How To Perform A Microscopic Particle Count Test For Particulate Matter Within Injectables
What is particulate matter testing, and why is particulate matter testing important for injectable products?
When it comes to particulate matter testing for injectables, the American USP aligns its guidelines with both the European and Japanese Pharmacopeia. Particulate matter refers to undissolved particles (other than gas bubbles) that are present in injectable solutions. This particulate matter is not an intentional addition to the parenteral formulation. Just as unintentional microbes in injectables are avoided and regulated to prevent patient illness, unintentional, non-biological particles are regulated in injectable formulations to prevent unwanted toxicity, disease, or side effects. For those creating parenteral products for veterinary use, solutions used for irrigation alone, or radiopharmaceutical preparations, particle matter regulation requirements of USP 788 do not need to be met. If your injectable product uses a filter before patient administration, your injectable may be exempt from particulate matter guidelines, provided supporting scientific data justify such an exemption. Note that this article will use parenteral and injectable synonymously. Thus, a small-volume injection and a small-volume parenteral are synonymous terms.
Particulate matter can be detected two ways, a light-obscuration particle count test and a microscopic particle count test. Light-obscuration particle count testing is preferred for subvisible particle detection. However, parenteral preparations with reduced clarity or increased viscosity (such as emulsions, colloids, and liposomal formulations) should be assessed using microscopic particle count testing. Microscopic particle count testing is also useful for products that tend to produce air or gas bubbles during sampling. In some cases, both particulate matter test methods will be needed to confirm subvisible particle counts. And select injectable formulations may be difficult to assay via both traditional particulate matter testing methods. In these select cases, often when the viscosity of the test preparation is high, a quantitative dilution with an appropriate diluent may be made to decrease viscosity, as necessary, to allow the particulate analysis to be performed.
How is microscopic particle count testing performed?
Microscopic particle count testing uses a binocular microscope, a filter assembly for retaining particulate matter, and a membrane filter for sample examination. The microscope will have an ocular micrometer with a graticule (see Figure 1 reproduced from Figure 1 of USP 788 below). The ocular micrometer is calibrated using a stage micrometer certified by either a domestic or international standard institution. Additionally, two illuminators (one internal and one external) are needed for the microscope assembly.
Prior to use, all glassware and testing equipment are rinsed with particle-free water. Samples of particle-free water are tested following equipment set-up to assure that no more than 20 particles of 10 μm or greater are identified in particle-free controls in replicate. If more particles are identified, the testing equipment will be cleansed again and recalibrated before sample testing.
For testing, each sample’s contents are mixed by slow inversion. For small-volume Parenterals (less than 25 milliliters in volume), the contents of 10 or more units are combined in a cleaned container to obtain a volume not less than 25 milliliters (mL). If a powder for parenteral use is tested, the powder is reconstituted with particle-free water or an appropriate particle-free solvent to ensure test accuracy. Injectables with a volume of 25 mL or more do not need to be pooled and are tested individually. Overall, the number of samples (pooled or not) must be adequate to provide a statistically sound assessment.
After the injectable samples have been prepared, the filter holder is wetted with particle-free water. The total volume from an injectable sample or pooled sample is transferred to the filtration funnel. Then a vacuum is applied until the surface of the membrane filter is free of liquid. Next, the membrane filter is placed in a Petri dish and allowed to air-dry. After the membrane filter has dried, the entire membrane filter is scanned under the microscope. The total number of particles on the membrane filter that are equal to or greater than 10 μm and the number of particles that are equal to or greater than 25 μm are counted. The particle sizing is determined by estimating the equivalent diameter of the particle in comparison with the 10 μm and 25 μm reference circles on the graticule.
In microscopic particle count tests, amorphous, semiliquid, or other indistinct materials that have the appearance of a stain or discoloration on the membrane filter cannot be sized. These materials show a gelatinous or film-like appearance. If thick or film-like materials appear, test a sample of the solution by the light-obscuration particle count test.
Once particles have been counted, calculate the mean number of particles for the sample examined. For microscopic particle count tests, there are two test criteria. For sample preparations with a volume of more than 100 mL, the first test criteria apply. Samples with a volume of fewer than 100 mL use the second test criteria. In the first test criteria, the sample complies with the particle count test if the average number of particles present does not exceed 12 particles per mL equal to or greater than 10 μm and does not exceed 2 particles per mL equal to or greater than 25 μm.
In the second test criteria, the sample meets the testing criteria if the average number of particles present in the units tested does not exceed 3000 per container equal to or greater than 10 μm and does not exceed 300 per container equal to or greater than 25 μm.
Summary
Overall, particulate matter testing is an important microbiology test for injectables. Unintentional, non-biological particles are regulated in injectable formulations to prevent unwanted toxicity, illness, or side effects in patients following treatment. Two tests can be used for particulate matter testing, light-obscuration particle count testing and microscopic particle count testing. Microscopic particle count testing is used when preparations are too cloudy or viscous for light obscuration particle count methods. All in all, when developing your injectable product, ensure you choose a contract testing organization that can support you with appropriate microbiology testing for your unique medical device or product needs.
Ethide Labs is a contract testing organization that specializes in Microbiology Testing. Ethide Labs also offers Bioburden Testing, Environmental Monitoring, Sterilization Validations, Bacterial Endotoxin Testing, EO Residual Testing, Package Integrity Testing & Cytotoxicity Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <788> Particulate Matter In Injections, In Vitro. Rockville, MD, USA. 2021. (USPC <788>).
