Controlling colony forming units in cleanrooms
By Dr. Brigitte Hauff
Colony forming units, including bacteria and bacterial spores, fungi and fungal spores, viruses, and pollen can wreak havoc on the cleanliness of cleanrooms. In order to ensure hygiene in a cleanroom, one must understand their properties in order to control contamination.
The cleanroom concept
How do the microorganisms (CFUs) find their way into the cleanroom and into or to the surface of the product? Obviously, the raw materials (including water) and the packaging may be important sources of CFUs and particles. Therefore, their quality and especially their purity must be closely monitored.
Once production has started, cleanroom technology becomes essential. This is a generic term for a production concept comprising:
The building and the equipment (i.e., the cleanroom itself);
cleaning and maintenance;
personnel; and
plant and product monitoring.
Only a combination of these points guarantees contamination control.
Building and equipment
Building and equipment planning must be done by an expert in line with the applicable rules and guidelines for cleanrooms. Some important laws, standards and rules are the World Health Organization Good Manufacturing Practice (GMP) (food and drugs), the EU Guide to the GMP, the German Epidemics Protection Act (medicine/research), the German Pharmacopeia and the VDI (Association of German Engineers) recommendations, e.g., 2083/2.
Cleaning and maintenance
Standard operating procedures for cleaning, disinfection (see Table 1), sterilization, and maintenance are mandatory and should be drawn up by the quality assurance department separately for each product.
Cleaning in place (CIP) is a special case: The production equipment is hermetically isolated from the surrounding atmosphere and cleaned in place, i.e., there are no major changes in their operating mode and the equipment is not disassembled into components. The machinery needed for CIP is adapted to the specific requirements and normally forms an integral part of the production equipment.
Plant and product monitoring
Quality assurance is of particular importance, and this type of manufacture must strictly follow carefully established and validated methods of preparation and procedure. Controls during production as well as controls of the end product are the be-all and end-all of contamination control. Additional microbiological monitoring is also required outside production operations, e.g., after validation of systems, cleaning and sanitization.
Apart from the product, the air and the surfaces, especially those in contact with the product, should also be monitored.
The EU GMP Guide for the microbiological monitoring of cleanrooms (see Table 3) recommends limits. They may also serve as a guideline for other industries. In addition to the monitoring of airborne microorganisms (sedimentation of airborne germs), two further parameters are controlled: microbiological contamination of surfaces and gloves.
Examples of operations to be carried out in the various grades in the pharmaceutical industry are given below.
Examples of operations for terminally sterilized products:
A. Filling of products, when unusually at risk
B. Preparation of solutions, when unusually at risk. Filling of products.
C. Preparation of solutions and components for subsequent filling.
Airborne germ count: Airborne germs are normally bound to particles. However, the ratio of particle count to germ count is not constant. Therefore, microbiological inspection is necessary to determine microbiological air quality.
Both qualitative and quantitative methods are available for airborne germ monitoring.
Sedimentation. Petri dishes containing a suitable solid culture medium are placed in the room for a specific time; the airborne germs adhered to particles will sediment on the surface of the culture medium. After culturing, the dishes can be evaluated.
Filtration. The air is passed through membrane filters, e.g., containing gelatin. The germs are retained and can be cultured.
Impaction. Air is sampled, accelerated and blown on solid culture media (Anderson Sampler, Reuter Centrifugal Sampler).
Impingement. The germs are absorbed by a liquid. A defined amount of air is passed through nutrient broth; this method is widely regarded as the gold standard, but rare in routine monitoring due to complexity.
In addition, particle counting has proven useful. The counter operates constantly, and the measurements are displayed on a screen. Both the facility and the personnel (see Table 2) can be monitored in this way. Particles are important for hygiene because they must be regarded as vehicles for germ transport. Kanz (1983) suggested that germs are nearly constantly bound to particles. Litonski (1974) showed that dust is also a source of germs.
Surface inspection (particularly surfaces in contact with the product): Surface monitoring methods include contact prints (culture medium is pressed directly onto the object), swabs (e.g., from locations that cannot be accessed by a dish), and adhesive tape.
Neutralizing substances should be added in all three cases since the surfaces are likely to hold disinfectant residues. In addition the focus of inspection should be on those surfaces which pose the greatest contamination risk for the product.
According to the EU Guide to GMP, appropriate alert and action limits should be set for the result of particulate and microbiological monitoring. If these limits are exceeded, operating procedures should prescribe corrective action.
An excellent management concept for contamination control is the Hazard Analysis of Critical Control Point (HACCP). The HACCP concept is an analysis of the weak points with respect to a possible health hazard for the consumer.
By this method, the microbiological risks, which vary from product to product, are identified. From them, the critical control points for production are derived. Maximum levels are then determined and monitored. In this way, the risk is minimized. A critical control point is a link in the production chain where lack of monitoring would pose an unacceptable risk.
The development of resistance
One obstacle in minimizing contamination is the problem of resistance to disinfectants. Problems may result from the formation of biofilms in the drinking water system, and fungal attacks in hard-to-clean moist niches such as corners, canals, pipes and air conditioning systems. Resistance develops mainly as a result of insufficient disinfectant concentrations. If the disinfectant solution falls short of the threshold of bacteriostatic concentration, a selection process for resistant germs will be initiated, particularly if the same disinfectant (i.e., active substance) has been used for a prolonged period. However, an expert cleanroom planner will take these problems into consideration at the planning stage.
Dr. Brigitte Hauff works with Hauff GmbH, a planning office for cleanroom technology in Reichenbach/Fils, Germany, Siegenbergstr. 39, 73262 Reichenbach. The company offers consultancy, training, planning, construction and assembly planning, site management and project monitoring services. The office has more than 25 years of experience in the planning of cleanrooms. For more information, Dr. Hauff can be reached at Tel: (011) 0049 / 7153 / 59834, Fax: (011) 0049 / 7153 / 59934, or E-mail: [email protected].
References:
1. H.P. Buzek, “Hygiene in Reinräumen,” Swiss Contamination Control 5/3 (1992), 7-9.
2. A. Grasshoff, “CIP-Anlagen in der Lebensmitteltechnik – eine Einführung,” Dr. Ing. Albrecht Grasshoff, Institut für Verfahrenstechnik der Bundesanstalt für Milchforschung, Kiel.
3. A. Hasenböhler, “Good Manufacturing Practice and Pest Control,” Swiss Contamination Control 5/9 (1992), 10-16.
4. E. Kanz, “Luftkeimanalyse in klimatisierten Operationsräumen,” Reinraumtechnik I (1983) 82.
5. B. Litonski, “Klimaanlage als Entkeimungseinrichtung der OP-Raumluft,” Zbl. Bakt. Hyg., I Abt. Orig. B 159 (1974) 244.
6. H. H. Schicht, “Neufassung des Anhangs 1 zur Herstellung steriler Arzneimittel im GMP-Leitfaden der Europäischen Union,” Swiss Pharma 19 (1997), 5-10.
7. H. Seyfarth, “Mikrobiologische Gesichtspunkte bei der Herstellung von Pharmaka unter der Berücksichtigung der GMP-Richtlinien,” Swiss Pharma 19/1-2 (1997), 7-20.
8. K.J. Steffens, “Contamination Control in der Medizinaltechnik; Partikuläre Kontamination medizinischer Einmalartikel – Ursache und Wirkung,” Swiss Contamination Control, 10/2 (1997), 11-17.
9. W. Stein, Vorratsschädlinge und Hausungeziefer- Biologie, Ökologie, Gegenmassnahmen, Stuttgart, Verlag Eugen Ulmer 1986.
10. W. Todt, “Reinraumtechnik in der Weichkäseherstellung,” Reinraumtechnik, April 1991.
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Microbiological basics of contamination control
Bacteria.
Bacteria are omnipresent.
Under ideal conditions, bacteria multiply very quickly.
Under certain conditions (lack of nutrients or accumulation of metabolites), certain species may develop endospores, i.e., permanent forms. These bacterial spores are so important because of their resistance to heat, radiation and chemicals, e.g.,, disinfectants.
Bacteria form toxins. They can be divided into bacterial excretions, called exotoxins, and toxic components of the cell wall of gram-negative bacteria called endotoxins. In many cases, the ability of bacteria to cause disease (their pathogenicity) depends on their ability to produce toxins. Both types of toxin may be relevant for contamination control.
Viruses.
Viruses differ from the other microorganisms in three respects. The factor most relevant for cleanroom production is that they cannot multiply outside living cells. Structurally, they consist of genetic material (DNA or RNA), protein shell and (for embedded viruses) lipids or the like.
Their sensitivity to environmental conditions (and disinfectants) differs greatly due to their wide structural variation. This is also reflected in their main routes of transmission.
Fungi.
Many types of fungi can spread quickly and readily via fungal spores (reproductive bodies) and can grow on a large variety of substances.
This paper was presented at CleanRooms Europe `98 (June 16 to 19). For a copy of the CleanRooms Europe conference proceedings (available for $95 ground shipping), please contact Libby Duggar at (603) 891-9462.