Control of Microbial Contamination During Manufacture: General Aspects

CONTROL OF MICROBIAL CONTAMINATION DURING MANUFACTURE: GENERAL ASPECTS

A pharmaceutical product may become contaminated by a number of means and at several points during manufacture. There are several ways in which this risk can be minimized. Any such measures require an understanding of the risks involved.

A)  Risk Assessment

GMP is informed by past mistakes and case studies have been valuable (Friedman, 2004). However a proactive approach is required. Nowadays a manufacturer is expected to demonstrate to the regulatory authorities that an extensive risk assessment has been carried out. Risk analysis must comply with ICH 9Q (EMEA, 2006) and is underpinned by a sound understanding of the process and of the microbial ecology of the environment and ingredients.

Several methods are employed (see EMEA, 2006; Kirupakar, 2007), including hazard analysis critical control points (HACCP), failure mode and effects analysis (FMEA), fault tree analysis (FTA), risk ranking and filtering (RRF) and hazard operability analysis (HAZOP). Only HACCP and FMEA are discussed here.

                   i)  Hazard analysis critical control points (HACCP)

HACCP has been widely used in the food industry and is becoming more commonly used in the pharmaceutical industry (McCullogh, 2007; Sharp, 2000; WHO, 2003; Whyte, 2010). The original HACCP had seven steps:

1.                       Conduct a hazard analysis and identify preventive measures for each step of the process

2.                       Determine the critical control points.

3.                       Establish critical limits.

4.                       Establish a system to monitor the critical control points.

5.                       Establish the corrective action to be taken when monitoring indicates that the critical control points are not in a state of control.

6.                       Establish a system to verify that the HACCP procedure is working effectively.

7.                       Establish a record-keeping system.

However, HACCP has been modified so that it can be applied quantitatively not only to microbiology but also to pyrogens and particles (Tidswell, 2004; Whyte, 2010).

               ii)  Failure mode and effects analysis (FMEA)

FMEA was first used in the engineering industry (Stamatis, 2003). It involves breaking the process down into many discrete steps. For each step scales are set for severity, occurrence and detection. The scores are multiplied and compared to an informed score at which risk becomes unacceptable.

B)   Environmental Cleanliness And Hygiene

Microorganisms may be transferred to a product from working surfaces, fixtures and equipment. Pooled stagnant water is a frequent source of contamination. Thus it is essential that all working areas are kept clean, dry and tidy. Any cracks where microorganisms may accumulate must be eliminated. All walls, floors and ceilings should be easy to clean. This entails impervious and washable surfaces, free from open joints or ledges. Coving should be used at junctions between walls and floors or ceilings. All services such as pipes, light fittings and ventilation points should be sited so that inaccessible recesses are avoided. A rigorous disinfection policy must be in place. All equipment must be easy to dismantle and clean and should be inspected for cleanliness before use.

Fall-out of dust-and droplet-borne microorganisms from the atmosphere is an obvious route for contamination. ‘Clean’ air is therefore a prerequisite during manufacturing processes and the spread of dust during manufacture or packaging must be avoided. Microorganisms may thrive in certain liquid preparations and creams and ointments. The manufacture of such products should, as far as possible, be in a closed system; this serves a dual purpose as it also prevents evaporative loss.

Personnel are another source of potential contamination. High standards of personal hygiene are essential. Operatives should be free from communicable disease and open lesions on exposed body surfaces. To ensure high standards of personal cleanliness, adequate hand-washing and hand-disinfecting facilities and protective garments, including headgear and gloves, must be provided. Staff should be trained in the principles of GMP and in the practice (and theory) of the tasks assigned. Staff employed in the manufacture of sterile products should also receive training in basic microbiology.

C)   Quality Of Starting Materials

Raw materials account for a high proportion of the microorganisms introduced during the manufacture of pharmaceuticals, and the selection of materials of good microbiological quality aids in the control of contamination levels in both products and the environment. It is, however, common to have to accept raw materials which have some non-pathogenic microorganisms present and this must be considered during risk assessment. Whatever the means of prevention of growth or survival by chemical or in-process treatment, it should be regarded as critical and controlled accordingly (Sharp 2000).

Untreated raw materials that are derived from a natural source usually support an extensive and varied microflora. Products from animal sources such as gelatin, desiccated thyroid, pancreas and cochineal may be contaminated with animal-borne pathogens. For this reason some statutory bodies such as the British Pharmacopoeia require freedom of such materials from Escherichia coli and Salmonella spp. At a stated level before they can be used in the preparation of pharmaceutical products. The microflora of materials of plant origin such as gum acacia and tragacanth, agar, powdered rhubarb and starches may arise from those indigenous to plants and may include bacteria such as Erwinia spp., Pseudomonas spp., Lactobacillus spp., Bacillus spp., streptococci, moulds such as Cladosporium spp., Alternaria spp. And Fusarium spp. And non-mycelated yeasts, or those introduced during cultivation. For example, the use of untreated sewage as a fertilizer may result in animal-borne pathogens such as Salmonella spp. Being present. Some refining processes modify the microflora of raw materials; for example, drying may concentrate the level of spore-forming bacteria and some solubilizing processes may introduce waterborne bacteria such as Escherichia coli. Synthetic raw materials are usually free from all but incidental microbial contamination.

The storage condition of raw materials, particularly hygroscopic substances, is important, and as a minimum water activity (A_w) of 0.70 is required for osmophilic yeasts, 0.80 for most spoilage moulds and 0.91 for most spoilage bacteria, precautions should be taken to ensure that dry materials are held below these levels. Some packaging used for raw materials, such as unlined paper sacks, may absorb moisture and may itself be subject to microbial deterioration and so contaminate the contents; for this reason polythene-lined sacks are preferable. Some liquid or semisolid raw materials contain preservatives, but others such as syrups depend upon osmotic pressure to prevent the growth of osmophiles, which are often present. With this type of material it is important that they are held at a constant temperature, as any variation may result in evaporation of some of the water content followed by condensation and dilution of the surface layers to give an A_w value which may permit the growth of osmophiles and spoil the syrup.

The use of natural products with a high non-pathogenic microbial count is possible if a sterilization stage is included either before or during the manufacturing process. Such sterilization procedures may include heat treatment, filtration, irradiation, recrystallization from a bactericidal solvent such as an alcohol, or for dry products, where compatible, ethylene oxide gas. If the raw material is only a minor constituent and the final product is adequately preserved either by low A_w, chemically or by virtue of its pH, sugar or alcohol content, an in-process sterilization stage may not be necessary. If, however, the product is intended for parenteral or ophthalmic use a sterilization stage is essential.

The handling of contaminated raw materials as described previously may increase the airborne contamination level, and if there is a central dispensing area precautions may be necessary to prevent airborne cross-contamination, as well as that from contaminated measuring and weighing equipment. This presents a risk for all materials but in particular those stored in the liquid state where contamination may result in the bulk being spoiled.

D)   Water

Many grades of water are used in pharmaceutical manufacturing (Table 23.1). Water for manufacturing may be potable mains water, water purified by ion exchange, reverse osmosis or distillation, or water for injection purposes (EMEA, 2002).

Most types of water are derived from municipal supplies. Such water is treated, sometimes by filtration, and always by chemicals, usually chlorine, to render it free from coliforms. This water is, however, not sterile. Its microbial and chemical content varies from region to region and the microbial count can increase on storage.

Water used for parenteral products, known as Water for Injections or Water For Injection (WFI), must be virtually apyrogenic. The British Pharmacopoeia (British Pharmacopoeia Commission, 2010) and the US Pharmacopeia (2009a) specify an endotoxin level of no more than 0.25 IU/ml for WFI. In Europe such water is usually produced in a still specially designed to prevent pyrogens from being mechanically carried over into the distillate. In other countries reverse osmosis may also be used (US Pharmacopoeia, 2009a), but in Europe reverse osmosis is not approved (EMEA, 2002). WFI can be used immediately for the preparation of injections, provided it is sterilized within 4 hours of water collection. Alternatively, the water can be kept for longer periods at a temperature above 65°C (typically 80°C) to prevent bacterial growth with consequent pyrogen production. Ultraviolet radiation may be useful for treating WFI in order to reduce the bacterial count, but this must not be regarded as a sterilization process . A more detailed account of water for pharmaceutical use may be found in EMEA (2002) and US Pharmacopeia (2009b).

E)   Process Design

The manufacturing process must be fully defined and capable of providing, with the facilities available, a product that is microbiologically acceptable and conforms to specifications. The process must be fully validated before starting to ensure that it is suitable for routine production operations. Processes and procedures must also be subject to frequent reappraisal and should be re-evaluated when any significant changes are made in the equipment or materials used.

F)  Quality Control And Documentation

The lower the microbiological count of the starting materials, the more readily the quality of the product can be controlled. Microbiological standards should be set for all raw materials as well as microbial limits for in-process samples and the final product. Microbiological quality assurance also covers the validation of cleaning and dis-infectant solutions and the monitoring of the production environment by microbial counts. This monitoring should be carried out while normal production operations are in progress. In addition, sterile manufacture requires extra safeguards. Operators must be adequately trained and their aseptic technique monitored both by observation and microbiological testing. Air filter and sterilizer efficiency must also be evaluated , whilst sterility testing  and, where necessary testing for pyrogens , are the final tests on the finished product.

Documentation is a vital part of quality assurance. Details of starting materials, packaging materials, and intermediate, bulk and finished products should be recorded so that the history of each batch may be traced. Distribution records must be kept. This information is of paramount importance in the event that a defective batch has to be recalled.

G) Packaging, Storage And Transport

Packaging serves a number of functions; it keeps the contents in, it should keep contaminants out and is labelled to permit identification of its contents. The product is contained within primary packaging. In industry these packages are then placed inside secondary packaging for storage and transport. This secondary packaging may take the form of cartons, boxes, trays or shrink wrapping.

Consideration must be given to both the fabric of the packaging and its cleaning, and to the actual process of packaging. Where terminal sterilization is carried out, the packaging must be suitable for the process. Packaging of aseptically processed products into a sterile container must be carried out in a grade A environment (Table 23.2).

Packaging material has a dual role and acts both to contain the product and to prevent the entry of microorganisms or moisture which may result in spoilage, and it is therefore important that the source of contamination is not the packaging itself. The microflora of a packaging material is dependent upon both its composition and storage conditions. This, and a consideration of the type of pharmaceutical product to be packed, determines whether a sterilization treatment is required.

Glass containers are sterile on leaving the furnace, but are often stored in dusty conditions and packed for transport in cardboard boxes. As a result they may contain mould spores of Cladosporium spp., Penicillium spp., Aspergillus spp. and bacteria such as Bacillus spp. and Micrococcus spp. which originate from the cardboard, although it can be treated to remove these contaminants. It is commonplace either to air-blow or wash glass containers to remove any glass spicules or dust which may be present, and it is often advantageous to include a disinfection stage if the product is a liquid or semisolid preparation. Plastic bottles that are either blowor injection-moulded have a very low microbial count and may not require disinfection. They may, however, become contaminated with mould spores if they are transported in a non-sanitary packaging material such as unlined cardboard. Packaging materials that have a smooth, impervious surface, free from crevices or interstices, e.g. cellulose acetate, polyethylene, polypropylene, polyvinyl chloride (PVC), and metal foils and laminates, all have a low surface microbial count.

Closure liners of pulpboard or cork, unless specially treated with a preservative, foil or wax coating, are often a source of mould contamination for liquid or semisolid products. A closure with a plastic flowed-in liner is less prone to introduce or support microbial growth than one stuck in with an adhesive, particularly if the latter is based on a natural product such as casein. Closures can be sterilized by either formaldehyde or ethylene oxide gas if required. In the case of injectables and ophthalmic preparations which are manufactured aseptically but do not receive a sterilization treatment in their final container the packaging has to be sterilized (Figure 23.2b). Dry heat at 170 °C is often used for vials and ampoules. Containers and closures may also be sterilized by moist heat, chemicals and irradiation, but consideration of the destruction or removal of bacterial pyrogens may be necessary.Regardless of the type of sterilization, the process must be validated and critical control points or other risk assessment parameters must be established.