Originally Published MDDI October 2002

STERILIZATION VALIDATION

By its nature, bioburden testing is an inexact science. The degree of inaccuracy in the testing procedure can be quantified, however, using bioburden recovery tests.

Trabue Bryans and Karen Alexander

Bioburden testing serves a variety of purposes, and the relevance of the data can range from relatively insignificant to extremely critical. Bioburden tests can be used for general tracking or trending, for accepting incoming materials, for evaluating changes, or for establishing sterilization processes. A bioburden test alone may not tell the whole story, however. Because it involves the determination of several types and levels of viable organisms that can be located on a variety of materials or components, bioburden testing is by its nature a less-than-exact science.

Most bioburden tests involve the removal, culture, and enumeration of viable organisms—three general steps that all have a degree of variability. Removal may not be complete, depending on the material being tested; culture may not be all encompassing, depending on the media and incubation conditions; and even enumeration can vary, depending on the technician. Thus, the collective variability of these steps can result in bioburden data that may be less than 100% accurate.

The degree of inaccuracy in a given test procedure can be quantified by a bioburden recovery test. The recovery test simply measures the ability of a specific bioburden procedure to detect microorganisms that are either naturally present on a product or introduced artificially.

BIOBURDEN TEST DESIGN

A bioburden procedure is typically designed by selecting appropriate parameters for the type of product to be tested and the level and types of organisms expected to be present on that product. A particular bioburden procedure may result in lower or higher recovery efficiency, based simply on the choice of parameters. Some of the parameters to be considered are:

The choice of parameters for the test design must take into account the test sample material, which can have a significant impact on the recovery of organisms. For example, the same test parameters would most likely not be chosen for a liquid injectable and a patient drape, simply because of the material differences. Furthermore, the choice of parameters should consider the expected types and levels of organisms. A bioburden procedure designed for an ultraclean pharmaceutical would not be optimum for a cotton surgical dressing. Lower levels of organisms require different extract-handling techniques (for example, procedures involving filtration versus those involving plating). And, of course, the types of organisms expected to be present on a particular item affect the selection of media type and incubation conditions.

RECOVERY TESTING TERMS

Once a particular bioburden procedure has been designed for a specific product, the procedure is evaluated for its overall ability to recover organisms. This assessment can be called several things—a bioburden recovery test, a recovery efficiency study, a bioburden validation—but it is most often simply called a validation. What then is the most appropriate classification or term for the recovery efficiency test? Title 21, part 820.3 of the Code of Federal Regulations and other FDA documents provide a whole list of candidates—validation, process validation, verification, calibration, calibration verification, and qualification (see the sidebar on page 75 for their specific definitions)—but which one of these terms really describes what is being accomplished by performing a bioburden recovery efficiency test?

The answer is complicated and depends on the test performed. If it is truly a validation or verification, then the recovery results from a particular bioburden procedure must meet predetermined specifications or requirements. On the other hand, if the recovery test is called a calibration, then comparison to a standard is expected in order to address accuracy variation. If the recovery efficiency test is a qualification, however, then it is essentially an assessment of a process to ensure the applicability or fitness of that process for use.

Of the terms listed in the sidebar, the definition for the term qualification seems to come the closest to describing the true purpose of recovery testing. Nonetheless, it is important to be aware that many terms can be associated with and correctly used for this same test, and that the term used might be contingent on the purpose or application of the test.

TEST APPLICATIONS

There are three reasons to conduct a bioburden recovery test: to evaluate the test procedure in order to improve recovery, to assess antimicrobial qualities of the product against certain organisms, or merely to establish a recovery factor for adjusting future bioburden data. Depending on the purpose and application of the recovery test, there may be a target recovery efficiency one is trying to achieve, or it may simply be that the recovery efficiency needs to be determined.

In the medical device industry, the products tested for recovery are generally solid items that can be made up of diverse materials. Such products have a broad range of bioburden levels, possess many environmentally associated organisms, and are unlikely to be antimicrobial or contain preservatives. These factors mean that recovery tests must be designed to physically extract organisms from material surfaces and difficult sites, to detect a large variety of natural organisms, and to adapt to diverse bioburden levels.

BIOBURDEN RECOVERY TESTING DOCUMENTS

Unfortunately, there are very few standards or guidelines available that address bioburden recovery testing. Nonetheless, each of these guidelines tends to be more applicable to either pharmaceutical industry products or the medical device industry and its products. A summary of the guidelines appears in Table I.

United States Pharmacopeia and PDA Technical Report. The methods outlined in the United States Pharmacopeia (USP) and Parenteral Drug Association (PDA) documents listed in Table I are intended for use with products that many times are, of themselves, antimicrobial in nature, or that contain inhibitory substances or preservatives. Rather than determining how effectively the bioburden technique recovers only the natural organisms from a product, these recovery methods are used mainly to ensure that the antimicrobial or preservative is effectively neutralized so selected organisms can be detected.

For both the USP and PDA methods, the use of low levels of organisms, such as <100 colony-forming units (CFUs), are indicated. The USP identifies five specific organisms to be used as inoculums, while the PDA report indicates that the organisms chosen should be representative of those likely to be detected by the bioburden test method. Both the PDA and USP guidelines have a target percent recovery, indicating that the method should be modified until that target can be met for the organisms used. The term bioburden recovery validation is more appropriate for this application because there are predetermined specifications that need to be met.

ISO 11737-1 and EN 1174-3. The methods outlined in the International Organization for Standardization (ISO) and Community of European Nations (EN) documents are virtually identical; in each case, they are specifically intended for use with medical devices or their components. Simply put, the purpose of these methods is to determine how effective the bioburden procedure is at recovering the natural organisms from a product.

The two recovery efficiency tests cited in the ISO and EN documents are the inoculation method and the repetitive method. These techniques are very different in their methodology, application, advantages, and disadvantages. The general guidance is to choose the inoculation method for products with very low bioburden levels or for products with potential antimicrobial properties, and to choose the repetitive method for products with typical bioburden levels or to determine the thoroughness of a method in recovering natural bioburden.

Inoculation Method for Medical Devices. The inoculation method is exactly what the term indicates—product samples are inoculated with a selected organism at a specified level, and the bioburden test designed for that product is run to determine what amount (or percent) of the inoculum is recovered. The inoculum used for medical device applications is typically Bacillus spores. They are used for two reasons: one, aerobic spore formers are a common isolate from medical devices; two, adherence properties of organisms on various materials—as opposed to antimicrobial properties—are a significant factor in the purpose of this test. The level of the inoculum can vary widely but should be appropriate for the product being tested. For example, the inoculum level for the fluid path of an intravenous set should be relatively low, such as 10² CFUs, whereas the inoculum level for a woven product might be as high as 10⁵ CFUs.

The inoculation method is quick, inexpensive, and easy to perform. It is certainly applicable in cases where antimicrobial properties are known or suspected, or in cases where the product actually dissolves or disintegrates, making a repetitive test impossible. But because this method uses an artificial inoculum that is not always similar in form or adherence properties to that of the natural organisms, recovery may tend to be somewhat artificial compared to that of the naturally occurring bioburden. The results between replicate samples are usually consistent, however, because the level of the inoculum is controlled.

Repetitive Method for Medical Devices. The repetitive method for medical devices was known in the past as the exhaustive method. As the term repetitive indicates, a bioburden test is run repeatedly on an individual sample to determine what additional organisms can be recovered in the repetitions. The assumption is that a single extraction does not always recover 100% of the natural bioburden; therefore, repetitions of the extraction on the same sample will recover what might have been left on the product from the previous test. Inoculation of the product is not applicable for this purpose because the object of the test is to recover the natural bioburden organisms as they exist on the materials of the product, in terms of location, adherence, age, etc.

The repetitive method is relatively labor intensive, since it consists of running multiple bioburden tests per sample. It is therefore more complex and more expensive to perform than the inoculation method. The results between replicate samples are not typically consistent. This is mainly because the samples themselves usually do not possess consistent initial bioburden populations—in either numbers or types of organisms. Selection of the appropriate parameters in the test design is much more critical for the repetitive method, since the initial population is not only variable but often unknown. The number of repetitions to be run on a sample can range from three to 10 or more, and must be determined based on experience. A comparison of inoculation and repetitive test properties for medical devices is shown in Table II.

USE OF RECOVERY DATA

A recovery test will yield a percent recovery or a correction factor (the ISO term). Bioburden counts that have not been adjusted for recovery may be called microbial counts, viable counts, presterilization counts, or a number of other terms. Once a count is adjusted for recovery efficiency, however, the term should be definitive. Bioburden estimate is the defined ISO term for adjusted bioburden counts related to medical devices.

In the medical device arena, a recovery efficiency test is viewed as a qualification because it is essentially an assessment of process applicability. In most cases, no predetermined specifications apply because of the diversity of materials and configurations that can be encountered. Naturally, it is possible to alter recovery efficiency somewhat by varying certain test parameters, but for many medical devices, a bioburden procedure cannot be forced to achieve a desired recovery efficiency.

The user decides whether or not a correction factor will be applied to bioburden data based on the application of the data. If one is comparing bioburden results for tracking, trending, corrective action, etc., the numbers that are compared should reflect either all unadjusted numbers or all adjusted numbers. If bioburden data are used to establish critical processes—such as sterilization—using the bioburden estimate, although not required in all cases, is the most logical and conservative approach.

A correction factor is not automatically transferable from one product to another, from one bioburden procedure to another, or from one lab to another. The correction factor applies specifically to the bioburden procedure designed for a particular product, and cannot be assumed to be applicable if changes are made to the procedure or the product. All changes should be evaluated to determine if the recovery test should be repeated.

A FINAL WORD

Bioburden testing is a tool for establishing and evaluating many aspects of a manufacturing process. For the bioburden data to be valid, there must be some measure of the ability of the test procedure to detect the organisms that are or may be present. Recovery testing provides this measure and, regardless of the method used or the application of the results, is necessary for a true understanding of a product's bioburden.

BIBLIOGRAPHY

1. ANSI/AMMI/ISO 11737-1, Sterilization of Medical Devices—Microbiological methods—Part 1: Estimation of Population of Microorganisms on Product (Geneva, Switzerland: 150, 1995).

2. EN 1174-1, Sterilization of Medical Devices—Estimation of the Population of Microorganisms on Product. Part 1: Requirements (Community of European Nations, 1996).

3. EN 1174-3, Sterilization of Medical Devices—Estimation of the Population of Microorganisms on Product. Part 3. Guide to the Methods for Validation of Microbiological Techniques (Community of European Nations, 1996).

4. Parenteral Drug Association, Technical Report No. 21, Bioburden Recovery Validation, vol. 44, no. S3 (PDA, 1990).

5. Quality System Regulation, Code of Federal Regulations, 21 CFR 820 (1996).

6. United States Pharmacopeia XXV, Validation of Microbial Recovery from Pharmacopeial Articles (U.S. Pharmaceutical Convention), 2259–2261.

Trabue Bryans is president of Atlanta operations at AppTec Laboratory Services. Karen Alexander is the company's director of microbiology.

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