How To Test For Subvisible Particles In Therapeutic Protein Injections?
What is particulate matter testing, and why is particulate matter testing important for therapeutic protein products?
Particulate matter refers to undissolved particles (other than gas bubbles) that are present in solutions. This particulate matter is not an intentional addition to a therapeutic protein formulation, and parenteral products are to be free from particles that can be visually observed. Just as unintentional microbes in injectables are avoided and regulated to prevent patient illness, unintentional and non-biological particles are regulated in products to prevent unwanted toxicity, illness, or side effects. Therapeutic protein injections are products containing proteins or peptides. Particulate matter in therapeutic protein injections may be (a) truly foreign, or “extrinsic,” e.g., unexpected foreign material, such as cellulose; (b) “intrinsic” resulting from an unexpected addition into the product such as dust due to insufficient cleaning during manufacturing; and (c) “inherent,” such the protein or formulation components themselves. All these particle types are detected and counted for therapeutic protein injectables. The light-obscuration particle count test is the preferred method for therapeutic protein injection subvisible particle evaluation. However, microscopic particle count testing may be useful to verify that the particles being analyzed are primarily non-proteinaceous. Products that are used with a final filter during in-line administration are exempt from the particle count requirements, providing that scientific data are available to justify the exemption. The techniques for light obscuration and microscopic particle count testing for therapeutic protein injectables follow USP 788 guidelines. Additional information is detailed below regarding how each test particle test is performed and the particle count limits for therapeutic protein injections. Subvisible particle range, testing, and subvisible particle analysis are covered in this article.
How is light obscuration Subvisible Particle testing performed?
Light-obscuration subvisible particle analysis uses a liquid particle counter machine that, based on the principle of light scattering, allows for an automated determination of the subvisible particle range (size) in a sample and the number of particles according to size. The liquid particle counter is calibrated using dispersions of spherical particles in particle-free water. The liquid particle counter will have a sensor capable of detecting particles within the anticipated particle size range and particle count volume. For traditional tests detailed in USP 788, particles are of known sizes between 10 microns (μm) and 25 μm. For therapeutic proteins, subvisible particle range is often from 2 μm to 100 μm.
For injectable products, a single subvisible particle analysis requires 0.2–5.0 milliliters (mL) of product. Product samples are pooled to generate the appropriate testing volume if needed. If a product requires reconstruction, the reconstitution or dilution must be performed with the appropriate amount of specified solvent. Subtraction of the solvent particle count from the total particle count is not allowed. The reconstituted products must meet therapeutic protein particle count requirements with their respective solvents included. Under certain circumstances, such as with high-concentration products that could saturate the particle counter instrument sensor or high-viscosity products, a dilution of the product samples may perform to obtain reliable results. If dilution is needed, additional studies exploring the effect of dilution on particle aggregates or changes in the protein ratio to excipients should be performed.
Care should be taken to minimize shaking, inversion, sonication, and other stresses to the product samples to prevent the introduction of air bubbles. Any air bubbles in the samples will result in false-positive subvisible particle ranges. Air bubbles can be further eliminated by allowing the product to rest under ambient pressure or by applying a gentle vacuum to the samples. Samples should undergo a slow mixing (swirling the container by hand) before four aliquots of the product undergo subvisible particle analysis in the particle counter.
What are the particle count limits for therapeutic protein injections?
Once particles have been counted, acquire the number of particles over the selected size range, including particles equal to or greater than 10 and 25 μm. For infusions or injections that have a volume of less than or equal to 100 mL, the average number of particles present in the units tested should not exceed 6000 particles per container equal to or greater than 10 μm and not exceed 600 particles per container equal to or greater than 25 μm. For therapeutic protein injections/infusions greater than 100 mL, the average number of particles in the units tested should not exceed 25 particles per mL equal to or greater than 10 μm and should not exceed 3 particles per mL equal to or greater than 25 μm. Additionally, with product volumes greater than 100 mL, the total particle load should meet the same limits as the particle load for products less than or equal to 100mL. In other words, the total particle load should not exceed 6000 particles per container equal to or greater than 10 μm and should not exceed 600 particles per container equal to or greater than 25 μm. For products first reconstituted in less than 100 mL and then diluted for infusion in a volume greater than 100 mL, particle content should be assessed before and after dilution and evaluated based on their final volume.
How is microscopic particle count testing of therapeutic protein solutions performed?
The light obscuration method is the preferred method for therapeutic protein injections and parenteral infusions. For actual particle count testing, the light-obscuration method should be used. However, the microscopic method may be used to determine extrinsic and intrinsic particle types. Note that the microscopic particle count test results are not equivalent to those of the light-obscuration particle count test. Results are not comparable because of differences in interference of some protein particles due to their fragile or translucent nature. Thus, you cannot interchange these two methods for the determination of extrinsic and intrinsic particle types.
Suppose you do perform a microscopic particle count test. In that case, the microscopic particle count test is performed by evaluating particulate matter from solutions after the solutions have been filtered through a microporous membrane filter. The filter assembly will retain the particulate matter from the filtered sample. This particulate matter can then be examined under a microscope. Particulate matter samples are assessed under a microscope using a graticule. The graticule is used to size any particulate matter found in the samples.
Summary
Overall, particulate matter testing is an important microbiology test for therapeutic proteins. Unintentional, non-biological particles are regulated in protein therapeutic formulations to prevent unwanted toxicity, illness, or side effects in patients following treatment. Two tests are traditionally used for particulate matter testing: a light-obscuration particle count test and a microscopic particle count test. The light obscuration particle count method is to be used for all therapeutic protein particle count tests for accurate particle detection. Microscopic particle count testing should only be used to verify intrinsic and extrinsic particle types. All in all, when developing your therapeutic protein product, ensure you choose a contract testing organization that can support you with appropriate microbiology particle count testing for your unique product needs.
Ethide Labs is a contract testing organization specializing in Particulate Matter Testing and Microbiology Testing. Ethide Labs also offers EO Residual Testing, Sterility Testing, Cytotoxicity Testing, Bacterial Endotoxin Testing, Bioburden Testing, Package Integrity Testing & Environmental Monitoring services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <787> Subvisible Particulate Matter In Therapeutic Protein Injections, In Vitro. Rockville, MD, USA. 2021. (USPC <787>).
United States Pharmacopeial Convention. <788> Particulate Matter In Injections, In Vitro. Rockville, MD, USA. 2021. (USPC <788>)
