What Are The Current Issues With Electronic Particle Counters?
What is particulate matter testing, and what test methods are available?
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 parenteral product, 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.
Particulate matter can be detected in two ways, a light-obscuration particle count test and a microscopic particle count test. Light-obscuration particle count testing uses an electronic particle counter and is the preferred method for subvisible particle detection. However, using light-obscuration particle count testing, traditional parenteral preparations with reduced clarity or increased viscosity (such as emulsions, colloids, and liposomal formulations) are difficult to assess. In cases 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 light obscuration testing performed?
Light-obscuration particulate matter testing uses an electronic particle counter machine that uses the principle of light scattering to determine the size of particles in a sample and the number of particles in a sample according to size. The electronic particle counter is calibrated using dispersions of spherical particles in particle-free water. The electronic particle counter has a sensor capable of detecting particles within the anticipated particle size range and particle count volume for each parenteral product. For traditional light-obscuration particle count tests detailed in USP 788, particles equal to or greater than 10 μm and 25 μm are assessed from four aliquots (no less than 5 milliliters each) of the parenteral product being tested. Care is taken not to introduce air bubbles into examined preparations, and samples can be pooled to create the desired testing volume when needed.
Electronic Particle Counter Issues
Light-obscuration counters regularly deliver inaccurate measurements of both particle number and particle size depending on the attributes of the parenteral product assessed and the particles present in the product. Inaccurate measurements are due to the principle on which light-obscuration counters operate and not because of a design flaw or engineering defect. Particle counts from electric particle counters occur from interactions between a moving particle and an intense light beam in the counter’s sensor. Whenever a particle crosses the light beam, the light intensity that reaches the light sensor (photodiode) is diminished. An amplified voltage pulse is created in response to the diminished light intensity. The amplitude of the voltage pulse is approximately proportional to the particle size. When there is a low particle density, large, spherical (>5 μm) particles can be detected with reasonable accuracy. However, inaccuracies occur when particle density is high, and there are many non-spherical (>5 μm) particles. Other issues with electronic particle counters are listed below.
#1: Solution Flow Rates
The flow rate of the liquid tested will significantly affect particle count accuracy. Slower flow rates (such as occur with highly viscous products) result in longer pulse durations. Longer pulse durations increase the probability of electronic noise on the count pulse, leading to an inaccurate increase in apparent particle size. Flow rates that are too fast can result in the under-sizing of particles.
#2: Non-Spherically Shaped Particles
Non-spherical particles produce significant errors in the sizing accuracy of electronic particle counters. Light obscuration measurements are based on the light obscured by the particle according to its orientation when it enters the counter’s view. Thus, if the particle isn’t spherical, its size will be based on a single view of the particle and not the entire particle itself, leading to inaccuracies. Typically, the size recorded will be less than the true particle size. For example, let’s assume that a rectangular particle has the dimensions of 1 μm by 2 μm by 4 μm. If the particle counter only captures the 1 μm by 2 μm plane of the particle, the size will be assumed to be 1 μm by 2 μm by 1.5 μm, which is smaller than the 1 μm by 2 μm by 4 μm size of the actual particle. Differences between the refractive index of the particle and the refractive index of the product solution containing the particle can also result in inaccuracies. Typically differences in particle and solution refractive indexes will increase the apparent size of the particle. For example, a particle in water will have a greater refractive index than the same particle in a solution of dextrose. Thus, the light obscuration sensor will measure any particles in water to have a greater size and a greater particle number than the same particles in the concentrated dextrose solution.
#3: Calibration Errors
Calibration errors may occur because calibration is done with mono-sized spherical latex particles. These mono-shaped particles provide a narrow range for detection and introduce calibration bias when measuring the actual samples with particles of unknown sizes and shapes.
The calibration error introduced nearly always results in particle measurements being smaller than they should be. However, this calibration bias is necessary as electronic counters cannot be calibrated with non-spherical particles due to their nonuniformity, dispersal difficulties, and differences in optical properties.
#4: Coincidence Effects
A coincidence effect occurs when two or more particles are incorrectly counted as a single larger particle. This problem can be detected by comparing dilutions of the same sample. If an increase in total particle count occurs with the diluted sample, coincidence counts are probably the cause. Coincidence effects are difficult to remove. The best way to confirm particle count and size is through microscopic analysis.
#5: Immiscible Fluids
Immiscible liquids in a parenteral production formulation can be incorrectly counted as subvisible particles. Silicone is the primary source of fluid immiscibility. Immiscible silicone often creates tiny particles, (1 μm) microdroplets. Other immiscible liquids detected and counted as particles by electronic particle counters include leachable agents from packaging (plasticizers), oils from manufacturing equipment, and lower polarity impurities from the active pharmaceutical ingredient. Inorganic silica, silicone fragments, and extracts from process tubing may also be present in the formulation and cause positive particle count data. Only large volumes of silicone microdroplets produce significant errors in particle measurement.
#6: Air Bubbles
Air bubbles are also counted as particles. The USP states gas bubbles can be eliminated by allowing the solution “to stand for 2 minutes or sonicating.” However, these methods do not remove all microscopic bubbles and don’t reduce the dissolved air content in a product solution.
#7: Product Storage
Particle contents vary considerably between the date of initial product filling and a later date when the product containers are tested. Particle content varies depending on product storage because product storage causes particle agglomeration. Thus, freshly prepared solutions give the most accurate and stable particle counts. Mechanical agitation breaks up the agglomerates, but the counts are not the same as the original count that would be obtained if particle count testing was performed on the day of product filling. Agitation or shaking will increase the number of particles in a parenteral solution. Agitation by 20 hand inversions, as required by the USP procedure, will remove any particulate matter from the surface of the container, thus increasing the total number of particles greater than 1 μm. However, the relative size distribution of particles should not be altered significantly with 20 hand inversions.
Summary
Overall, particulate matter testing is a vital microbiology test for parenteral products. Unintentional, non-biological particles are regulated in parenteral product 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 uses an electronic particle counter. While light obscuration is the most common method for accurate particle count testing of parenteral products, electronic particle counters have several issues that can lead to inaccurate particle counts. For this reason, some parenteral products should not be assessed using an electronic particle counter. The top seven factors resulting in electronic particle count inaccuracies are solution flow rates, non-spherical particles, coincidence effects, immiscible fluids, air bubbles, and parenteral product shelf life. Make sure you consider these factors when performing particle count testing yourself, or ensure you choose a contract testing organization that can support you with appropriate 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
Michael J. Akers. Sterile Drug Products Formulation, Packaging, Manufacture, and Quality. Drugs and the Pharmaceutical Sciences. Informa Healthcare. 2010.
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>).
