Carry out the test under conditions that limit ingress of particulate matter, preferably in a laminar-flow cabinet. The glassware and equipment used should be properly cleaned and handled to remain particle free. Care should be taken to minimize shaking and other stresses to the preparations (for liquids or reconstituted powders) and to prevent introduction of air bubbles into the preparation under examination. This is particularly important when preparations are combined or transferred to the container in which the determination will be carried out.

Glassware selected for the method should accommodate the volume of blank and sample aliquots, allow easy mixing, and be amenable to repeated cleaning used to prevent any significant contribution to particle counts. Typically, separate vessels are utilized and retained specifically for this test only. Cleaning and rinsing of the method glassware should be in close proximity to the counting area.

The work area and equipment should be separated from general lab traffic. A good supply of filtered water (to comply with Particle-Free Water) and support equipment should be maintained and available for the routine work. Refer to general information chapter 1788.2

Two types of system controls are recommended: a Blank Test and System Suitability Verification.

In order to check that (a) the environment is suitable for the test, (b) the glassware is properly cleaned, and (c) the water or suitable solvent that will be used is particle free (b and c constitute the “blank”), the following test is carried out. Determine the count in five aliquots of degassed particle-free water or suitable solvent, representative of the product test volume (but NLT 1 mL). If the number of particles 10 µm or greater in size exceeds 1 particle per mL for the combined aliquot volume, the precautions taken for the test are not sufficient. Repeat the preparatory steps until the environment, glassware, and water are suitable for the test determination. The Blank Test must be run initially each day (environment) and is recommended throughout the daily routine, especially between product families (procedural).

To verify system suitability, Particle-Free Water and the USP Particle Count RS or equivalent commercial standard aqueous suspension are tested from the same or similar type of container as product samples, are degassed in the same manner, and generally are handled like the test article. Ensure that the system can meet the Blank Test requirement. Determine the particulate matter in a sample of the USP Particle Count RS that corresponds to the product test volume, according to the method described below. Results from the Standard preparation should fall within the stated specifications of the Reference Standard.

Product samples are tested in the manner that most suitably represents delivery (e.g., expelled syringe contents). For parenterals that have a sufficient volume (i.e., volume large enough to facilitate testing), testing of individual units is often diagnostic. For parenteral products that do not have a sufficient volume, carefully and thoroughly mix each unit, then combine the contents of a suitable number of units in a separate container to obtain the volume required for a single test (generally 0.2–5.0 mL). If a dilution is performed, ensure that the blank container has sufficient volume for product and diluents and that few, if any, particles are introduced in the process. Open each unit cautiously, remove the sealing closure, and avoid contamination of the contents before sampling, diluting, or, if necessary, pooling.

When a product solvent is specified, e.g., for lyophilized solids or powders for parenteral use, the reconstitution or dilution must be performed with the appropriate amount of specified solvent. In this case, the solvent itself may be tested to ensure that it is not a significant source of particles. Subtraction of the solvent particle count from the total count is not allowed.

Eliminating gas bubbles is a key step, especially for proteinaceous products that readily entrain gas. Two methods are recommended: either allowing the product fluid to stand under ambient pressure or applying a gentle (e.g., 75 Torr) vacuum. Other methods may be used when demonstrated to be suitable. Sonication should be avoided.

Once the samples have been degassed, they must be remixed gently to suspend all particles by mixing the contents of the sample gently but thoroughly by an appropriate means, e.g., slow swirling of the container by hand. Inversion to mix the product fluid is not recommended at any time.

Immediately after mixing, withdraw NLT four aliquots, each of a volume appropriate for the instrument's capacity (generally 0.2–5.0 mL). Count the number of particles over the selected size range, including particles equal to or greater than 10 and 25 µm. Disregard the result obtained for the first aliquot, and calculate the mean number of particles at each size range for the remaining aliquots of the preparation being tested. A tare volume also can be applied to control sampling that should be representative of the sensor dynamic range and needle volume.

Dilution is allowed as long as the diluent and methods are demonstrated to be appropriate and the smallest level of dilution that allows for reproducible testing is used. Under certain circumstances dilution of samples may be required to obtain reliable results. This may be the case with high-concentration products and/or high-viscosity products that cannot be drawn into the instrument properly; products that cause saturation of the instrument sensor due to high counts, especially in the smaller than 10-µm size ranges; and cases where the high concentration results in very small differences in refractive index between the solution and the protein aggregates, etc. In situations where sample dilution is conducted, the underlying rationale for the dilution as well as the suitability of the selected dilution scheme including choice of diluent must be demonstrated. Suitability of the dilution scheme and of the diluent may be assessed by demonstrating consistency of results over a dilution range. Bridging studies with and without dilution at size ranges intended for particle counting should be considered. Studies exploring the ability of dilution to decrease the number of aggregates, or to increase particles due to changes in the ratio of protein to excipients, should also be performed. An orthogonal technique may be used to justify the suitability of the method by verifying the impact of dilution.

The values below are derived historically from general chapters 788 and 789. If specifications are different from those stated below, they will be indicated in an individual monograph where available.

For parenteral products that are therapeutic protein injections for infusion or injection supplied in containers with a nominal content of less than or equal to 100 mL:

For therapeutic protein injections supplied in containers with a nominal content of more than 100 mL, and parenteral infusion preparations or injections with a nominal content of more than 100 mL:

Products that are used with a final filter during administration (in-line) are exempt from these requirements, providing that scientific data are available to justify the exemption. However, filtrates are expected to comply with the guideline. For products supplied or first reconstituted in <100 mL, and then diluted for infusion in a volume >100 mL, particle content should be assessed both before and after dilution and evaluated based on their final volume.