Particular about particles

Cleanroom environments are crucial areas for the process and manufacture of pharmaceutical products. These areas are controlled and maintained using very stringent protocols and guidelines outlined by organisations such as the FDA¹ and ISO².

Quality control and assurance within the cleanroom has become a science in itself, and part of this is the monitoring of particulate matter. This monitoring verifies that the cleanroom is operating within the required classifications and that the level of particulate matter is such that it will not have an adverse effect on the sterility of the product. The cleanroom environment is monitored to verify the existence or non existence of particles, both viable and non viable. A viable particle is a particle that contains one or more living micro-organisms. These can affect the sterility of the pharmaceutical product and generally range in size from ~0.2µm to ~30µm.³ The method of monitoring these particles is by capturing, colonising and counting. Two technologies are used: (1) Settled Plates; which are used to measure the number of micro-organisms settling from the air onto a surface area over a period of time. These plates are placed in positions of interest around the cleanroom, collected after several hours, incubated and the resulting colonies then counted, based on the area of the surface on which they were collected in addition to the period of time over which the sample was taken. Refer to ISO 14698 Annex C for specific guidelines.⁴ (2) Air Samplers are used to sample micro-organisms in air. The volume sampled is usually 1 m³ or cfu/m³.⁵ The method of collecting the micro-organisms is impaction.⁶ The sampling head of the air sampler is engineered so that the sample is delivered to the contact plate or agar strip in such as way as to not disrupt normal ambient airflow and, more importantly, without rendering the micro-organism non viable. This has become an area of interest as it had been previously overlooked and was recently outlined in ISO 14698-1:2003. The contact plates, agar strips or filter membrane are then incubated and the resulting colonies are counted. Under microscope the type of bacteria is also identified.

Transportation means A non viable particle is one that does not contain a living micro-organism but acts as transportation for viable particles. They are monitored using particle counters that do not distinguish between viable and non viable particles but are much more technically advanced than air samplers. Particle counters were developed by the US military in the 1960s for use in the aerospace industry and were then further developed for use in the semiconductor and pharmaceutical industries. They consist of a dark chamber, or sensor, containing a discrete laser that uses mirrors and optics to view the particle, and a pump to pull the required sample through the sensor.

Simple principle The principle behind the detection and sizing of particles is simple. The vacuum pump sucks the particle through the sensor and the laser beam. At this stage it deflects the light from the laser onto mirrors which are focused onto a photo detector; this reflected light is then converted into an electrical pulse by the photo detector. The pulses are counted and sized by the electronics within the particle counter. The bigger the particle the more light it reflects and therefore the bigger the electrical pulse converted by the photo detector. The particles are sampled using a selected volume of air. Because particle counters were first developed and manufactured in the US, the first standards⁷ developed were imperial; a sample volume of 1 cubic foot became accepted internationally as the standard as it conveniently took one minute to complete. However, many countries developed their own standards - European countries, for example, use a 1m³ sample. The sample is taken and the concentration of particles per unit of volume is displayed on the screen of the particle counter. Normally the size of particle is shown with the corresponding number of particles. The pharmaceutical industry is mostly interested in the reporting of two sizes: equal to or greater than 0.5µm and equal to or greater than 5.0µm, as these ranges contain the micro-organisms that will have an adverse effect on the sterility of the product. This information is then recorded, printed or downloaded and the data interpreted based on the standards used. Since 1999, ISO 14644-1 has been adopted as the international standard, so that all countries follow the same guidelines and use the same parameters. In September 2003 the EC Guide to Good Manufacturing Practice revision to Annex 1 came into operation.

Widespread confusion This has had a big impact on the pharmaceutical industry as it requires continuous sampling in Grade A/B areas and has caused confusion regarding sampling a cubic metre of air. Generally, one minute samples were taken but a 1m3 sample would take more than 35 minutes, as the flow rate on particle counters has traditionally been 1 cf/min.⁸ This has pushed current technology. One manufacturer has responded by introducing a particle counter with a 'higher than normal' flow rate of 1.77 cf/min (50 litres/min), which cuts the 1m³ sampling time from 35 to 20 minutes while keeping the size of the particle counter extremely small and allowing full portability.

Continuous sampling For continuous sampling, facility monitoring systems are usually used. These systems incorporate remote sensors situated near to the point of fill or where production is taking place. The sample data is then relayed to software on a PC where the operator can view the data in real time and take any corrective actions required immediately. These systems are fully automated (and adhere to 21 CFR part 11), collect data continuously and have alarms built into the software so they can dramatically improve quality control and data collection. Particle detection is likely to become increasingly automated. Facility monitoring systems will become more adaptable, to the point where viable and non viable particle detection, in addition to other environmental parameters such as temperature, pressure and relative humidity, will all be controlled via one system. The aim is to keep human contact to the minimum, as people are the major contributors to cleanroom contamination. Many of these systems are already in place, but viable testing still needs human intervention to incubate and physically count the samples collected. Quality systems will also need to be reconsidered in line with advances in technology. One example is to correlate results from particle counters to that of air samplers to obtain a trend of viable particles per concentration, thereby giving the microbiologist more information in order to make improvements in overall quality. New technology will soon be available to enable the creation of a hybrid of an air sampler and particle counter. The only thing holding back such an innovation would be the incubation time, but it may be possible to overcome this. For example, TSI Inc's ultraviolet aerodynamic particle sizer (UV-APS) measures the aerodynamic diameter, light scattering intensity and intrinsic fluorescence of an aerosol particle more or less in real time. This instrument is commercially available, but it is expensive and not yet widely used in cleanrooms. However, it does illustrate that the technology - although in its infant stages - exists and will become more mainstream as companies strive to improve safety and quality of products.