Introduction

This paper, in its third revision, discusses the particle counter requirements laid out by US (USP), European (EP), and Japanese (JP) Pharmacopoeia standards and includes the most recent USP 788 (April 2007), EP 5.1 and JP 15 release information. These standards demand that injectable solutions are effectively monitored for microcontamination, specifically non-soluble particles.

Notable changes have occurred to all of the three standards, namely:

1. USP recently updated in April 2007 and undertakes a new course on testing
2. EP changed to include SVI products in 2005
3. JP updated to JP in 2006

Injectable Solutions

Pharmaceutical companies are manufacturers of both solid and liquid formulations. Solid formulations are tablets, dry powers, confectionery, and some solid injectables. Liquid formulations, historically known as parenteral solutions, are now described as injectable solutions or injectables. They include opthalmics, ointments, I.V., vaccines, and others. Injectable solutions are packed as Large Volume Injectable (LVI) solutions, Small Volume Injectable (SVI) solutions, and dry powders requiring reconstitution as either LVI or SVI but most commonly as SVI.

LVIs are typically packaged as bags or bottles containing large volumes of intravenous (IV) solutions. Common uses of LVI solutions without additives include: 1) correction of electrolyte and fluid balance disturbances; 2) nutrition; 3) a vehicle for administering other drugs. Large volume injectable solutions are packaged in containers holding 100 mL or more (≥ 100 mL) and can be packaged in one of the three types of containers: glass bottle with an air vent tube, glass bottle without an air vent tube, or plastic bags.

Small volume injectable (SVI) solutions are usually less than 100 mL (< 100 mL) and are packaged depending on the intended use. SVIs are typically packed as ampoules, vials, small bags, and pre-filled syringes.

If the solution is a sterile formulation it must be free of all visible particulate material as well as of smaller particles. Particulate material refers to mobile solids unintentionally present in parenteral products. These solids may consist of individual components or mixtures of cellulose, glass, or rubber cores from vials, metal, or plastic fragments. Sterile suspensions may have particulate material but these are usually the active drug or an ingredient, not contaminants.

Potential sources of particulate contamination:

• manufacturing environment and equipment
• manufacturing personnel
• packaging components

History of control over Particles in Injectables

When we look for the rational as to why are we controlling the limits and number of particles a patient may be exposed to we must ask, "What effect might the particles have?" and "How many can a patient safely be subjected to?"

Primarily, it is important to have control over the limits of these non soluble particles as they can prove to be delitorius to human health for several reasons, including:

1. Chemical Reaction The burden of particles are chemically incompatible with the arterial system of the body, essentially poisoning the patient.
2. Pulmonary aneurism The particle is of sufficient size that it becomes entrapped in the arterial system, causing a physiological effect.
3. Hypertension The body rejects the foreign matter and encourages the immune system to work excessively causing secondary effects.

We must gauge how many particles can be deemed to be a safe limit. Two factors are important, the physiological form of the patient and the anticipated tolerances to extraneous particles and the testing limits of the available technology to make such measurements. The physiological form is a huge variable and difficult to realize and may be in excess of the tolerance posed by the instrumentation. Therefore, let us consider the tolerances on measurement of the injectable solution.

There have been several studies reviewing the testing limits of liquid particle counters [Sizing accuracy of Particle Counters: Fujishita, Sendo, Hisazumi, Otsubo, Aoyama & Oishi. Coincidence Model for Particle Counters: Knapp, Lieberman & Abramson]. These papers looked at defining the maximum concentration allowed by particle counters and anticipated accuracies of measuring devices. The conclusions of these particle counter studies were:

• Particle counter sizing accuracy defined by either a half count value of the voltage threshold (sensor resolution) or by determining the Gaussian distribution of particles by computer program
• Maximum permitted concentration is a function of Doublet/Triplet particles resident in the laser simultaneously

The effects of the above are twofold:

• Over particle counting and possible false failure of limit testing due to doublet/triplet particles encroaching on upper channel
• Under counting of particles and false passing of smaller size channel as multiple particles are counted as single, larger particles

Limits were defined based upon the original particle counter efficiencies of available technology. USP 22 originally defined the limits for test <788> as: - 10,000 particle counts per container @ 10 µm

• 1,000 particle counts per container @ 25 µm

Following the studies, the particle limits were reviewed for maximum particle counting efficiency for optical particle counters and determined that due to false counts and allowing for acceptable errors, the limits for USP 23 <788> should be:

• 6,000 particle counts per container @ 10 µm
• 600 particle counts per container @ 25 µm

Counting Particles to Specifications

The devices used to administer IV products also create potential particle contamination and fall under a different requirement for medical device testing and proof of control. Particle measurements of 50 µm or larger can be detected by visual inspection. To detect particles less than 50 µm an APSS 200 parenteral particle counter is recommended. International limits apply to the number of particles which can be present in parenteral formulations (USP Test Section <788>).

Figure 1 APSS 200 Particle Counter System with Validation Documentation (Download pdf for all tables and figures)

The United States Pharmacopoeia (USP) Test Section <788> defines the allowable limits of noninfectious contaminants, i.e., those particulate materials which may be present and, therefore, considered safe for IV administration. Specific limits are set forth for particles above 10 and 25 µm. The USP directive applies to all large volume solutions intended for single-dose infusion that is ready for use from the manufacturer.

The original European Pharmacopoeia (EP) standard 5.0 states in section 2.9.19 that preparations greater than 100 mL must also be tested to meet defined limits of particulate concentration. These limits are defined in Table 1. This definition created questions about small volume injectables. SVIs had defined limits in the EP 5.0 for preparation but had no requirement for testing. This was, therefore, done as a function of quality control validation and not because it was specified in EP 5.0. This changed in April 2005 with the release of EP 5.1. The section now requires that "For preparations for human use, solutions for infusion or solutions for injection (must) comply with this test." Therefore, all preparations (LVI and SVI) must meet the EP 5.0 requirement. It also identified that intramuscular and subcutaneous products are allowed higher limits, and that radiopharmaceuticals and those with final filtration are exempt from testing.

Japanese Pharmacopoeia (JP) requires that insoluble particles are tested for either in the finished products or the transportation media is tested independently. But all large and small volume injectables must conform to the test.

Table 1: Pharmacopoeia Particle Standards (Download pdf for all tables and figures)

Apparatus and Methodologies

The Pharmacopoeia tests allows for the determination of the particulate content of LVI samples to be performed by two different methodologies, The Light Obscuration Particle Count Test and The Microscopic Particle Count Test. Not all injection formulations can be analyzed by both these methods; light obscuration is not always applicable to solutions having a different color, viscosity or clarity than water and may give erroneous results. The Microscopic method is unsuitable for solutions which may contain gelatinous constituents that agglomerate on a filter paper. Additional evaluation may be required in these instances to support the release of a product. When a product is unsuitable for testing by Method 1 (light blockage) it is allowable to dilute the sample to achieve either a clarity or viscosity to achieve testing.

The automation of particle counting predominantly is performed by an optical laser particle counter system. Two criteria are defined for the performance of an automated system.

1. Sensor Concentration Limits: the concentration at which the sensor coincidence count rate is 10% at the 10 µm size limit. The APSS 200 System has a maximum concentration of 10,000 /mL.
2. Sensor Dynamic Range: the dynamic range of the instrument which must include the smallest size to be enumerated. For the USP this is 10 and 25 µm. The APSS 200 System is typically configured to sample dynamically from 2 to 125 µm, and can be configured for particles as small as 0.1 µm.

Figure 2 LiQuilaz Liquid Particle Counter

The LiQuilaz liquid particle counter in the APSS 200 system operates on the principle that the light extinguished by a particle in a liquid within a classical laser beam is a direct function of its area. Particles obscure the laser beam during transit through the beam. The pulses produced by electronically detecting the total laser light minus the light obscured by the particle are used by the particle counter to size the particle. These pulses are measured by an analog to digital converter in the particle coutner. The liquid is presented to the optical particle counter system through a rectangular capillary.

The particle counter capillary has a window attached to both the front and back sides, which are coated to reduce reflections. The whole system is controlled by a central software application, which controls the hardware, analyzes the data, and stores data for future interpretation.

The variation in light caused by the passing of a particle is electronically detected by the particle counter photodetector. This signal is then amplified and converted to its digital equivalent. The value of this digital signal is converted into an equivalent particle size in a microprocessor. The different size particles are counted and stored in the microprocessor and made available for transmission to the particle counter data display system upon request.

To ensure that the LiQuilaz particle counter, the LS-200 Sampler, and the SamplerSight-Pharma Control System Software are acceptable and validated to perform the Pharmacopoeia tests they are challenged to various tests with defined acceptance criteria. These IQ and OQ (Installation and Operational Qualification) tests challenge the following areas:

Particle Counter Sample Volume Accuracy. As the total number of particles is based upon a known volume of sample measured, it is critical that the sample volume is accurate. The LS-200 Syringe Sampler is available with syringes of 1, 5, 10, and 25 mL and has sample accuracy greater than the required 5%.

Particle Counter Sample Flow Rate. The sample flow rate importance is based upon the speed at which a particle moves through the optical particle counter chamber. This determines the duration of the shadow the particle presents to the photodiode. The LiQuilaz particle counter is calibrated at two sample flow rates, 10 and 20 mL/min. The sample flow rate is timed, for a fixed volume, from when the syringe starts moving to the point at which it resets to its origination point.

Running the internal self-calibration program performs the calibration of the APSS-200 system. The program systematically requests particles sizes of 2, 5, 10, 15, 20, 25, and 30 µm. These particles include the USP specified 10, 15, and 25 µm sizes (EP specifies 10 and 25 µm). The algorithm built into the system micro-controller comes with a Particle Measuring Systems' certificate of conformity. JP15 requires the system meet a specified tolerance for the instrumentation, where as EP and now USP require only that the instrument be "suitably" calibrated. The specification for JP15 is:

Table 4 (Download pdf for all tables and figures)

Once the sizing capabilities have been challenged in the calibration, the counting accuracy is performed during the site PQ (Performance Qualification) procedures. A known concentration of particles is sampled by the system and the results verified as being within 10% the total count in solution at both 10 and 15 µm.

Table 5 (Download pdf for all tables and figures)

Taking Measurements

The system is now ready to use and measure the particle count levels in the parenteral sample. The sample measurement is performed by first preparing a sample of known volume of the solution for analysis. This may be a single large volume parenteral, or an agglomeration of smaller vials into one single sample. In either case it is representative of the released product.

Each Pharmacopoeia has defined testing requirements for sampling products, and with the changes to the USP test these have again been brought into a more harmonized position, and follow the same as the EP had historically undertaken. A summary of the expected test method is found below.

• Test using a suitably calibrated light blockage particle counter
  • Calibrated at sizes between 10 & 25 um
  • Care taken to avoid aggregation of particles
• Laminar flow cabinet environment
• Ensure glassware is clean
  • 5 samples each of 5mL, 10 um <25 counts (total)
• Test method
  • Invert 20 times
  • Degas by standing or sonication
  • Take 4 samples (min 5mL); discard 1; average 3
• Limits unchanged

The LiQuilaz particle sensor and LS-200 liquid sampler are easily configured in the SamplerSight-Pharma software to match the criteria of testing required and a recipe can be defined and saved for future runs of a similar product or batch. The sample is presented to the APSS-200 system and a magnetic stirrer is used to constantly agitate the sample to ensure that any potential particulate contamination is evenly distributed and does not settle to the bottom of the sample vessel. The syringe is used to draw a known volume of sample for analysis through the LiQuilaz particle sensor. The particle count data is recorded and a number of measurements taken for the sample. Data can be interpreted as either raw counts, counts per mL, counts per container, a ratio of counts in each channel, or as an average of pooled measurements for a single sample. The average of the results is then assessed by the software for compliance to known standards.

The data is stored in a database managed by SamplerSight-Pharma software and results can be printed immediately or saved for future data interpretation. To comply with 21CFR Part 11 regulatory guidelines the data files are stored in an encrypted format to ensure that data cannot be modified. Software security features built into the sampling system restrict system operation and data interpretation to authorized system personnel only.

The data can then be used to prove the particle matter contamination values for the product. When this data is used in conjunction with additional data from other sources proving sterility, pyrogen-free or otherwise, stability, pH, and osmotic pressure the solution can be released for approval and use.

Conclusion

USP, EP and JP have strict requirements for parenteral sterility. The APSS-200 particle counter system from Particle Measuring Systems clearly meets these standards and provides a simple methodology for users to demonstrate compliance. It has been globally proven to meet the challenging requirements from all three pharmacopoeia standards which demand proven technology, the legacy of the standards are all light obscuration based and this is the only proven technology that can reliably and repeatably meet the tolerances required.

Author

Mark Hallworth is the Pharmaceutical Manager for Particle Measuring Systems, Boulder, CO, USA. He has managed the installation and validation of almost 200 facility-monitoring particle counter projects into pharmaceutical production facilities for over 8 years and has managed the company transition to 21 CFR Part 11 compliant software. He currently lectures for pharmaceutical societies throughout Europe and the US on non-viable particulate monitoring, cGMP compliance (both for FDA and EU approval processes), and facility monitoring systems and the implications of validating those systems to GAMP®.

GAMP® is a registered trademark of ISPE. To learn more about GAMP to place an order, visit www.ispe.org

LiQuilaz® is a registered trademark of Particle Measuring Systems, Inc.

Contact us if you need more information or have questions regarding particle counting.