Microbiological Testing
Please note that all microbiological testing requires product-specific validation, known as method suitability. As the name implies, method suitability provides evidence that the method used by Pharmetric on the product tested was sufficient to neutralize any antimicrobial properties of the drug substance or finished product being tested. Please contact Pharmetric Laboratory to have your method suitability tests scheduled today!
USP <71> Sterility Test
The sterility test is a test which assesses whether there are microbial contaminants in a product. There are two approaches to the sterility test: membrane filtration method and direct method, the former being the preferred method. The test assesses whether viable cells are present through a change in turbidity of growth media. Trained technicians monitor the growth media over a 14-18 day time period to assess whether the media are turbid from microbial contamination.
Pharmetric uses Sartorius Sterisart® sterility testing technology during membrane filtration.
Rapid Sterility Test using ATP Bioluminescence Method
A Rapid Microbiological Method (RMM) is a microbiological method which utilizes a biomarker to assess the key sterility test parameter, viability, and provide quality results in shorter timeframe when compared to the traditional method (e.g., 14-18 days for sterility test). Pharmetric completes rapid sterility tests using ATP bioluminescence technology, and the test results are provided in 5-7 days rather than 14-18 days. An additional benefit is that the results are objective, reported as Relative Light Units (RLU), indicative of the presence of ATP when results are elevated when compared to controls.
Pharmetric uses Charles River Celsis Advance II technology for ATP bioluminescence readings.
USP <51> Antimicrobial Effectiveness Testing
Preservatives are added in sterile multi-dose parenteral products to inhibit or kill the growth of microorganisms that may be accidentally introduced during multiple withdrawals of the product from its container. Examples of common preservatives used in injectables are benzyl alcohol, chlorbutanol, and methyl-propyl-parabens. How well these antimicrobial preservatives inhibit or kill microorganisms is evaluated during USP <51> Antimicrobial Effectiveness Testing.
Also known as the preservative effectiveness test, the test is commonly carried out during formulation development and stability testing of multi-dose product. The test procedures and acceptance criteria are described in the USP chapter. The testing is carried out over 28 days, not including incubation periods for the petri dishes.
USP <60> Microbial Examination of Nonsterile Products – Tests for Burkholderia Cepacia Complex
Burkholderia cepacia complex (BCC) is a group of gram-negative bacteria comprising more than 20 species which has been linked to multiple instances of opportunistic infections caused by contaminated purified water systems. Patients exposed to BCC contamination may be at increased risk for illness, particularly those who are immunocompromised.
USP <60> testing allows for the determination of the absence of BCC, which can be detected under the conditions described. The tests are designed to determine whether a substance or preparation complies with an established specification for microbiological quality and/or to evaluate whether products—especially those for inhalation use or aqueous preparations for oral, oromucosal, cutaneous, or nasal use—contain members of the BCC.
USP <61> Microbial Examination of Nonsterile Products – Microbial Enumeration Tests
USP <61> is a quality test carried out to ensure that drug substance or a finished drug product preparation complies with specific microbial count limits which are established a priori. Like the sterility test, the USP <61> test assesses whether there are microbial contaminants in a product. However, it differs from the sterility test in that the expectation is not complete absence of microorganisms. Rather, we count the microbial
colonies, known as Colony Forming Units (CFUs), and compare the results to a set limit. If the number of colonies is below the set limit, the product meets the specification set forth in the USP chapter.
Rapid Microbial Enumeration Test using ATP Bioluminescence Method
Many non-sterile products are functionally sterile due to the added preservatives. Functionally sterile non-sterile products are candidates for utilizing ATP bioluminescence as a rapid microbial enumeration test. If the product passes the ATP bioluminescence test, then the traditional microbial enumeration test is unnecessary. However, if the test fails the ATP bioluminescence test, then the traditional microbial enumeration test is required.
USP <62> Microbial Examination of Nonsterile Products – Tests for Specified Microorganisms
USP <62> tests allow for the determination of the absence of, or limited occurrence of, objectionable microorganisms that may be detected in the product, under the conditions of the test. The tests are designed to determine if a drug substance or finished non-sterile drug product complies with the specification of “absence of” for specific objectionable microorganism(s). The tests are carried out with growth media which select for the specific microorganism(s) in question.
Growth Promotion Testing of Growth Media
Growth promotion is a quality control test which assesses the ability of a specific batch of growth media can support growth of representative microorganisms defined in the compendia, and may include additional isolates of interest, such as environmental isolates. All media used at a cGMP facility should be go through growth promotion testing prior to use at the facility for tasks such as environmental monitoring. Growth promotion testing requirements apply to in-house and purchased culture media.
Disinfection Validation Studies
The effectiveness of antimicrobial agents used in disinfectants, sanitizers, and sporicidal agents is measured by its ability to kill microorganisms within a specified contact time. The accurate determination of antimicrobial effectiveness requires the complete and immediate inactivation or neutralization of the antimicrobial agent. Incomplete neutralization will lead to overestimation of the antimicrobial activity of the disinfectant, sanitizer, or sporicidal agent. Thus, disinfection validation studies are carried out in two stages.
The first stage requires validation of the neutralization step, and the second stage requires the use of small coupons of the surface material, and high concentrations of microorganisms dried onto the coupons. The ability of the microbial agent to kill microorganisms is assessed by comparing the microbial counts from coupons (containing dried concentrations of specific microbes) treated with the antimicrobial agent to coupons which were not treated. The counts are log10-transformed, and log reductions determined. Specific log10-reductions are established a prior.
Biological Indicators
A biological indicator (BI) is a well-characterized, often commercially available, microorganism preparation from a specific species resistant to steam or dry heat. BIs are commonly used to assess the effectiveness of a sterilization step of either finished product or materials and equipment utilized during the compounding process. The incubation and monitoring of the BIs should follow specific instruction from the manufacturer, and include control BIs tested to ensure the BIs would grow under the conditions of the test.
Media Fill – 14 days
A media fill test is a process simulation, and it is a microbiological test where the product is replaced with a sterile culture media, often soybean casein digest media (also known as trypticase soy broth, TSB). The purpose of the media fill test is to assess the sterility of an aseptic process, and media fill is also used to assess aseptic technique of specific compounding technician(s). All units compounded during the media fill are incubated at 20-25C and 30-35C minimally for 14-days. The vials pass the media fill if there is no evidence of microbial growth over the duration of the test, and the media meet growth promotion requirements.
