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The Top 8 Most Asked Questions about Microbial In-Use Studies

According to Dr. John Metcalfe, “pharmaceutical companies go to great lengths to manufacture a drug product which is sterile, whether by terminal sterilization or aseptic processing. Equally important to the microbiological quality of the product at the time of product release is its microbiological quality at the time of patient administration.” This microbial in-use studies Q & A with microbiological expert, Brent Harlow, will address the most common questions asked about these studies.

What is a microbial in-use study and why is this testing necessary?
A microbial in-use study is intended to evaluate the growth of low-level microorganisms inoculated into the drug product over the prescribed hold time. The growth represents inadvertent contamination during rehydration or dilution of the product prior to administration. These studies are performed to determine if the product will support microbial growth and/or proliferate that growth in the event of inadvertent microbial contamination during the holding period prior to patient administration.

Do I need to perform a microbial in-use study on my drug product?
Yes, if the drug product does not contain any antimicrobial preservatives and is rehydrated or diluted at the hospital/clinic and is not administered immediately (typically defined as held for more than four hours) after penetration of the container closure system. If these conditions are met, the US Food and Drug Administration (FDA) expects a microbial in-use study to be performed.

What chapter of the compendia can I find information on this testing?
Microbial in-use testing is not currently explained in any compendia. The basis of the study is described in an industry journal authored by Dr. John Metcalfe of the FDA.

Additionally, International Conference of Harmonisation (ICH) guidance documents reference this testing. ICH Q8 Pharmaceutical Development states the following in Microbial Attributes, Section 2.5 (2): “where relevant, microbial challenge testing under testing conditions that, as far as possible, simulate patient use should be performed during development and documented in this section.”

How does in-use testing differ from Antimicrobial Effectiveness Testing (AET) in USP <51>?
United States Pharmacopeia (USP) <51> provides guidance for testing multi-use drug products that contain antimicrobial preservatives that are added to inhibit any growth caused by inadvertent contamination during use. AET testing is performed to demonstrate the capabilities of the preservative containing drug product to kill off high levels of introduced challenge microorganisms.

A microbial in-use study is performed on drug products that require rehydration or dilution prior to being administered and do not contain a preservative. A low level of challenge microorganism is introduced to the diluted product so as to simulate inadvertent contamination during preparation in the clinic prior to administration. Testing is then performed over several time points to determine how the introduced microorganism reacts with diluted product.

At what point in the development of my drug product should microbial in-use testing be performed?
The final formulation of the drug product, as well as proposed product hold times, storage conditions, and admixture diluents should all be established prior to performing microbial in-use testing.

What should the testing parameters be for my drug product?
Testing will need to be performed for each concentration, admixture diluent, and storage condition (i.e. refrigeration and or room temperature). The length of the study should be two to three times the acceptable length at which the diluted drug product can be used per the product insert. Microbial in-use testing uses the five challenge microorganisms listed in USP <51>, and sometimes additional environmental type challenge microorganisms are recommended and utilized.

What are the acceptance criteria for this test?
Prior to initiating the in-use study, the method for recovery of the challenge microorganisms must be shown to be suitable.

For the in-use study, the starting inoculum level for each challenge microorganism when measured at time point zero should be not more than 100 CFU/mL of diluted product sample. Also, any increase of the challenge microorganism population in the diluted product samples greater than 0.5 log10 throughout the duration of the test will be evaluated on a case by case basis

Do generic drugs need this testing performed as well?
Yes, the FDA expects all drugs that fit the criteria explained above to undergo this testing. This is because microbial in-use testing may not have been performed on the reference listed drug. If the reference listed drug has not been previously challenged, the FDA may request comparison studies between the generic drug product and the reference listed drug product.

John Metcalfe, P. (n.d.). Microbiological Quality of Drug Products after Penetration of the Container System for Dose Preparation Prior to Patient Administration. American Pharmaceutical Review.

About the Author
Brent Harlow has experience performing microbiological testing in the pharmaceutical industry since 2009. He joined Alcami in 2014 and has considerable experience in performing validation and suitability work for Microbial Limits Testing Antimicrobial Effectiveness Testing and Sterility Testing and is the subject matter expert for Microbial Genetic Identification testing and Microbial In-Use Studies. Brent has a Bachelor of Science degree in Biology from the Ohio State University.