, the baseline of the bioburden values for the product needs to be determined.

Together with the sterilisation company, the sterilisation temperature, time, speed of gas blowing, and suction are first tested in a fractional/sublethal cycle and the resistance of the microorganisms to the EO. The three next rounds are half cycles, using half of the full cycle EO, followed by three rounds of a full cycle.

These tests are done with a fully loaded pallet with products (maximum density of products). Optionally a minimum load can be tested to allow flexibility in the number of products sent for sterilisation.


At the end of the validation, the monitoring frequency must be determined. Often this is (initially) done 4x per year but depends on risk and previous outcomes.

The bioburden level of the product must be tested on, e.g., ten samples. You usually want the average bioburden value below 1.5 times the value measured during validation.

Also, e.g., ten samples must be sterilised at the verification dose (irradiation) or half cycle (EO) and tested on sterility.