The current demands in today’s pharmaceutical industry for quality control and quality assurance have been driven by the aspiration to deliver consistently high quality and safe products to the consumer. The standards that are set to meet this goal are exorbitant and tightly controlled both internally and externally.
Cleanroom environments are a crucial area for the process and manufacture of pharmaceutical products. Cleanrooms are controlled and maintainedusing very stringent protocols and guidelines outlined by organizationssuch as the FDA [1] and ISO [2]. Quality control and assurance withinthe cleanroom has become a science in itself; part of this relatesto monitoring particulate matter in the cleanroom. This monitoring verifies that the cleanroom is operating within the required classificationsand that the level of particulate matter is at such a level that it will not have an adverse effect on the sterility of the product, which further down the line could possibly be a matter of life or death. Let us take a closer look at the way the cleanroom environment is monitored to verify the existence or nonexistence of particles both viable and non-viable.
What is a Viable Particle?
A viable particle is a particle that contains one or more livingmicroorganisms. These can affect the sterility of the pharmaceuticalproduct and generally range from ~0.2µm to ~30µm in size[3].
How are They Monitored?
The method of monitoring these particles is by capturing, colonizing and counting them. Two technologies are used.
(1) Settled Plates: These are used to measure the number of microorganisms 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 counted, based on the area of the surface on which they were collected in addition to the period of time the sample was taken. Refer to ISO 14698 Annex C [4] for specific guidelines.
(2) Air Samplers: Used to sample microorganisms in air. The volume sampled is usually 1 m3 or cfu/m3 [5].
The method of collecting the microorganisms is by impaction [6]. The sampling head of the air sampler is en gineered in such a way that the delivery of the sample to the contact plate, or agar strip, is delivered so as not to disrupt normal ambient airflow and more importantly without rendering the microorganism 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 a microscope and the type of bacteria is also identified. (See Figure 1)
What is a non-viable particle?
A non-viable particle is a particle that does not containa living microorganism but acts as transportation for viable particles.
How are they monitored?
Non-viable particles are monitored using particle counters which do not distinguish between viable and non-viable particles but are much more technically advanced than air samplers. Particle counters were developed by the U.S. 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 which uses mirrors and optics to view the particle, and a pump to pull the required sample through the sensor. The principal 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. (See Figure 2)
How is the sample taken?
The particles are sampled using a selected volume of air. Because particle counters were first developed and manufactured in the U.S., the first standards [7] developed were imperial and a sample volume of 1 cubic foot became accepted internationally as the standard volume, as it conveniently took 1 minute to complete. However, a lot of countries developed their own standards and countries in Europe used metric volumes so the 1 cubic meter sample came about. Depending on what standard is followed, the sample is taken and the concentration of particles per 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: =0.5µm and =5.0µm, as these ranges contain the microorganisms that will have an adverseeffect 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. 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 m3 of air. Generally, 1 minute samples were taken but a 1 m3 sample would take over 35 minutes, as the flow rate on particle counters has traditionally been 1 cf/min. 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 L/min) which cuts the 1 m3 sampling time from 35mins to 20mins while keeping the size of the particle counter extremely small and allowing full portability. (See Figure 3)
For continuous sampling, facility monitoring systems are usually used. These systems incorporate remote sensors situated near 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 immediately take required corrective actions. 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 anddata collection.
Looking to the future
Particle detection will become more and more automated. Facility monitoring systems will become more adaptable, where viable and non-viable particle detection, in addition to other environmental parameters such as temperature and relative humidity pressure, will all be controlled via one system. The aim is to keep human contact to a minimum as that is the major contributor to cleanroomcontamination.
A lot of these systems are already in place, but viable testing still needs human intervention where the samples collected need to be incubated and physically counted. Also quality systems will need to be reconsidered with the advances of technology. One example is to correlate results from particle counters to that of air samplers so that a trend of viable particles per concentration of particles can be obtained. This will enable the microbiologist to have moreinformation allowing improvements on overall quality.
New technology will soon be available where a hybrid of an air sampler and particle counter will be available; but the only thing holding back such an innovation would be the incubation time. Maybe this can be overcome, but if this happens, we will all be using instrumentation as seen in Star Trek. For example, one particular company has recently made this innovation possible. 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. While this instrument is commercially available, it is expensive and not yet widely used in cleanrooms, but it does illustrate that the technology, although in its infant stages, is available and will become more mainstreamas companies strive to improve safety and quality of products.
References:
Standards References: ISO 14698-1:2003; ISO 14698-2:2003; ISO 14644-1; ISO 14644-2; EC GMP Annex 1
1 FDA: Food and Drug Administration
2 ISO: International Standards Organization
3 Bacteria (viables) don’t fly—but they will hitch rides on particles floating about in the room. They attach themselves to particles of ~1.0µm. Hence, if you have very few 0.5µm particles you are unlikely to have many viables floating about. Particles of size =5.0µm offer the same opportunity for viables to ride on, but in addition, also present a threat to blocking veins or capillaries if they get into your blood stream. If the pharmaceutical customer is preparing a product that will ultimately be injected into a human body, they need to be assured that the product doesn’t contain particles that will cause blockages. Veins tend to be ~ 7-50µm and capillaries ~ 4-7µm.
4 ISO 14698-1: General Principals and Methods for Bio-contamination Control.
5 cfu/m3: Colony forming unit per cubic meter outlined in EC GMP Annex 1. See recommended limits for microbiological monitoring, part 5.
6 Impaction: Collision with a solid surface.
7 Military Standard, Federal Standard 209
Jason Kelly is Director of Optical Sciences at OptiCal Sciences (Ireland) Ltd. Pembroke, Carlow, Co. Carlow, Ireland. He canbe reached at +353 (0)59 91 82948 or [email protected].
