Cleanroom requirements for liquid particle testing
Contaminating particles in injectable or infusion drug therapies can be microbiological resulting in infection, allergic reaction or, in extreme cases, anaphylactic shock. With patient safety on the line, Good Manufacturing Practice (GMP) guidelines require injections and infusions be produced in a controlled environment to contain any risk of product contamination due to airborne particulates.
This same risk exists when testing for particulate levels in the final product. USP chapters <787> recommend liquid particle testing be done in a controlled environment to reduce the risk of contamination during sample testing and help prevent “false positives” that can happen should particles be introduced into the drug product during testing phase. Protecting the sample during testing is critical to avoiding a costly contamination event that may prevent release of the entire batch. More on Light Obscuration particle testing for USP <787>.
Classifying particulate matter
Overall, any semi-solid to solid material may be counted and considered hazardous upon identification. Examples include air, liquid, gel, singular solid, aggregate, agglomerate, drug solid, salt, polymorph, lubricant and plasticizer.
Common types of particulate matter are extrinsic, intrinsic and inherent.
USP chapter <787> allows for the presence of insoluble protein particles as an intrinsic part of protein-based therapies, labeling them as inherent particles. The standard provides a clear distinction between inherent protein particles and ‘contaminating’ particles that should not be present. To ensure the final product complies with allowable particle limits, it is essential to understand the identification, origin and characterization of particulate matter.
USP <787> requires manufacturers to test for particles at ≥10 microns and ≥25 microns. Inherent protein particles are typically found in the sub-micron or nanometer size range and therefore go undetected by the tests. However, proteins tend to “fold” or aggregate over time forming particles large enough to impact a pass/fail result. Protein aggregation is undesirable as it causes the therapeutic part of the protein to become occluded, making it less effective for patients.
Sample preparation for protein-based formulation
- Draw a light vacuum on the material to eliminate entrained gas
- Avoid sonication when working with proteins
- Inverting to mix product is not recommended at any time
- Gently remix; slow swirl of container by hand
- Dilution must be carefully done to avoid disassociation and/or aggregation
- Reconstitution must be performed with proper amount of specific solvent.
- Solvent itself should be tested to ensure it’s not a source of particles. NOTE: Subtracting the diluent count from total count is not allowed
USP <787> and light obscuration system preparation
When working with a light obscuration counter, run a couple of test solutions to verify the system is functioning correctly. Consider doing these health checks at shift or operator changes or other defined intervals.
- Blank Test 1 (‘particle free’ water)
- System Suitability for test of materials
- Particle Count Reference Standard or USP PCRS (material of known quantity at specific size)
- or suitable alternate
Testing and sample volumes
USP <787> also addresses the volume of the injections, which are typically smaller than those of small molecule drug products. The USP <788> per batch sample volume requirement (25.0 mL) poses a challenging and costly test mandate for protein-based therapies. With <787>, manufacturers are allowed to test with 0.2 mL and 5.0 mL sample volumes provided a body of evidence is provided and the sample tested is a good statistical representation of the manufactured batch.
Total particle content: Limited to 6,000 particles
Particle-size thresholds: Similar limits USP <787>/<788> of ≥10 μm & 600 particles ≥ 25 μm (all dose formats)
USP <787> recommends populations below 10 μm be routinely monitored to characterize the stability of the final drug formulation.
USP <787> encourages manufacturers of protein-based therapies to perform particle testing with a light obscuration particle counter and orthogonal techniques2 to better reveal the structure and stability of the biologic over time. Although the primary technique, not all preparations can be examined for subvisible particles by light obscuration.
2An orthogonal method can be employed to evaluate/validate the primary method. An example: two methods may be used to investigate protein aggregate – method 1: size-exclusion chromatograph / orthogonal method 2: analytical ultracentrifugation.