Factors influencing the growth of spoilage organisms

In addition to water activity, which was considered earlier in this chapter, factors influencing the rate and extent of growth of a contaminant within a pharmaceutical raw material or manufactured medicine include:

  • nutrient availability;
  • temperature;
  • pH;
  • redox potential; and
  • the presence and concentration of antimicrobial chemicals.

Microorganisms differ enormously in their metabolic capabilities. Some, such as Escherichia coli, Pseudomonas aeruginosa and several Bacillus species, can synthesize all the amino acids and vitamins they need from a variety of simple carbon and nitrogen sources.

The minerals that they require are often present in sufficient concentration as impurities in the ingredients of the medicinal product. Thus, in the absence of antimicrobial chemicals, organisms of this type may grow to concentrations of 104 per millilitre or gram, or even higher in products such as syrups, linctuses and
creams.

Products containing glycerol, sugars, amino acids or proteins would clearly represent such ideal media for microbial growth that their preservation is sometimes difficult to achieve despite the addition of preservatives. Even in the absence of these nutritionally rich materials, many bacteria and fungi are still able to utilize other components of the formulation as food sources. Several of these have already been mentioned, but in addition to surfactants and various viscosity-raising agents, the volatile and fixed oils used as flavourings or emulsion components are particularly
suitable as nutrients for microorganisms.

The rate of spoilage progression will vary with temperature, although the period for which a manufactured medicine is usually stored before use is normally so long that the difference in bacterial growth rate between, say, 15°C and 20°C may become insignificant in the context of a 2-year shelf life.

However, there is the possibility of organisms growing during the course of manufacture, and so it is important for production scientists to be aware just
how rapidly the population of contaminants may rise.

Fig. 1: The survival and recovery ofPseudomonas aeruginosa exposed to benzethonium chloride during a preservative efficacy test.

 

Fig. 1 above shows that the concentration of Pseudomonas aeruginosa rose 10000-fold in 44 hours at ambient room temperature in a multidose veterinary injection that was supposedly preserved with benzethonium chloride. Clearly, the potential for a rapid increase in numbers is even greater when there is nobantimicrobial agent present at all.

Most bacteria have an optimum pH for growth that is near neutrality, but most fungi favour slightly acidic conditions and grow best at pH 5–6. Although
the product pH may markedly influence the growth rate itself, it also has a bearing on the activity and stability of any antimicrobial chemicals present, so
the magnitudes of these various effects may have to be considered at the product formulation stage, and a compromise value may have to be selected for the
product pH.

Redox potential (oxidation–reduction potential; Eh) is a term that indicates whether oxidizing or reducing conditions exist in a liquid. It is expressed as a positive or negative value on a scale in millivolts.

Oxidizing conditions (favouring the growth of aerobic organisms) prevail in culture media or liquids with positive Eh values, and reducing conditions (favouring anaerobes) apply at negative values.

Facultative anaerobic organisms, such as Escherichia coli and many similar intestinal pathogens, will grow under both conditions from +300 mV to −100 mV (Food and Drug Administration, 2015).

Most pharmaceutical products possess positive redox potentials, and so
anaerobic growth is not common, but there is the potential for aerobic primary contaminants to utilize the available dissolved oxygen and so render the
product vulnerable to secondary spoilage by anaerobes.

Antimicrobial chemicals are usually added as preservatives in multidose sterile medicines and in nonsterile medicines. However, it is not uncommon for other ingredients of the product to possess antimicrobial activity or enhance the activity of recognized preservatives.

Alcohols used as cosolvents are good examples: ethanol, 2-propanol, propylene glycol and glycerol all possess useful antimicrobial activity, although, with the exception of glycerol, their use tends to be limited to topical products.

Ethylenediaminetetraacetic acid
(EDTA) has a dual role in many pharmaceutical products. It is a chelating agent used to remove metal ions that may catalyse the oxidation of certain active ingredients and, although it possesses little antimicrobial activity in its own right, it may markedly potentiate the action of many established preservatives Indeed, EDTA is present in most proprietary multidose anti-inflammatory eye drops currently
available in the UK. In every case, one of its functions is to potentiate the activity of benzalkonium chloride used as the preservative.

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