A Risk-Based Approach to the Use of Biological Indicators in the Development and Control of Steam-Sterilization Processes - Pharmaceutical Technology

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A Risk-Based Approach to the Use of Biological Indicators in the Development and Control of Steam-Sterilization Processes
Validating the sterilization process is extremely important in pharmaceutical manufacturing. The authors explore different types of sterilization processes and discuss the importance of finding the worst-case positions of loads or equipment to be sterilized and the worst-case conditions for each sterilization cycle. Biological indicators (BIs) can be used to simulate worst-case scenarios and determine the effectiveness of a particular sterilization process.


Pharmaceutical Technology


When commercially available BIs are used, the D-value should be chosen in correlation with the sterilization process. The European Pharmacopoeia defines a standard steam-sterilization process of 15 min at 121 C. It also is specified in Ph.Eur. chapter 5.1.1 that BIs used should have a D-value that exceeds 1.5 min (13). Following Ph.Eur., it must be verified that "exposing the biological indicators to steam at 1211 C for 6 min leaves revivable spores, and that there is no growth of the reference microorganisms after the biological indicators have been exposed to steam at 1211 C for 15 min"(14).

Kill times are expressed in a more general way in USP 30: "Kill time (in minutes) = not more than (labeled D value) (log labeled spore count per carrier + 4)". For a viable spore count of 106/carrier and a labeled D-value of 1.5 min, the kill time is easily calculated to be 15 min, identical with the Ph.Eur. standard sterilization time. Following the logic of Ph.Eur., BIs with a lower kill time than the process to be tested would not present a sufficient challenge to the standard process.

It is interesting to note that no upper limit for the D-value is mentioned in Ph.Eur. For a BI with a D-value of 2 min and a viable spore count of 106/carrier, the kill time would be 20 min, which means that occasional positives are expected to occur after 15 min of exposure. A manufacturer obtaining a positive BI after exposure usually would be considered an indication of sterilization-cycle failure, while the true reason is inappropriate resistance of the BI.

Spore suspensions. Spore suspensions form the basis for the manufacture of commercially available BIs on carriers. They are also commercially available for use in the inoculation of products and surfaces to evaluate the effect of sterilization processes and to prepare customized BIs. Spore suspensions are addressed in USP 30 as a type of BI.

The quality of commercially available spore suspensions, however, is neither addressed in ISO 11138 (12) nor is there a monograph on BI suspensions in USP (15). Typically, manufacturers certify the D-value of their suspensions when tested on paper strips. The D-value of the spores in suspension is usually different from the certified D-value. The stability of the viable spore count in suspension also is of concern. Some spore suspensions are supplied in alcoholic suspensions, and others are supplied in water. The purity of spore suspensions with regard to cell debris that may cover spores during drying is not clearly specified anywhere. There is no international standard that could be used to qualify the spore suspensions available on the market.

More questions may arise regarding the methods applied in direct inoculation of test pieces. Test pieces may have a modulating effect on spore resistance caused by the release of ions or other substances, surface roughness that may provide local shielding of spores from the access of saturated steam, or local temperature effects. Other factors are spore distribution on the inoculated surface, the accessibility of the sterilizing agent, or the adhesiveness of the spore layer. Spores may be difficult to recover from the surface of inoculated test pieces because of strong adherence that may increase during heat exposure, meaning that the determination of an initial recovered spore count is very difficult. Standardized procedures for inoculation and testing of inoculated test pieces and recovery of spores from surfaces are not available.

When product solutions are inoculated, there are questions concerning the volume of liquid used and the kinetics of heating. Is the temperature profile in the test volume a square wave, or are there shoulder conditions that must be taken into consideration?

To determine the influence of pharmaceutical preparations on the resistance and growth ability of spores, the inactivation effect during sterilization must be evaluated separately from the inhibiting effect on the growth of spores surviving after sterilization. Vice versa, the product can be influenced or altered by introducing the BI—such as in the case of the inoculation of an anhydrous product with aqueous spore suspensions.


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