(BQ) Part 2 book “Hugo and russell’s pharmaceutical microbiology” has contents: Pharmaceutical biotechnology, manufacture of antibiotics, factory and hospital hygiene, sterile pharmaceutical products, sterilization procedures and sterility assurance,… and other contents.
Part Microbiological Aspects of Pharmaceutical Processing Chapter 15 Ecology of microorganisms as it affects the pharmaceutical industry Elaine Underwood Introduction Atmosphere 2.1 Microbial content 2.2 Reduction of microbial count 2.3 Compressed air Water 3.1 Raw or mains water 3.2 Softened water 3.3 Deionized or demineralized water 3.4 Distilled water 3.5 Water produced by reverse osmosis 3.6 Distribution system 3.7 Disinfection of water 3.7.1 Chemical treatment 3.7.2 Filtration 3.7.3 Light 3.7.4 Microbial checks Introduction The microbiological quality of pharmaceutical products is influenced by the environment in which they are manufactured and by the materials used in their formulation With the exception of preparations which are terminally sterilized in their final container, the microflora of the final product may represent the contaminants from the raw materials, from the equipment with which it was made, from the atmosphere, from the person operating the process or from the final container into which it was packed Some of the contaminants may be pathogenic while others may grow even in the presence of preservatives and spoil the product Any microorganisms that are destroyed by in-process heat treatment may still leave cell residues which may be toxic or pyrogenic (Chapter 3), as the pyrogenic fraction, lipid A, which is present in the cell wall is not destroyed under the same conditions as the organisms Skin and respiratory tract flora 4.1 Microbial transfer from operators 4.2 Hygiene and protective clothing Raw materials Packaging Buildings 7.1 Walls and ceilings 7.2 Floors and drains 7.3 Doors, windows and fittings Equipment 8.1 Pipelines 8.2 Cleansing 8.3 Disinfection and sterilization 8.4 Microbial checks Cleaning equipment and utensils 10 Further reading In parallel with improvements in manufacturing technology there have been developments in Good Manufacturing Practices to minimize contamination by a study of the ecology of microorganisms, the hazards posed by them and any points in the process which are critical to their control This approach has been distilled into the concept of Hazard Analysis of Critical Control Points (HACCP), with the objective of improving the microbiological safety of the product in a cost-effective manner, which has been assisted by the development of rapid methods for the detection of microorganisms Atmosphere 2.1 Microbial content Air is not a natural environment for the growth and reproduction of microorganisms, as it does not contain the necessary amount of moisture and nutrients 251 Chapter 15 in a form that can be utilized However, almost any sample of untreated air contains suspended bacteria, moulds and yeasts, but to survive they must be able to tolerate desiccation and the continuing dry state Microorganisms commonly isolated from air are the spore-forming bacteria Bacillus spp and Clostridium spp., the non-sporing bacteria Staphylococcus spp., Streptococcus spp and Corynebacterium spp., the moulds Penicillium spp., Cladosporium spp., Aspergillus spp and Mucor spp., as well as the yeast Rhodotorula spp The number of organisms in the atmosphere depends on the activity in the environment and the amount of dust that is disturbed An area containing working machinery and active personnel will have a higher microbial count than one with a still atmosphere, and the air count of a dirty, untidy room will be greater than that of a clean room The microbial air count is also influenced by humidity A damp atmosphere usually contains fewer organisms than a dry one, as the contaminants are carried down by the droplets of moisture Thus, the air in a cold store is usually free from microorganisms and air is less contaminated during the wet winter months than in the drier summer months Microorganisms are carried into the atmosphere suspended on particles of dust, skin or clothing, or in droplets of moisture or sputum following talking, coughing or sneezing The size of the particles to which the organisms are attached, together with the humidity of the air, determines the rate at which they will settle out Bacteria and moulds not attached to suspended matter will settle out slowly in a quiet atmosphere The rate of settling out will depend upon air current caused by ventilation, air extraction systems, convection currents above heat sources and the activity in the room The microbial content of the air may be increased during the handling of contaminated materials during dispensing, blending and their addition to formulations In particular, the use of starches and some sugars in the dry state may increase the mould count Some packaging components, e.g card and paperboard, have a microflora of both moulds and bacteria, and this is often reflected in high counts around packaging machines Common methods for checking the microbiological quality of air include the following: 252 The exposure of Petri dishes containing a nutrient agar to the atmosphere for a given length of time This relies upon microorganisms or dust particles bearing them settling on the surface The use of an air-sampling machine which draws a measured volume of air from the environment and impinges it on a nutrient agar surface on either a Petri dish, a plastic strip or a membrane filter which may then be incubated with a nutrient medium This method provides valuable information in areas of low microbial contamination, particularly if the sample is taken close to the working area The type of formulation being prepared determines the microbiological standard of the air supply required and the hazard it poses In areas where products for injection and ophthalmic use which cannot be terminally sterilized by moist heat are being manufactured, the air count should be very low and regarded as a critical control point in the process, as although these products are required to pass a test for sterility (Chapter 20), the test itself is destructive, and therefore only relatively few samples are tested An unsatisfactory air count may lead to the casual contamination of a few containers and may be undetected by the test for sterility In addition, if the microbiological air quality is identified as a critical point, it may also give an early warning of potential contamination and permit timely correction The manufacture of liquid or semi-solid preparations for either oral or topical use requires a clean environment for both the production and filling stages While many formulations are adequately protected by chemical preservatives or a pH unfavourable to airborne bacteria that may settle in them, preservation against mould spores is more difficult to achieve 2.2 Reduction of microbial count The microbial count of air may be reduced by filtration, chemical disinfection and to a limited extent by ultraviolet (UV) light Filtration is the most commonly used method and filters may be made of a variety of materials such as cellulose, glass wool, fibreglass mixtures or polytetrafluorethylene (PTFE) with resin or acrylic binders There are standards in both the UK and USA for the quality of moving air, in the UK there is a grading system from Ecology of microorganisms as it affects the pharmaceutical industry A to D and in the USA, six classes from class to class 100 000 For the most critical aseptic work, it may be necessary to remove all particles in excess of 0.1 mm in size using a high efficiency particulate air (HEPA) filter, but for many operations a standard of < 100 particles per 3.5 litres (1.0 ft3) of 0.5 mm or larger (grade A in the UK — class 100 in the USA) is adequate Such fine filtration is usually preceded by a coarse filter stage, or any suspended matter is removed by passing the air through an electrostatic field To maintain efficiency, all air filters must be kept dry, as microorganisms may be capable of movement along continuous wet films and may be carried through a damp filter Filtered air may be used to purge a complete room, or it may be confined to a specific area and incorporate the principle of laminar flow, which permits operations to be carried out in a gentle current of sterile air The direction of the airflow may be horizontal or vertical, depending on the type of equipment being used, the type of operation and the material being handled It is important that there is no obstruction between the air supply and the exposed product, as this may result in the deflection of microorganisms or particulate matter from a non-sterile surface and cause contamination Airflow gauges are essential to monitor that the correct flow rate is obtained in laminar flow units and in complete suites to ensure that a positive pressure from clean to less clean areas is always maintained The integrity of the air-filtration system must be checked regularly, and the most common method is by counting the particulate matter both in the working area and across the surface of the filter For systems which have complex ducting or where the surfaces of the terminal filters are recessed, smoke tests using a chemical of known particulate size may be introduced just after the main fan and monitored at each outlet The test has a twofold application, as both the terminal filter and any leaks in the ducting can be checked These methods are useful in conjunction with those for determining the microbial air count as given earlier Chemical disinfectants are limited in their use as air sterilants because of their irritant properties when sprayed However, some success has been achieved with atomized propylene glycol at a con- centration of 0.05–0.5 mg/L and quaternary ammonium compounds (QACs) at 0.075% may be used For areas that can be effectively sealed off for fumigation purposes, formaldehyde gas at a concentration of 1–2 mg/L of air at a relative humidity of 80–90% is effective UV irradiation at wavelengths between 240 and 280 nm (2400 and 2800 Å) is used to reduce bacterial contamination of air, but it is only active at a relatively short distance from the source Bacteria and mould spores, particularly those with heavily pigmented spore coats, are often resistant to such treatment It is however, useful if used in combination with air filtration 2.3 Compressed air Compressed air has many applications in the manufacture of pharmaceutical products A few examples of its uses are the conveyance of powders and suspension, providing aeration for some fermentations and as a power supply for the reduction of particle size by impaction Unless it is sterilized by filtration or a combination of heat and filtration, microorganisms present will be introduced into the product The microbial content of compressed air may be assessed by bubbling a known volume through a nutrient liquid and either filtering through a membrane, which is then incubated with a nutrient agar and a total viable count made, or the microbial content may be estimated more rapidly using techniques developed to detect changes in physical or chemical characteristics in the nutrient liquid Water The microbial ecology of water is of great importance in the pharmaceutical industry owing to its multiple uses as a constituent of many products as well as for various washing and cooling processes Two main aspects are involved: the quality of the raw water and any processing it receives and the distribution system Both should be taken into consideration when reviewing the hazards to the finished product and any critical control points Microorganisms indigenous to fresh water in253 Chapter 15 clude Pseudomonas spp., Alcaligenes spp., Flavobacterium spp., Chromobacter spp and Serratia spp Such bacteria are nutritionally undemanding and often have a relatively low optimum growth temperature Bacteria which are introduced as a result of soil erosion, heavy rainfall and decaying plant matter include Bacillus subtilis, B megaterium, Enterobacter aerogenes and Enterobacter cloacae Contamination by sewage results in the presence of Proteus spp., Escherichia coli and other enterobacteria, Streptococcus faecalis and Clostridium spp Bacteria which are introduced as a result of animal or plant debris usually die as a result of the unfavourable conditions An examination of stored industrial water supplies showed that 98% of the contaminants were Gram-negative bacteria; other organisms isolated were Micrococcus spp., Cytophaga spp., yeast, yeast-like fungi and actinomycetes 3.1 Raw or mains water The quality of the water from the mains supply varies with both the source and the local authority, and while it is free from known pathogens and from faecal contaminants such as E coli, it may contain other microorganisms When the supply is derived from surface water the flora is usually more abundant and faster-growing than that of supplies from a deep-water source such as a well or spring This is due to surface waters receiving both microorganisms and nutrients from soil and sewage while water from deep sources has its microflora filtered out On prolonged storage in a reservoir, waterborne organisms tend to settle out, but in industrial storage tanks the intermittent throughput ensures that, unless treated, the contents of the tank serve as a source of infection The bacterial count may rise rapidly in such tanks during summer months and reach 105–106 per ml One of the uses of mains water is for washing chemicals used in pharmaceutical preparations to remove impurities or unwanted by-products of a reaction, and although the bacterial count of the water may be low, the volume used is large and the material being washed may be exposed to a considerable number of bacteria The microbial count of the mains water will be re254 flected in both softened and deionized water which may be prepared from it 3.2 Softened water This is usually prepared by either a base-exchange method using sodium zeolite, by a lime-soda ash process, or by the addition of sodium hexametaphosphate In addition to the bacteria derived from the mains water, additional flora of Bacillus spp and Staphylococcus aureus may be introduced into systems which use brine for regeneration and from the chemical filter beds which, unless treated, can act as a reservoir for bacteria Softened water is often used for washing containers before filling with liquid or semi-solid preparations and for cooling systems Unless precautions are taken, the microbial count in a cooling system or jacketed vessel will rise rapidly and if faults develop in the cooling plates or vessel wall, contamination of the product may occur 3.3 Deionized or demineralized water Deionized water is prepared by passing mains water through anion and cation exchange resin beds to remove the ions Thus, any bacteria present in the mains water will also be present in the deionized water, and beds which are not regenerated frequently with strong acid or alkali are often heavily contaminated and add to the bacterial content of the water This problem has prompted the development of resins able to resist microbiological contamination One such resin, a large-pore, strong-base, macroreticular, quaternary ammonium anion exchange resin which permits microorganisms to enter the pore cavity and then electrostatically binds them to the cavity surface, is currently being marketed The main function is a final cleaning bed downstream of conventional demineralizing columns Deionized water is used in pharmaceutical formulations, for washing containers and plant, and for the preparation of disinfectant solutions 3.4 Distilled water As it leaves the still, distilled water is free from Ecology of microorganisms as it affects the pharmaceutical industry microorganisms, and contamination occurs as a result of a fault in the cooling system, the storage vessel or the distribution system The flora of contaminated distilled water is usually Gram-negative bacteria and as it is introduced after a sterilization process, it is often a pure culture A level of organisms up to 106 per ml has been recorded Distilled water is often used in the formulation of oral and topical pharmaceutical preparations and a low bacterial count is desirable It is also used after distillation with a specially designed still, often made of glass, for the manufacture of parenteral preparations and a post-distillation heat sterilization stage is commonly included in the process Water for such preparations is often stored at 80°C to prevent bacterial growth and the production of pyrogenic substances which accompany such growth 3.5 Water produced by reverse osmosis Water produced by reverse osmosis (RO) is forced by an osmotic pressure through a semi-permeable membrane which acts as a molecular filter The diffusion of solubles dissolved in the water is impeded, and those with a molecular weight in excess of 250 not diffuse at all The process, which is the reverse of the natural process of osmosis, thus removes microorganisms and their pyrogens Post-RO contamination may occur if the plant after the membrane, the storage vessel or the distribution system is not kept free from microorganisms 3.6 Distribution system If microorganisms colonize a storage vessel, it then acts as a microbial reservoir and contaminates all water passing through it It is therefore important that the contents of all storage vessels are tested regularly Reservoirs of microorganisms may also build up in booster pumps, water meters and unused sections of pipeline Where a high positive pressure is absent or cannot be continuously maintained, outlets such as cocks and taps may permit bacteria to enter the system An optimum system for reducing the growth of microbial flora is one that ensures a constant recirculation of water at a positive pressure through a ring-main without ‘dead-legs’ (areas which due to their location are not regularly used) and only very short branches to the take-off points In addition there should be a system to re-sterilize the water, usually by membrane filtration or UV light treatment, just before return to the main storage tank Some plumbing materials used for storage vessels, pipework and jointing may support microbial growth Some plastics, in particular plasticized polyvinylchlorides and resins used in the manufacture of glass-reinforced plastics, have caused serious microbiological problems when used for water storage and distribution systems Both natural and synthetic rubbers used for washers, O-rings and diaphragms are susceptible to contamination if not sanitized regularly For jointing, packing and lubricating materials, PTFE and silicone-based compounds are superior to those based on natural products such as vegetable oils or fibres and animal fats, and petroleum-based compounds 3.7 Disinfection of water Three methods are used for treating water, namely chemicals, filtration or light 3.7.1 Chemical treatment Chemical treatment is applicable usually to raw, mains and softened water, but is also used to treat the storage and distribution systems of distilled and deionized water and of water produced by reverse osmosis (section 3.5) Sodium hypochlorite and chlorine gas are the most common agents for treating the water supply itself, and the concentration employed depends both upon the dwell time and the chlorine demand of the water For most purposes a free residual chlorine level of 0.5–5 ppm is adequate For storage vessels, pipelines, pumps and outlets a higher level of 50–100 ppm may be necessary, but it is usually necessary to use a descaling agent before disinfection in areas where the water is hard Distilled, deionized and RO systems and pipelines may be treated with sodium hypochlorite or 1% formaldehyde solution With deionized systems it is usual to exhaust the resin beds with brine before sterilization with formaldehyde to prevent its inactivation to 255 Chapter 15 paraformaldehyde If only local contamination occurs, live steam is often effective in eradicating it During chemical sterilization it is important that no ‘dead-legs’ remain untreated and that all instruments such as water meters are treated sodium thiosulphate Although an incubation temperature of 37°C may be necessary to recover some pathogens or faecal contaminants from water, many indigenous species fail to grow at this temperature, and it is usual to incubate at 20–26°C for their detection 3.7.2 Filtration Membrane filtration is useful where the usage is moderate and a continuous circulation of water can be maintained Thus, with the exception of that drawn off for use, the water is continually being returned to the storage tank and refiltered As many waterborne bacteria are small, it is usual to install a 0.22-µm pore-size membrane as the terminal filter and to use coarser prefilters to prolong its life Membrane filters require regular sterilization to prevent microbial colonization and ‘grow through’ They may be treated chemically with the remainder of the storage/distribution system or removed and treated by moist heat The latter method is usually the most successful for heavily contaminated filters 3.7.3 Light UV light at a wavelength of 254 nm is useful for the disinfection of water of good optical clarity Such treatment has an advantage over chemical disinfection as there is no odour or flavour problem and, unlike membrane filters, it is not subject to microbial colonization One of the newer technologies suitable for disinfecting water is UV-rich high intensity light pulses in which 30% of the energy is at wavelengths of