Why Perform a D-value Study?

Introduction

It is important to understand how the resistance of microorganisms is affected by the product with which they are associated. This is accomplished by performing product D-value studies which are a necessary activity when developing a sterilization process and are discussed in detail in the Spore News publications referenced below. These studies require the use of a resistometer (specialized equipment not widely available to organizations other than biological indicator (BI) manufacturers or third-party labs).

Standards & References

ISO 11138-7:2019 Sterilization of health care products - Biological indicators - Guidance for the selection, use and interpretation of results
5.3.2 The resistance characteristics of a test organism in suspension can be considerably changed upon deposition on or in carriers. Several factors can influence the resistance characteristics, such as the surface on to which the suspension is inoculated (e.g., solid materials, viscous products or fluids), the way the spores are dispersed and otherwise treated, the method of drying, etc.

European Pharmacopoeia 10
5.1.2 Biological Indicators and related microbial preparations used in the manufacture of sterile products
2 Biological Indicators for sterilisation processes
Spores inoculated into a product or onto surfaces are known to react differently to sterilising conditions as compared to biological indicator units. In these cases, commercially available biological indicator units may not be suitable to test sterilisation effectiveness and an inoculated test product/item prepared from a well-characterised  spore suspension may be a better model to evaluate the effectiveness of the sterilisation cycle.

United States Pharmacopoeia 2022
GENERAL CHAPTER <1229.5> BIOLOGICAL INDICATORS FOR STERILIZATION
User-prepared Biological Indicators
The biological challenge is either directly inoculated into a liquid-filled container or is introduced via self-contained units provided there is adequate correlation between their resistance and the resistance that would occur in the process fluid. The liquid can be either the product or a surrogate fluid. The resistance of the indicator in the product (and surrogate fluid, where used) must be known. 

 ISO 11138-7:2019 Sterilization of health care products - Biological indicators - Guidance for the selection, use and interpretation of results

5. Characteristics of biological indicators
5.2.4. The D value and, when appropriate, the z value, are constant values only under defined conditions. The resistance characteristics of a spore suspension provided by a biological indicator supplier might not correspond to the resistance characteristics for direct product inoculation studies. The resistance characteristics should be measured for the carrier employed (solid carrier material or fluid) as well as for the specific sterilization cycle employed.

European Pharmacopoeia 10
5.1.2. Biological Indicators and related microbial preparations used in the manufacture of sterile products
2-1-4. Custom-made Biological indicators
Custom-made Biological indicators are test Items (e.g., rubber stoppers), or products, inoculated with a suitable test micro-organism...
The D-value (…) and, when appropriate, the z-value of the spore suspension must be determined. Also, the D-value and z-value (if appropriate) of the spores of the inoculated test items/products must be determined as this may be different from the spores in suspension.

United States Pharmacopoeia 2022
GENERAL CHAPTER <1229.5> BIOLOGICAL INDICATORS FOR STERILIZATION
User-prepared Biological Indicators
A user of BIs may elect to propagate spore crops of a single species for use as a suspension. Alternatively, these spore suspensions may be purchased from a BI manufacturer. When liquid suspensions are applied to a substrate, it is the user’s responsibility to determinate the population and resistance of the microorganism used. The resistance determined for liquid suspensions relates only to other lots of the same suspension and is not representative of how that microorganism will perform on a substrate or in a different suspending medium. In these circumstances, the BI resistance and population should be re-established.

Mesa Labs’ Recommendations

1. Perform product D-value studies on each product or family of products as part of the sterilization cycle development activities.
2. Perform product D-value studies utilizing appropriate methods such as Survivor Curve testing for liquid pharmaceutical products and fraction-negative testing for smaller closure components such as stoppers.
3. Utilize the Contract Studies Lab at Mesa Labs for product D-value studies. Mesa Labs Contract Studies Lab has decades of experience in conducting these studies and possesses all of the necessary equipment (e.g. resistometers) and materials (spore crops) required for such testing. The Contract Studies Lab is ISO 13485 certified and licensed by the US Drug Enforcement Agency to test controlled substances.

Additional Resources

1. Spore News SN003 Product D-value Studies: A Critical Tool When Developing a Sterilization Process 
2. Spore News SN040 Custom Biological Indicators 
3. Spore News SN060 Contract Studies
4. Spore News SN069 Why Perform a Product D-value Study? 
5. Spore News SN070 Product D-Value Study – Part 2, – Heat-sensitive & Atypical Aqueous Liquids 

SN079-V1