USP Microbial Examination of Non-Sterile Products

A road map to the world of non-sterile testing regulations

By James E.T. Gebo, technical representative, Laboratory Services, and Kimberly Kitch, laboratory technician, Microtest Laboratories Inc.

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When a product is submitted for Microbiological Examination of Non-Sterile Products, USP <61> and <62>, the examination process is initiated with many steps being performed concurrently. The microorganisms that are used for the assay are cultivated and their populations are determined; this process is accomplished within a couple of days from start to finish. The current USP guidelines emphasize the need to use fresh cultures; therefore the microorganisms are grown and used within 24 hours of harvesting.
The required media for testing is procured and the quality of the media is assessed. To accomplish this, growth promotion tests, inhibitive properties tests and indicative properties tests are all performed on every new batch of media used. This ensures that each batch of media is capable of supporting or inhibiting growth of the microorganisms.

Stock sample mixtures are prepared using aseptic practices in a Class 100 Laminar Flow Hood or a Biosafety cabinet to avoid contamination of endogenous organisms. For microbial enumeration, USP <61>, appropriate volumes of the sample preparation are plated to Soybean Casein Digest Agar and Sabouraud Dextrose Agar.

USP <62> outlines methods used to encourage the growth of existing microbes present in the product that could go unnoticed without enhancing the organism’s growth. Therefore, the sample and suitable portions of the prepared sample stock solution are transferred to separate, appropriate mediums to begin the enrichment phase of the test. These mediums include, but are not limited to Soybean Casein Digest Broth, Sabouraud Dextrose Broth, or Reinforced Medium for Clostridia.

The initial sample-broth mixtures used for enrichment are then sequentially subcultured to selective media one or two additional times before final results are obtained and documented. Although the media used is relatively selective to the organism screened for, a positive result is confirmed using microbial identification methods.

If the product has not undergone suitability testing, then each of the challenge organisms are combined with the sample stock preparation to validate both USP test methods. Suitability Testing validates that the microorganisms will grow in the presence of the product. A satisfactory result indicates that the product does not inhibit growth of the challenge organism and the methods employed for testing have been appropriately designed.

The USP requires that the testing method used is proven suitable for its intended purpose. The primary purpose of suitability testing is to establish that the product being tested does not have any adverse effects on the test, specifically inhibition of the control microorganisms. Some products may have antimicrobials or preservatives in them which could interfere with the detection of organisms. To demonstrate that there is no interference in the test, sample preparations are spiked with a known concentration of control microorganisms, incubated and the recovery of the microorganism is determined. If growth of each challenge microorganism is not inhibited by the product, then the method followed is deemed suitable.

There are two ways of showing system suitability differing primarily on the stage of product development. In the earlier stages of development, rather than performing the validation at this time, manufacturers may consider performing the suitability test with each new lot produced. The reasoning for this is because changes may need to be made during the development of a product which would, in turn, require re-validation. If a suitability test is performed after changes have been made to the product, it ensures that the test method used is still acceptable. This allows the manufacturer to maintain some level of flexibility while performing research and still certifies the quality of each product.

After a final product is manufactured and is ready for release to market, then a validation should be performed. According to USP <1227> a validation is performed on three independent lots of identical product. This should exhibit that the product does not interfere with the test method, while also showing that the method is repeatable between different lots of material. The benefit to this approach is that, after completion of the validation, the sample screening alone would be needed to release your product. A validation essentially locks the manufacturer into the final formulation of each product; therefore, if changes are made to the product, it would need to be re-validated.

Before submitting samples for testing, manufacturers should be prepared to answer several questions related to each product’s specific requirements. Although the answers to these questions are easily obtainable, an inability to provide them may cause delays in testing. Manufacturers should supply as much information about their product as possible to the testing laboratory to lessen delays in processing. Some of the information that should be provided will be discussed in the following paragraphs.

There are inherent differences in some products that require different means of handling. This is addressed in the USP by providing different methods for sample preparations based on which category the samples fall under. These categories are water soluble, non-fatty products insoluble in water, fatty products, fluids or solids in aerosol form and transdermal patches. Each of these categories has a different method to prepare the samples for testing. Therefore, it is important to know which categories each of your products belong to so they are handled properly during testing.

Manufacturers should also be prepared to provide the product’s Material Safety Data Sheet (MSDS). The MSDS will be helpful to the testing laboratory when they are preparing to test the product. It contains important safety information about the product, including handling procedures, which will determine how the product is managed when tested. Also included in the data is solubility information that will assist the laboratory in determining how to prepare your sample for testing. Many laboratories will require an MSDS up front to determine if the manufacturer’s product is something they can work with safely. Being able to provide this information as soon as possible will help prevent costly delays.

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