Factors Affecting Microbial Spoilage of Pharmaceutical Products

FACTORS AFFECTING MICROBIAL SPOILAGE OF PHARMACEUTICAL PRODUCTS

By understanding the influence of environmental parameters on microorganisms, it may be possible to manipulate formulations to create conditions which are as unfavourable as possible for growth and spoilage, within the limitations of patient acceptability and therapeutic efficacy. Furthermore, the overall characteristics of a particular formulation will indicate its susceptibility to attack by various classes of microorganisms.

a)    Types And Size Of Contaminant Inoculum

Successful formulation of products against microbial attack involves an element of prediction. An understanding of where and how the product is to be used and the challenges it must face during its life will enable the formulator to build in as much protection as possible against microbial attack. When failures inevitably occur from time to time, knowledge of the microbial ecology and careful identification of contaminants can be most useful in tracking down the defective steps in the design or production process.

Low levels of contaminants may not cause appreciable spoilage, particularly if they are unable to replicate in a product; however, an unexpected surge in the contaminant bioburden may present an unacceptable challenge to the designed formulation. This could arise if, for example, raw materials were unusually contaminated; there was a lapse in the plant-cleaning protocol; a biofilm detached itself from within supplying pipework; or the product had been grossly misused during administration. Inoculum size alone is not always a reliable indicator of likely spoilage potential. Low levels of aggressive pseudomonads in a weakly preserved solution may pose a greater risk than tablets containing fairly high numbers of fungal and bacterial spores.

When an aggressive microorganism contaminates a medicine, there may be an appreciable lag period before significant spoilage begins, the duration of which decreases disproportionately with increasing contaminant loading. As there is usually a considerable delay between manufacture and administration of factory made medicines, growth and attack could ensue during this period unless additional steps are taken to prevent it. On the other hand, for extemporaneously dispensed formulations some control can be provided by specifying short shelf-lives, for example 2 weeks.

The isolation of a particular microorganism from a markedly spoiled product does not necessarily mean that it was the initiator of the attack. It could be a secondary opportunist contaminant which had overgrown the primary spoilage organism once the physicochemical properties had been favourably modified by the primary spoiler.

b)   Nutritional Factors

The simple nutritional requirements and metabolic adaptability of many common spoilage microorganisms enable them to utilize many formulation components as substrates for biosynthesis and growth. The use of crude vegetable or animal products in a formulation provides an additionally nutritious environment. Even demineralized water prepared by good ion-exchange methods will normally contain sufficient nutrients to allow significant growth of many waterborne Gram-negative bacteria such as Pseudomonas spp. When such contaminants fail to survive, it is unlikely to be the result of nutrient limitation in the product but due to other, non-supportive, physicochemical or toxic properties.

Acute pathogens require specific growth factors normally associated with the tissues they infect but which are often absent in pharmaceutical formulations. They are thus unlikely to multiply in them, although they may remain viable and infective for an appreciable time in some dry products where the conditions are suitably protective.

c)    Moisture Content: Water Activity (A_w)

Microorganisms require readily accessible water in appreciable quantities for growth to occur. By measuring a product’s water activity, A_w, it is possible to obtain an estimate of the proportion of un-complexed water that is available in the formulation to support microbial growth, using the formula A_w = vapour pressure of formulation/vapour pressure of water under similar conditions.

The greater the solute concentration, the lower is the water activity. With the exception of halophilic bacteria, most microorganisms grow best in dilute solutions (high A_w) and, as solute concentration rises (lowering A_w), growth rates decline until a minimal growth-inhibitory A_w, is reached. Limiting A_w values are of the order of 0.95 for Gram-negative rods; 0.9 for staphylococci, micrococci and lactobacilli; and 0.88 for most yeasts. Syrup fermenting osmotolerant yeasts have spoiled products with A_w levels as low as 0.73, while some filamentous fungi such as Aspergillus glaucus can grow at 0.61.

The A_w of aqueous formulations can be lowered to increase resistance to microbial attack by the addition of high concentrations of sugars or polyethylene glycols. However, even Syrup BP (67% sucrose; A_w = 0.86) has occasionally failed to inhibit osmotolerant yeasts and additional preservation may be necessary. With a continuing trend towards the elimination of sucrose from medicines, alternative solutes which are not thought to encourage dental caries such as sorbitol and fructose have been investigated. A_w can also be reduced by drying, although the dry, often hygroscopic medicines (tablets, capsules, powders, vitreous ‘glasses’) will require suitable packaging to prevent resorption of water and consequent microbial growth (Figure 17.2).

Tablet film coatings are now available which greatly reduce water vapour uptake during storage while allowing ready dissolution in bulk water. These might contribute to increased microbial stability during storage in particularly humid climates, although suitable foil strip packing may be more effective, albeit more expensive.

Condensed water films can accumulate on the surface of otherwise ‘dry’ products such as tablets or bulk oils following storage in damp atmospheres with fluctuating temperatures, resulting in sufficiently high localized A_w to initiate fungal growth. Condensation similarly formed on the surface of viscous products such as syrups and creams, or exuded by syneresis from hydrogels, may well permit surface yeast and fungal spoilage.

d)    Redox Potential

The ability of microbes to grow in an environment is influenced by their oxidation–reduction balance (redox potential), as they will require compatible terminal electron acceptors to permit their respiratory pathways to function. The redox potential even in fairly viscous emulsions may be quite high because of the appreciable solubility of oxygen in most fats and oils.

e)    Storage Temperature

Spoilage of pharmaceuticals could occur potentially over the range of about −20°C to 60°C, although it is much less likely at the extremes. The particular storage temperature may selectively determine the types of microorganisms involved in spoilage. A deep freeze at − 20 °C or lower is used for long-term storage of some pharmaceutical raw materials and short-term storage of dispensed total parenteral nutrition (TPN) feeds prepared in hospitals. Reconstituted syrups and multidose eye drop packs are sometimes dispensed with the instruction to ‘store in a cool place’ such as a domestic fridge (2–8 °C), partly to reduce the risk of growth of contaminants inadvertently introduced during use. Conversely, Water for Injections (EP) should be held at 80 °C or above after distillation and before packing and sterilization to prevent possible regrowth of Gram-negative bacteria and the release of endotoxins.

f)      pH

Extremes of pH prevent microbial attack. Around neutrality bacterial spoilage is more likely, with reports of pseudomonads and related Gram-negative bacteria growing in antacid mixtures, flavoured mouthwashes and distilled or demineralized water. Above pH 8 (e.g. with soap-based emulsions) spoilage is rare. In products with low pH levels (e.g. fruit-juice-flavoured syrups with a pH 3–4), mould or yeast attack is more likely. Yeasts can metabolize organic acids and raise the pH to levels where secondary bacterial growth can occur. Although the use of low pH adjustment to preserve foodstuffs is well established (e.g. pickling, coleslaw, yoghurt), it is not practicable to make deliberate use of this for medicines.

g)     Packaging Design

Packaging can have a major influence on microbial stability of some formulations in controlling the entry of contaminants during both storage and use. Considerable thought has gone into the design of containers to prevent the ingress of contaminants into medicines for parenteral administration, because of the high risks of infection by this route. Self-sealing rubber wads must be used to prevent microbial entry into multidose injection containers following withdrawals with a hypodermic needle. Wide-mouthed cream jars have now been replaced by narrow nozzles and flexible screw-capped tubes, thereby removing the likelihood of operator introduced contamination during use of the product. Similarly, hand creams, previously supplied in glass jars, are now packed in closed, disposable dispensers. Where medicines rely on their low A_w to prevent spoilage, packaging such as strip foils must be of water-vapour-proof materials with fully efficient seals. Cardboard outer packaging and labels themselves can become substrates for microbial attack under humid conditions, and preservatives are often included to reduce the risk of damage.

h)    Protection Of Microorganisms Within Pharmaceutical Products

The survival of microorganisms in particular environments is sometimes influenced by the presence of relatively inert materials. Thus, microbes can be more resistant to heat or desiccation in the presence of polymers such as starch, acacia or gelatin. Adsorption on to naturally occurring particulate material may aid establishment and survival in some environments. There is a belief, but limited hard evidence, that the presence of suspended particles such as kaolin, magnesium trisilicate or aluminium hydroxide gel may influence contaminant longevity in those products containing them, and that the presence of some surfactants, suspending agents and proteins can increase the resistance of microorganisms to preservatives, over and above their direct inactivating effect on the preservative itself.