Our modern isolator-equipped test laboratory meets GMP and DIN EN ISO 14644 requirements

The methods of spore counting and D-value determination are common practice to characterize and control the quality of biological indicators

Sterilization processes preceding the aseptic filling of pharmaceuticals must be proven to be highly effective (EU GMP, Vol. 4, Annex 1). Biological indicators (BIs) are used to demonstrate the efficacy of decontamination or sterilization processes.

Both sterilization processes in autoclaves and decontamination processes using vaporous hydrogen peroxide (H₂O₂) in isolators usually use spores of Geobacillus stearothermophilus. These spores are an appropriate model system due to their high resistance. Furthermore, Geobacillus stearothermophilus is cultivated at relatively high temperatures (55 - 60°C), which specifies the evaluation of the test.

BIs for decontamination processes consist of spore monolayers applied on stainless steel carriers. These carriers are sealed in Tyvek® envelopes for better handling. Although this membrane is permeable for H₂O₂, it provides a diffusion barrier for the spore-inoculated carrier (worst case scenario).

After the application, carriers are transferred from the Tyvek® envelopes to nutrient media. If no growth occurs after seven days of incubation, all spores on the carrier have been inactivated by the treatment process.

Transfer of spore carriers into nutrient medium

Decontamination processes are supposed to provide a 6-log reduction of microorganisms. To the deliver proof, a BI must be occupied with at least 10⁶ spores.

Additionally, the spores should have a defined resistance. This resistance is prone to variations since spores represent a non-calibratable biological system. The D-value (decimal reduction time) can be used to determine a characteristic value for the resistance of a specific BI batch. D-values of different batches are comparable if they are determined in one and the same system. Moreover, this provides insights about the inactivation behavior of a batch and possible hyper-resistant BIs („rogue BIs“, „late survivors“, or „anomalous BIs“). A batch containing such indicators should not be used for validations, as this may produce false positive results.

DIN EN ISO 11138-7:2019-11 defines the modalities to determine of valid spore counts and D-values for the characterization and quality control of BIs.

Our isolator experts can optionally support you in the following topics:

Spore count

Spore count is determined according to DIN EN ISO 11138-7:2019-11: Sterilization of health care products – Biological indicators – Part 7: Guidance for the selection, use and interpretation of results (ISO 11138-7:2019); German version EN ISO 11138-7:2019, from four single samples using the heat shock method.

Determination of system D-value

The system D-value is determined following DIN EN ISO 11138-7:2019-11: Sterilization of health care products – Biological indicators – Part 7: Guidance for the selection, use and interpretation of results (ISO 11138-7:2019); German version EN ISO 11138-7:2019, from 30 sample groups, each with 10 or 20 single samples and a sampling interval of one minute.

The D-value is calculated according to the Limited Holcomb-Spearman-Karber Procedure (LHSKP) and is determined in a standardized system. Additionally, customers can choose from a set of predefined values of H₂O₂ concentrations and relative humidities to adapt the D-value determination as closely as possible to their conditions.

Spore identification by API 50

Spore identification is performed by means of a bioMérieux API® 50 CHB/E test. In parallel, KWIK STIK™ of the strain to be identified is entrained.

Initial qualification and quality testing of the BIs is performed in the ValicareLab using a pharma-grade sterilization isolator.

Please contact us in due time if you plan to use BIs for process development and validation of your isolator system. With a preliminary lead time, we can provide the necessary number of “qualified” ready-for-use BIs. In combination with a joint project, we will gladly take over complete review and characterization of your batch of BIs or supplement missing partial aspects.

Take advantage of BIs tested and characterized under real conditions in an isolator. If you also use H₂O₂ for decontamination in your isolator, you will get the D-value for your BIs reliably determined in your application scope together with a statement on anomalies.

This saves you a lot of time during process development and validation.

Microbiological decontamination effects by hydrogen peroxide of surfaces varies strongly depending on the material used

In a study conducted by experts from Syntegon, published in 2007 under the title "Suitability of different construction materials for use in aseptic processing environments decontaminated with gaseous hydrogen peroxide", 49 materials potentially used in aseptic processing environments were inoculated with spores of Geobacillus stearothermophilus ATCC®12980TM. To be able to record individual inactivation kinetics, the inoculated materials were exposed to reproducible hydrogen peroxide biocontamination processes for defined periods of time and the D-values were repeatedly determined.

The study found that both the chemical composition of the material and the nature of the surfaces affected the resistance of the spores. The calculated D-values show some correlation with the roughness and wettability of the materials. The full article can be found here.

Valicare offers exactly this kind of material studies. Materials inoculated with spores of Geobacillus stearothermophilus are exposed to reproducible biodecontamination processes with hydrogen peroxide in our own isolator over defined periods of time. The determination of the D-value is used as a measure of the material-dependent resistance of the spores.

Contact our experts if you have any questions about the procedure or would like to know more about our material studies.