PRESERVATION OF STERILITY

INTRODUCTION

Employing good manufacturing practices to limit microbial contamination in the first place is the preferred strategy, rather than, for example permitting contaminants to enter and proliferate in the product. This include proper design, cleansing and microbial monitoring of building and equipment. Personnel to observe health, hygiene, proper clothing and training. Proper storage of product and raw materials. Another factor utilized mostly in preservation of sterility include use of preservatives.

Characteristics of an ideal preservative

      It should have a broad spectrum of activity against gram negative and positive bacteria.

      It should be effective and stable over the range of pH values encountered in pharmaceutical products. In addition, it should be chemically stable so that there is no loss of preservative efficacy during the expected shelf life of the product.

      It should be compatible with other ingredients in the formulation and with packaging materials. This attribute would prevent loss of preservative potency as a result of interactions with formula components or packaging materials.

      It should not affect the physical properties of the product (i.e., color, clarity, odor, flavor, viscosity, texture).

      It should have a suitable O/W partition coefficient to ensure an effective concentration of the preservative in the aqueous phase of the product. Biological reactions take place in aqueous systems or at the interface of O/W systems; consequently, it is necessary to have sufficient preservative in the water phase to ensure adequate preservation of the product.

      It should inactivate microorganisms quickly enough to prevent microbial adaptation to the preservative system.

      It should be safe to use i.e. non-irritating, non-sensitizing. Safety includes handling of pure or concentrated materials in the manufacturing plant as well as the effect of preservatives in the finished formulation on the consumer.

      It should be cost-effective to use. From a commercial perspective, an effective concentration should add little to the cost of the formulated product.

Sterile products contain preservatives that designed to kill or limit the growth of micro-organisms that may gain entry. Two basic types of preservative system exist: chemical agents of either natural or synthetic origin; or approaches based on altering the products physical conditions to limit microbial growth.

Physical preservative system 

Microorganisms require certain physical conditions to survive; the deliberate adjustment of these to kill or suppress microorganisms can be used as a mode of preserving sterility. They include the following

a)      Available Water

Most organisms require over 70% water to grow, so dry products are unlikely to suffer colonization unless water is added. The amount of water available to an organism in a product is given by:

A_w=Vapour pressure of product/ vapour pressure of water (always less than 1) 

Certain organisms will only survive and grow at specific A_w levels. If A_w is lowered by adding solutes microbial growth can be prevented thus a variable level of preservation can therefore be achieved by a simple manipulation of a products A_W value. For example, a product with an A_w of 0.9 is unlikely to be affected by Pseudomonas species but could be colonized by other bacteria or fungi and moulds. This technique is limited by the large quantities of solute required to reduce A_w sufficiently.

b)      pH value

Majority of organisms grow best at pH 7 but survival is known at pH values from 3-11. Lowering or raising the product pH from neutrality provides a degree of preservation. However the scope for pH variation in pharmaceutical products is limited by physiological acceptability and formulation stability.

c)      Temperature

Microbial growth and majority of contaminants in pharmaceutical products are mesophilic organisms that grow best at ambient temperatures (15-45 degrees Celsius). A reduced storage temperature can be used as a means of inhibiting growth but has the disadvantage that the conditions are not integral to the product.

Chemical preservative systems

Include both synthetic and natural preservative agents.

1.      Natural preservative agents

i)        Essential oils

The antimicrobial properties of essential oils can be ascribed to their chemical constituents; alcohols, aldehydes, esters, ketones and terpenes. Sage oil for example is bactericidal and fungicidal; thyme oil which has been used as an antimicrobial contains 40% of phenolic compounds. Use of essential oils is limited due to high concentrations required for activity, which impart unusual organoleptic properties to the product.

ii)      Enzymes and proteins

The iron binding proteins lactoferrin and ovotransferrin reduce free iron concentration to around 10^-18M which inhibits the growth of many organisms with exception of Pseudomonas aeruginosa. These types of agents have been used primarily in the food industry but have not found common use in pharmaceuticals. Their protein nature precludes them from use in parenteral products.

2.      Synthetic preservatives

Biggest and the most common used group in the preservation of pharmaceutical products. They are classified based on their chemical structure. Within an individual chemical group several agents may be used as preservatives at low concentration and as disinfectants or antiseptics at higher concentrations.

Examples of the various groups include:

      Organic acids and salts: Include weak carboxylic acids such asbenzoic or sorbicacid and are used as preservatives in oral products. They are uncoupling agents that prevent the uptake of substrates requiring a proton motive force to enter the cell. Sorbic acid and benzoic acid have sufficiently low toxicity thus find application for oral use. Their main disadvantage-: Their activity is greatest at acid pH values thus the use of these preservatives is limited to products with pH less than 5.

      Parabens or hydoxybenzoates: Include methyl, ethyl, butyl, propyl, benzyl parabens and their salts. Preservatives used principally in topical and oral products and in some injections. They have relatively good activity against fungi and low toxicity. Unlike benzoic acid, the parabens retain their activity at raised pH values (pH 7 to 9). Disadvantages-: They have poor water solubility and a tendency to partition into the oily phase of emulsions. Relatively weak activity against gram-negative bacteria.

      Quaternary ammonium compounds: Include Benzalkonium chloride, cetrimide. Mode of action is by cell membrane damage resulting in loss of essential chemicals from the cell. They find use as preservatives in ophthalmic (in contact lens cleansing and soaking solutions), topical and some injectable products. They are very water soluble and effective at neutral and alkaline pH. Good stability, non-corrosive and generally non-hazardous. Their disadvantages is that they are usually incompatible with many negatively charged materials. Also, Benzalkonium chloride causes skin and ophthalmic sensitization.

      Alcohols: Only the arylalkyl and the substituted aliphatic alcohols are used as preservatives and include benzyl alcohol, phenoxyethanol, phenylethanol, chlorbutol, bronopol. The effect of aromatic substitution is to produce a range of compounds which are less volatile and less rapidly active and find use as preservatives as eye drops and contact lens solutions. Propylene glycol has preservative activity at concentrations of 10% in syrups and 20% in gelatin capsules. Bronopol find use in medicated shampoo solutions. They act by disrupting the bacterial cytoplasmic membrane, protein denaturation and cell lysis.

      Phenols: Include phenol and chlorocresol. The activity of phenolic compounds is pH dependent and is greatest below pH 9. Their mechanism of antimicrobial activity is by cell lysis. Phenolics find use as preservatives in topical and parenteral products. They have relatively low toxicity. Phenolics are usually absorbed by rubber and plastics.

      Biguanides: The only agent in this series is chlorhexidine. The Biguanides act on the cytoplasmic membrane causing leakage of cell contents Chlorhexidine is mainly used as preservatives in eye drops. Chlorhexidine is more active in the pH range 5 to 8. It has good activity against gram positive bacteria but less activity against gram negative bacteria and fungi. Biguanides are usually incompatible with many negatively charged materials.

      Mercurials: They include the phenylmercuric salts (phenylmercuric acetate, nitrate and borate) and thiomersal. They act through cell wall disruption, cytoplasmic coagulation and bind to thiol groups of enzymes and proteins. Thiomersal is used as a preservative in biological products and certain eye drops. Because of the toxicity of mercury, they are not recommended where prolonged administration is necessary.

Preservative choice is controlled by the physicochemical properties of the formulation and the processes involved during manufacture. Factors affecting activity of antimicrobial activity of preservatives-:

      Effect of concentration-: The activity of a preservative depends on the free concentration of the active form of the molecule in the aqueous phase. Partitioning between water and oil phase, micellar solubilization, and dissociation may all decrease the efficacy of a preservative.

      Effect of pH-: The pH of the formulation may affect the efficacy of the preservative system in a number of ways: pH affects the activity of preservatives, for example, sorbic acid is only active as the undissociated molecule. pH affects the oil–water partition coefficient of the preservative, the micellar solubilization, and the interaction with cyclodextrins, because the undissociated form is more hydrophobic than the dissociated form. Some preservative-component interactions may be pH-dependent because of ionization effects on components.

      Effect of temperature-: An increase in temperature enhances the activity of a preservative. Temperature affects the interaction of preservatives with other formulation ingredients for example, adsorption of preservatives by solid ingredients decreases with increasing temperature.

      Sorption of the preservative to packaging For example hydoxybenzoates, benzyl alcohol and phenol interact with olefin based plastics such as polyethylene and polypropylene. These interactions reduce free preservative concentration in a product

      Interaction between preservative and other ingredients. Surfactants that are present in concentration above their critical micelle concentration can solubilize lipophilic preservatives resulting in reduced free aqueous concentration and reduced antimicrobial activity, for example cyclodextrins form inclusion complexes with hydoxybenzoates

In conclusion, preservatives, either singly or in synergistic combinations remain necessary to prevent microbial contamination of multi-use liquid or semi-solid medicinal products, particularly from opportunistic pathogens. Non-inclusion can result in serious patient health consequences.

REFERENCES

1)      Hodges, N.A. and Hanlon, G. (2000). Antimicrobial preservative efficacy testing. In Handbook of Microbiological Quality Control: Pharmaceuticals and Medical Devices. Baird, R.M., Hodges, N.A., and Denyer, S.P., Eds. Taylor & Francis, London, pp.168–189.

2)      Hodges, N.A. and Denyer, S.P. (1996). Preservative testing. In Encyclopedia of Pharmaceutical Technology, Swarbrick, J. and Boylan, J.C., Eds. Vol. 13. Marcel Dekker, NewYork, pp. 21–37.

3)      Chapman, D.G. (1987). Preservatives available for use. In Preservatives in the Food, Pharmaceutical and Environmental Industries. Board, R.G., Allwood, M.C., and Banks, J.G., Eds. SAB Technical Series 22. Blackwell Scientific Publications, Oxford, pp.177–195.

4)      Denyer, S.P., Hugo, W.B., and Harding, V.D. (1985). Synergy in preservative combinations. Int. J Pharm., 25, 245–253.