An important aspect of the use of cleanrooms (Figure 1) is the ability to monitor the quality of the manufacturing environment and thus detect changes, which may be deleterious to product quality. Those aspects of product quality, which concern us here, are the levels of microbial and particulate contamination at successive stages in the manufacturing procedure. To a certain extent, particulate levels in a product reflect the particulate burden in the manufacturing environment. Additionally good manufacturing practice (GMP) requires that certain minimum environmental standards be maintained in certain manufacturing areas. Products that are terminally sterilized are required to be manufactured under environmental conditions equivalent at least to class C (MCA, Rules and Guidance for Pharmaceutical Manufacturers, 1997, often described as the Orange Guide), and products that are aseptically manufactured are filled under class A conditions, situated in a class B environment . In the United Kingdom, the details of construction of rooms that will meet these conditions are stipulated in British Standard 5295. Some information will also be found in Annex 1 of the Orange Guide. Table 1 lists the relevant information for particulate and microbiological contamination.
Particulate levels in the atmosphere of the cleanrooms was determined by using a particle counting system which continuously
samples air at a constant flow rate through a light-scattering sensor.The degree of scatter is proportional to the size of the particles present so that processing of the signals from the scattering unit provides a figure for the number of particles sampled in a known time and also the size of these particles. Normally the filling and sterility testing rooms are class B and the preparation room is class C. Class A conditions are achieved locally by the use of laminar air flow (LAF) hoods which are used for aseptic manipulations and filling operations. Sampling of the filling and sterility testing room atmospheres was done from preparation room. The air flow rate into the microcount sensor is 1 ft/min-1 so that the sampling time gives a measure of the volume of air sampled and the particle number at the 5.0 and 0.5 µm levels. A sampling time setting of 1 min is suggested so that the display gives particles per cub. ft. Convert this value to particles per cubic meter using the conversion.
1 m3 = 35.3 ft3
Two types of sampling method, active and passive, were used to determine the microbiological quality of the atmosphere. Both methods rely on the fact that microbial cells, which are invisible to the naked eye, will reveal themselves as colonies after incubation of the nutrient agar plates used for sampling. A colony may form as the result of the growth of a single organism. A single organism colony will also arise from a clump of a few organisms of the same strain. Because of this, the results of colony units are noted as colony-forming units (CFUs) and not as simple numbers of organisms. Two types of agar used in these tests, Nutrient agar for the detection of bacteria after incubation at 31°C and malt extract agar for the detection of bacterial and fungal organisms (moulds and yeasts) after incubation at 25°C.
The simplest method of enumeration is passive sampling by the use of a settle plate. This involves exposing the plate at the location of interest for a known period of time (sampling time), which is terminated by replacing the lid of the plate. The result is expressed as a number per unit area per unit time (cfu/cm2/h). The second technique is active sampling, which involves measuring the number of microorganisms in a known volume of air, e.g. the concentration of microorganism in the air. Generally, a measured volume of air is drawn through the sampling apparatus and the microbes present impinged on or into a nutrient media usually agar. The result can therefore be expressed as a number of cfu's per unit volume of air. It is usually considered that this is a more precise way of estimating the microbiological quality of the environment and is the measure for example in Table 1.
Surface sampling: Contact plates (containing either type of agar, nutrient or malt extract) for surface contamination. As with the passive and active sampling choose sampling sites of interest or area that present a microbiological risk to the product. (Note: ensure that any excess agar is removed from the surface after sampling using an alcohol wipe. It should be noted that the greatest microbial risk to a product comes from the operators). Contact plates taken before and after hand washing and putting on gloves can be used to assess the efficiency of clothing procedures prior to entry into the cleanest, critical areas. The efficiency of any sterilization process is directly proportional to the bio burden or challenge to that process. An increasing approach to sterilization in the pharmaceutical industry is the use of F° to control a sterilization process, rather than the set cycle times listed in British Pharmacopoeia (BP). To employ control via F° implies that the bio burden and likely heat sensitivities of any organisms contaminating the product before sterilization are known. One feature of microbiological monitoring is therefore the measurement of microbiological contamination during the production process. This provides information on the quality of raw materials, handling procedures and the ability of the various steps employed (e.g. filtration) to remove any contaminating organisms present.
Additional Measures of Environment Quality
Before, during and after use of the class A rooms (e.g. filling and sterility testing rooms) record values for temperature and humidity within the rooms and also values for over-pressure. The required values are laid down in British Standard (BS) 5295 and are temperature, 20°C ± 2°C; humidity, 35-50%; over-pressure, 25Pa (2.5 mm water gauge).
Sterility Testing of Pharmaceuticals
Sterility testing attempts to reveal the presence of viable forms of bacteria, fungi and yeasts which may occur in or on pharmaceutical products, and in simple terms this is done by exposing portions of the material under test to media capable of supporting the growth of these different microorganisms. After 14 days incubation at a suitable temperature, the media are examined for the presence, shown by turbidity or pellicle formation, or absence of growth. If no growth occurs in the test media, the material is assumed to be sterile. Since this is a destructive test, only a sample of the batch can be analyzed, and this introduces the problem of sampling statistics. Regulatory or Licensing Authorities require a product contamination rate within a batch of 1 in 1,000,000 or less, it can be easily demonstrated that the sensitivity of the sterility test is several orders of magnitude lower than this. The sterility test is therefore suspect and of little value in products that are terminally sterilized where the possible contamination rate (based on an Fo approach) is very much lower than 1 in 1,000,000. Terminally sterilized products can therefore be exempt from the test as long as bio burden and autoclave monitoring (physical measurements) are applied. However, the sterility test is still mandatory for aseptically produced products. One additional problem is contamination of test material during the sterility test itself, this invalidates the results and it is vitally important that the procedure is carried out under specific conditions. Usually under class A environmental standards or increasingly in isolator units which can be maintained to levels of microbiological cleanliness far greater than class A.
As stated above, the test requires the product to be exposed to a suitable nutrient medium to support microbial growth; this is difficult if large volumes (500 ml) are being tested. There are currently two approaches to this problem. The classical technique was to use direct inoculation (BP method 11) of the product into the medium, but this limits the volume that may be tested and can be problematical if the product contains antimicrobial agents (e.g. preservatives). The modern method of handling large liquid (or readily soluble) samples is to use membrane filtration (BP method 1). In this procedure, the entire contents of the containers under test are divided and passed through one of two sterile bacteria-proof membranes (0.45µm pore size) set up in a special filtration assembly. After filtration of the contents of all the test containers through the filter units these latter become containers for sterile media (one for fluid thioglycollate and the other for soya casein digest) and are then incubated accordingly. This has the dual advantage of testing the complete contents of the container and eliminating any antimicrobial substances, which may have interfered with the test. If viable organisms had been present in any of the test containers, the growth of these in the units can be detected in the usual manner. There are several feathers of the sterility test that are common irrespective of the method used and these are considered below.