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

May 1, 2007
Pharmaceutical Technology
Volume 2007 Supplement, Issue 2

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.

In discussions between colleagues dealing with steam-sterilization processes in the pharmaceutical industry, the medical device industry, or in hospitals, it frequently becomes obvious that sterility assurance and the use of biological indicators (BIs) as tools for the validation of sterilization cycles is not a commonly well understood and clear concept. Although it may not be surprising that sterilization is regarded differently in hospitals than in the canning industry, the differences in the healthcare sectors are more difficult to understand. Validation of sterilization processes is regarded quite differently in hospitals and in the manufacture of medical devices than in the manufacture of pharmaceutical products. It is even more confusing that within the pharmaceutical industry, the view on validation of sterilization processes and the use of BIs is not the same on both sides of the Atlantic. This article elucidates some reasons for the surprising variations in understanding the verification of sterilization effectivity by BIs.

It is not entirely clear to everybody why BIs are used at all. BIs intended for monitoring and controlling sterilization processes are preparations of bacterial endospores that are highly resistant to a particular sterilization process. They are used to demonstrate the sterilizing effect of the process. As such, BIs contain endospores that are much more resistant and present in a far larger number than the microorganisms encountered in the presterilization bioburden of any product to be sterilized. For that reason, bioindicator studies often are considered irrelevant, especially for so-called overkill processes. Is this correct, and, if so, why are we using such irrelevant sterilization procedures?

Another issue concerns the significance of the BI results obtained when monitoring or validating a sterilization cycle. Is the killing of BIs the ultimate proof of cycle validity? Or is a sterilization cycle invalid when a BI has survived this sterilization cycle? If the validation of sterilization cycles is truly simple, why are we going through a huge effort to develop and validate sterilization cycles?

One question frequently asked in the European pharmaceutical industry is: Why is it not sufficient to use qualified equipment and utilities and run a cycle that is effective enough to kill every microorganism present? When this overkill effect is verified by the routine addition of a few BIs, why should there be a need to validate specific cycles? This approach is typically taken in hospitals and in the medical device industry. The logical reverse argument also is frequently raised: Do we need biological indicators at all, or isn't it sufficient to simply use physical measurements of temperature, pressure, time, and steam quality to characterize a steam-sterilization process?

Sterilization processes, like all other processes, can be validated only when their possible problems and pitfalls are well understood. BIs and other methods or tools can be correctly used only with a clear understanding of what is intended by their use and with the identification of the strengths and limitations of the tool. There are many different steam-sterilization processes that require different validation strategies, and understanding the use of BIs is much more complicated than it may initially appear.

Sterilization processes

Sterilization processes using saturated and nonsaturated steam. In the generally accepted scientific opinion, the full effect of steam sterilization is achieved only by saturated steam in a process where heat is effectively transferred by condensation at the surface of the autoclaved products or on the surface of sterilized equipment in combination with the hydrating effect of the condensate. Although this is a correct description of the general physical phenomena that occurs in steam-sterilization processes, it is not always what happens in an autoclave. It also is an oversimplification of the real process in many cases.

In cases in which product is autoclaved in the final sealed containers, condensation of saturated steam may be a very effective method of transferring energy to the surface of the containers, but this is not the primary sterilization process. The relevant sterilizing conditions for the product itself will be generated inside the sealed containers. As an extreme example, dry-heat conditions always will be achieved in empty fused ampules regardless of how they are heated. For the same reason, it does not make sense to use self-contained spore preparations in sealed glass ampules to evaluate a process that relies on steam saturation. The degree of steam saturation is irrelevant for the sterilizing effect in this case. The device will react to heat input no matter how the heat is supplied. There can be no differentiation among dry heat, heating in an oil bath, or saturated steam. Any thermoelement would do the same job, be easier to handle, and give immediate and more-accurate results.

For sealed containers, it is important to verify during the development of the sterilization cycle that sterilizing conditions are achieved in all parts of the containers when they reach the sterilization temperature.

In cases in which porous goods are sterilized with the direct access of steam, it is important to verify full penetration of the steam through the pores of the product. Because sterilization conditions in this case are achieved by the direct action of the steam, steam saturation is a critical parameter.

Penetration of steam also can be notoriously difficult to achieve, for example, with filling equipment that has pipes or tubing that is sterilized in place (SIP). Even in processes where air is removed by evacuation, complete air removal may be difficult.

There also may be positions in a load to be steam sterilized that are occluded from the access of steam; for example, the space between the barrel and piston of a filling pump, or the interface between elastomeric stoppers and the necks of glass vials.

Effect of the microenvironment on sterilization efficiency. In addition, the effect of sterilizing conditions can be strongly modulated by the microenvironment encountered by bacterial endospores during sterilization. The decimal reduction time (D-value) of a spore preparation is notoriously different when the spores are presented on a paper strip, suspended in water, or attached to a polymeric surface (1). D-values of spores in solutions can depend on the composition of the solution (2–4). For example, the presence of divalent cations has significant influence on endospore resistance, and D-values are lower in solutions containing high concentrations of glucose (5). Thus, spore inactivation is not dependent solely on the conditions in the autoclave. There are additional chemical and possibly other surface effects that may strongly influence the D-values of suspended or attached endospores.

Such influences of the microenvironment cannot be measured by any physical probes. Using BIs is the only method to directly measure the sterilizing effect, and, therefore, an ideal BI should indicate any effect of product and microenvironment.

Definition of the worst-case position in a sterilizer load. The sterilization effect is achieved as a result of the combined influence of temperature, heat transfer, surface hydration, and all other protecting or inactivating factors that influence endospores during the sterilization process. Therefore, the most difficult-to-sterilize position (the worst-case position) in a sterilizer load should be defined as the position where the sum of all influences on microorganisms results in minimal inactivation.

The relevance of the worst-case position to product safety also should be considered. Although there may be occluded positions in a piece of SIP equipment that are never reached by steam during the sterilization process, such positions also may never come in contact with product. As long as there is no potential to jeopardize the sterility of any product manufactured with that equipment, there would be no good reason why the position should be sterilized. This, however, can be correctly judged only with a thorough understanding of the equipment and the process.

The effect of steam sterilization on microorganisms trapped between the polymeric stopper and the vial in terminally sterilized pharmaceuticals has been discussed (6). The relevance of that position to the sterility of the contents of the vial is critical for the decision of whether that is, in fact, the worst-case position of the load. As long as deformation of elastomeric stoppers during the cooling phase of autoclaves cannot be excluded, such a position certainly would have to be considered as relevant for the sterilization effect.

Overkill processes. The necessary sterilization assurance is described in United States Pharmacopeia: "A sterilization process must result in a biologically verified lethality sufficient to achieve a probability of obtaining a nonsterile unit that is less than one in a million" (7).

In cases in which the product to be sterilized is very heat resistant, sterilization processes are usually designed to achieve inactivation of microorganisms by a wide margin of safety. Such overkill processes are frequently defined on the basis of their ability to inactivate a given number of microorganisms. Overkill processes are defined in USP 30 as follows:

In general, all overkill processes are built upon the assumption that the bioburden is equal to one million organisms and that the organisms are highly resistant. Thus, to achieve the required probability of a nonsterile unit that is less than one in a million, a minimum 12 D process is required. A 12 D process is defined as a process that provides a lethality sufficient to result in a 12 log reduction, which is equivalent to 12 times a D value for organisms with sufficiently higher resistance than the mean resistance of bioburden.

Endless discussions have been led on the number of orders of magnitude by which a resistant spore preparation must be inactivated for a sterilization process to be called an overkill process. Is there a requirement for 12 logs of inactivation as defined in USP or are 8 logs sufficient? And what is the prerequisite D-value of the resistant organisms or BI at 121 °C? Is it 1 minute or 1.5 minutes? Or should the definition of an overkill process be based on the theoretical effectiveness of a sterilization process, the F-value, or the standardized F0-value (8)?

The outcome of these discussions can be meaningful only if the subject is precisely defined and clearly understood by everybody. Several points must be clarified that are also not precisely stated in USP (see sidebar "Matters to consider when testing and selecting a BI").

Matters to consider when testing and selecting a BI

To characterize an overkill sterilization process, the desired (and claimed) effectiveness of the process must be defined. If the overkill effect is defined from inactivation of a large number of resistant spores in a reference position, it is essential to understand how this correlates to the sterilizing effect in worst-case positions. For example, how does the effect seen on paper-strip BIs distributed in the autoclave chamber correlate to the effect on spores in sealed containers or on the surface of polymeric stoppers? How does the effect on paper-strip BIs distributed in easily accessible vessels correlate to the effect on spores on a difficult-to-reach valve in complex SIP equipment?

Bioburden-oriented or combination sterilization processes. Other sterilization processes than overkill processes are targeted to achieve complete inactivation of the bioburden at minimal heat input. This approach is chosen especially to sterilize heat-labile products. Targeted bioburden-oriented or combination processes rely on the experience that presterilization intermediates in pharmaceutical production can be manufactured under stringent precautions to achieve a very low bioburden. In addition, the environmental isolates of heat-resistant microorganisms are, in all experience, far less heat resistant than spores of Geobacillus stearothermophilus, the microorganism whose endospores are most frequently used in BIs.

For targeted sterilization processes, it is most important to ensure the target bioburden is not higher than expected. It is even more critical for these processes than for overkill processes to characterize the sterilizing effect of the cycle at worst-case positions in the load to be sterilized. BIs containing customized spore preparations (e.g., the most resistant spore preparations harvested from the product or production environment) are sometimes used to demonstrate sterilization effectiveness at worst-case or reference positions.

Commercially available BIs on carriers

In addition to characterization of the sterilizing effect at worst-case positions, the properties of the actual most-resistant bioburden isolates compared with those of the customized spore preparations must be considered in detail. Is the process bioburden well-enough characterized to ensure that the most resistant isolates are indeed known, and what are the cultivation and harvesting conditions needed to produce the most resistant spores from these isolates? What is the difference when resistant spores of these isolates are tested on paper or at a worst-case position? How do spores from process isolates react in a reference suspension in product or on the surface to be tested?

Using biological indicators

Use in product or process development. Ease of sterilization should be a criterion during the development of sterile products. It is the position of the European authorities that a heat-labile container-closure system alone is not a sufficient justification to choose a sterilization cycle other than the Standard Sterilization Cycle defined in the European Pharmacopoeia (10, 11). Although product reformulation may not be an option in many cases, primary packaging materials such as stoppers or delivery systems should at least be chosen in due consideration of their influence on sterilization effectiveness.

During product development, bacterial endospores should be inoculated as the model bioburden to evaluate the influence of sterilizing conditions on microorganisms suspended in the product relative to the effect obtained in a reference spore suspension in water or saline. There is no other way to measure the effect of product on spore inactivation. It is certainly not less important to characterize the influence of a sterilization procedure on suspended spores than to investigate its effect on product stability. Both studies are vital for the correct choice of a sterilization process.

In addition, the primary packaging (container-closure) system should be evaluated with respect to possible problem points during sterilization. In the experience of the authors, spore inactivation is not the same on all types of stoppers. Specific effects may be attributed to the material, the surface finish, or both.

For vials with elastomeric stoppers, the space in between the stopper and the vial is always a critical position because this is a position where steam does not easily penetrate. For BI studies, it is necessary to ensure that the bacterial endospores are in contact with the elastomeric surface during sterilization.

During the development of processes for equipment sterilization (e.g., SIP), it is important to ensure steam penetration throughout all parts that may affect the sterility of any product processed with that equipment. Product-delivery systems also may contain positions that are very difficult to penetrate during steam sterilization. As a general rule, the more complicated the geometry of equipment or a system, the more difficult steam penetration will be. Any equipment or system to be sterilized should be analyzed to define worst-case positions, and, wherever possible, the effect of sterilizing conditions should be tested on model systems simulating worst-case conditions as closely as possible.

Studies conducted to investigate the specific effect of sterilization conditions must be quantitative and the results must be seen relative to the effect obtained under reference conditions. It is scientifically correct and easier to conduct these studies under scale-down conditions in a laboratory using a precision autoclave that delivers heat exposure with square-wave characteristics.

Process and cycle validation. Not all steam-sterilization processes are the same. Whether a prevacuum process (also called a porous-load process), a hot-water-shower process, or a steam-air mixture process is used will depend on the properties of the product, load, or equipment to be sterilized. Depending on the configuration, there will be various considerations as to where worst-case positions are to be expected and what cycle is needed to achieve the expected sterilizing conditions in the worst-case position.

A scientifically ideal procedure would be to place bacterial endospores during cycle development at worst-case positions. The inactivation characteristics of the spores at that position could then be correlated to the inactivation of the same spore preparation achieved at a reference position. For such studies in theory, it is vital that the worst-case positions are well defined and bacterial endospores are correctly positioned without alteration of worst-case conditions. Most sterilization processes, however, are not easily amenable to such an analysis. Worst-case positions tend not to be freely accessible or easily inoculated with endospores, and it can be difficult to recover endospores from worst-case positions. In addition, such studies must be performed in production autoclaves or production equipment because various large-scale sterilization processes cannot be simulated with a biological indicator evaluator resistometer (BIER) vessel. Production autoclaves do not deliver heat with square-wave characteristics and, therefore, precise quantitative studies of D-values are not feasible therein.

To evaluate the biological effect of large-scale sterilization processes, test pieces with a defined number of microorganisms and defined resistance to saturated-steam exposure (D-value) should be exposed to actual or simulated worst-case conditions. Test pieces can be paper strips inoculated with resistant spores, units of inoculated product, vials with inoculated stoppers inserted, or pieces of tubing of a length simulating a worst-case exposure. The inactivation characteristics of the test pieces under reference conditions should be determined in a laboratory using a BIER-vessel. The requirements for manufacturing quality control of such test pieces (confectioned BI) are standardized in ISO 11138 (12).

Unfortunately, the definition of true worst-case positions at which BIs are to be exposed is not well understood by many users. The necessary availability of saturated steam or any possible influence of the microenvironment of spores are neglected. Standard paper strips or self-contained BIs are frequently used to simulate various worst-case conditions, and the coldest position measured in a temperature-mapping study of a load is often assumed to be the worst-case position, although this is not necessarily a valid assumption.

To correctly validate a sterilization cycle, it is necessary to use data gathered during product or process development to identify the conditions or positions where inactivation of spores is most difficult to achieve. These conditions should be simulated as closely as possible by suitable BIs. Whether this can be achieved by using a spore preparation on paper strips or a self-contained BI must be decided in each case. In many cases, a better simulation will be achieved with a customized BI that uses units or assembled parts of the product to be sterilized.

Cycle monitoring. Although using BIs as an additional means of monitoring autoclave cycles is recommended in hospitals, this practice is not common in the manufacture of pharmaceuticals or medical devices. Once a sterilization cycle has been validated for standardized defined loads, manufacturers typically rely on physical measurements for cycle control.

Again, the approach taken should be governed by what is intended with the process. In a hospital setting it is impossible to define reproducible loads and, thus, by convention a defined pack of tissue is considered the standard worst-case position. Defined loads are common practice in the manufacture of medicinal products. A pack of tissue would not be a good representation of a worst-case for typical pharmaceutical sterilization processes for the reasons discussed previously.

The situation may again be different in pharmaceutical laboratories engaged in development or quality work, where standardized sterilizer loads also are difficult to define. The less defined a sterilization process is with regard to worst-case positions, the higher the advantage of arriving at a conventional definition of a standard worst-case model. For production processes governed by the rules of good manufacturing practices (GMP), validation of load-specific cycles is generally required, and reliance on standard worst-case models is not accepted in most cases.

Properties and quality of bacterial endospore preparations and biological indicators

D-value of biological indicators. An important prerequisite for the suitability of endospore preparations is their D-value in correlation with the theoretical effectiveness of the process. When BIs are used to validate a sterilization cycle, the normal expectation is that all BIs exposed during the cycle are completely inactivated at the end of the exposure time. For quantitative determination of the sterilizing effect, it is necessary to apply reduced-exposure conditions that leave a fraction of viable endospores that can be quantified. The resistance of the endospore preparations used must be such that meaningful exposure times can be applied to obtain fraction-negative results. Graded fraction-negative conditions typically are used to evaluate the resistance of BIs.

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 121±1 °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 121±1 °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.

Conclusions

Models of sterility assurance. There are several models that can be applied to achieve sterility assurance. In the ISO approach, (mainly applied in hospitals and in the manufacture or treatment of medical devices) conventional worst-case devices are defined. For example, stacks of tissue of defined dimensions or hollow tubes of defined diameter and length are loaded with BIs or chemical indicators of saturated steam. These devices are placed at arbitrary positions in ill-defined sterilizer loads. When BIs are inactivated after a sterilization cycle, the cycle is considered effective.

The approach taken under GMP regulations is different. It is expected that each product-specific sterilization cycle is validated separately. The sterilizer load must be defined and the worst-case position must be characterized for each process. Sterilization effectiveness of the cycle should be correlated to the effect obtained at the true worst-case position and not to the effect obtained in a conventional worst-case device.

A third option seems to be favored by some regulators at present. The product to be sterilized is expected to be manufactured under extremely stringent conditions to ensure an extremely low presterilization bioburden. This is apparently seen as important because of the lack of confidence in the validation of the sterilizing effect obtained under worst-case conditions. The goal of this approach is to minimize the probability of survival at ill-defined worst-case conditions by minimizing the presterilization bioburden.

Definition of worst-case positions and worst-case conditions. The effectiveness of steam sterilization is influenced by a number of critical factors. Sterilization temperature and exposure time are the only factors that are considered in F-value or F0-value calculations of sterilization processes. It must be clear that such calculations are valid only when all other factors that influence the inactivation of microorganisms are duly considered. Steam quality is a critical factor in all cases in which steam comes in direct contact with the product or surface to be sterilized. Steam quality may be of minor significance where steam is used only as a means of heat transfer and where heat exchange is achieved rapidly by conduction or radiation.

The worst-case position in a sterilizer load is where the sum of all the influences on microorganisms, including the effect of the product or the influences of the microenvironment results in minimal inactivation. The conditions achieved at that worst-case position are the worst-case conditions for the sterilization process.

Worst-case positions can be determined only in studies using bacterial endospores during product and process development because the worst-case positions are difficult to predict. The worst-case conditions should be simulated in BI studies as closely as possible and the sterilizer conditions needed to achieve the required effect therein should be reflected in the parameters to be measured when the sterilization process is monitored.

Overkill cycles. The term overkill cycles for highly effective sterilization cycles is misleading and should be abandoned. Following the USP definition, the Ph.Eur. standard cycle for steam sterilization is an overkill cycle. It is sufficient to inactivate 15-log scales of a resistant microorganism with a D-value of 1 min. If the D-value of the BI is 1.5 min (as defined in Ph.Eur.), then the inactivation is only 10 logs, which means that it is just sufficient to deliver the kill time for a BI with 106 viable spores/unit. If the area between the stopper and the glass wall of a vial is taken as the worst-case position, then the cycle might not even kill 6 logs of endospores of the most resistant environmental isolate, and the cycle may qualify for a bioburden-oriented cycle at best.

A sterilization cycle in of itself cannot be considered an overkill cycle unless the effect is related to a given situation in which a given maximum number of organisms of a given maximum resistance under defined worst-case conditions is considered.

A risk-based approach. Worst-case positions of loads or equipment to be sterilized and the worst-case conditions achieved therein must be specified for each sterilization cycle. Because these are the conditions in which the least biological effect is achieved, quantitative studies on inoculated bacterial endospores are needed to investigate and determine the minimal lethal effect achieved by a sterilization cycle.

Such studies are difficult because many parameters may influence results, and very little work has been done to develop control procedures to verify the quality of marketed or self-grown spore suspensions or to standardize the procedures for the inoculation of product or equipment, the exposure to sterilization conditions, and the recovery of survivors.

Once the effect of a sterilization process at the worst-case position is known, a sterilization cycle can be defined in consideration of the heat sensitivity of the product, the expected bioburden, and the necessary biological effectiveness to be achieved. It may be that a process that is considered an overkill process in most parts needs special precautions to reduce the bioburden at worst-case positions.

Test pieces that simulate worst-case positions (e.g., vials inoculated between the stopper and the glass) may then be used to verify that the sterilization processes used in the production of pharmaceuticals correctly deliver the conditions needed to achieve the necessary sterilizing effect. Such customized test pieces are product and process oriented but otherwise similar to the conventional worst-case devices used in the ISO approach. Whether commercially available BIs on carriers are suitable to simulate worst-case conditions must be decided for each specific case.

A process characterized and validated with such an approach would then be routinely monitored by physical tests, and the biological effectiveness could be deduced from the measured physical parameters.

Klaus Haberer, PhD,* is the managing director and Korinna Vreden is the head of laboratory, both at Compliance Advice and Services in Microbiology GmbH, Robert-Perthel-Str. 49, D-50739 Cologne, Germany, tel. +49 (0) 221 957457 0, fax +49 (0) 221 957457 25, k.haberer@compliance-asim.de

*To whom all correspondence should be addressed.

Keywords: sterilization processes, biological indicators, steam sterilization

References

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14. European Pharmacopoeia, 5.7th ed. (Council of Europe, Strasbourg, 2007).

15. The United States Pharmacopeial Convention, Monograph Section, United States Pharmacopeia 30, CD edition (2007).