Preventing Contamination with Preservatives

Q What are the main considerations regarding the use of antimicrobial preservatives?

Microbiological safety is important for all pharmaceutical products, and should be considered during development, manufacturing and, importantly, in the product's subsequent storage and use. Any product that contains water offers an environment in which microorganisms can proliferate in from very low levels. A product that is susceptible to microbiological growth is deficient in several ways:

• It may pose a risk to the patient if it supports the growth of pathogenic microorganisms.

• The API may degrade due to microbial metabolism, leading to loss of product efficacy.

• Microbial growth may render the product organoleptically unacceptable to the patient.

• The metabolites of microorganisms may modify the API chemically, resulting in physical instability.

Greg Snowdon

Pharmacopoeias require the manufacturer to show that the product will both not support the growth of organisms if exposed to microbiological contamination and reduce the magnitude of contamination to acceptable levels. The necessary test is called a challenge test or a preservative efficacy (PE) test.

If the preparation itself does not possess sufficient anti-microbial properties, then preservatives must be added. In these cases, preservatives can extend a product's shelf life and are essential to prevent the growth of microbes and stop product degradation. Common preservatives in use in the pharma industry include parabens (such as methyl or propyl), benzyl alcohol, chlorobutanol and a variety of acids such as benzoic or salicylic.

However, there is an upward trend in the industry to reduce the level of preservatives. This makes the requirement to show that the preparation itself is effective in preventing microbial contamination even more important.

Q Is simply adding a recognised preservative sufficient to control contamination?

Even if a preservative is known to be efficacious, it is still important to consider its potential interactions with the preparation. The API and the formulation into which the preservative is incorporated can decrease, or possibly increase, the preservative's efficacy. For example, the preservative may react with the formulation in such a way that it reduces the concentration over time or renders it unavailable to interact with the microorganisms. Both instances could result in contamination being uncontrolled.

There is also a consideration around the expected activity of the preservative against different types of microorganism. Some preservatives may not be as efficacious against fungi as they may be against bacteria (or the other way round), in which case a combination may be necessary.

Q How can packaging material affect a preservative?

Packaging can also affect a preservative, particularly if a volatile preservative is used. If the preservative leaches into the packaging it can reduce the preservative levels below active concentrations. This factor is especially important to consider when moving a product to a new container material, such as from glass to PET or from PET to something like PPE.

Q And what about storage conditions?

Although it is true that a large number of pathogenic organisms will not proliferate at low temperatures, there are still plenty that aren't as inhibited by refrigeration as you may think. If these start metabolising, then you can end up with some interesting by-products that may not interact particularly well with the preparation—not to mention the risk introducing high levels of microorganisms to the patient.

There's also the human factor to consider. Just because it says 'keep in the fridge' doesn't guarantee that someone won't leave it out! Some bacteria have impressively fast growth cycles. E.coli, for example, can double every 20 minutes.

Q How can a preservative's efficacy be shown?

Efficacy of an antimicrobial preservation is commonly performed at two points in the product development process. The first point is during the formulation stage, where developers are looking at a range of the most promising preparations. The preservative is incorporated and the test carried out. This should narrow down the field of potentials, or confirm those that have already been chosen.

The second point is during stability storage, usually at the beginning and end of storage. A middle time point, however, can also be beneficial to catch failures earlier in the cycle. Testing during stability storage is perhaps the most important testing point because it highlights potential efficacy problems with the preservative. For example, it will show if the preservative degrades over time, which would require a higher starting level. Even if the preservative doesn't degrade, it may not remain biologically available. The test will show this. Different temperatures or humidity may cause the preservative to fall out of suspension or solution. The efficacy test will show any microbiological problems, the physical and chemical tests that are carried out concurrently can often then tell you why.

The actual testing method is very straightforward. The preparation is contaminated with a high level of chosen microorganisms and, at specific time points, the remaining level of each of the organisms is determined.

The Pharmacopoeias prescribe a set of test organisms that cover the basic types: Staphylococcus aureus for the Gram positives, Pseudomonas aeruginosa for the Gram negatives, Candida albicans for the yeasts and Aspergillus brasiliensis (formerly Aspergillus niger) for the moulds. The US Pharmacopeia includes Escherichia coli in its standard challenge organism list, while the European Pharmacopoeia reserves E.coli for use in oral preparations.

There is also the option of including organisms of interest, such as Zygosaccharomyces rouxii (anosmotolerant spoilage yeast) for high-sugar preparations, environmental isolates (either from a known contamination issue, or one that keeps appearing when you check the manufacturing area).

There is currently no requirement to test against antibiotic-resistant "super bugs". Although these organisms have evolved a method for protecting themselves from a variety of antibiotics, most preservative systems work along different pathways and so will have the same effect against both normal and 'super' varieties.

The Pharmacopoeias have a list of the minimum required efficacy based on the product type, be it oral, topical or parenteral. The requirement is expressed as a Log₁₀ reduction from the microbiological challenge level after a defined period of contact. Depending on the preparation type the fungi may only be required not to increase in numbers from the previous value. In addition, there are tests against a variety of organisms to ensure as wide an effect as possible. If the preparation doesn't meet these requirements, it's back to the formulation stage!

A common pitfall at this point is not realising that the Pharmacopoeias are not harmonised on efficacy testing. There may be different requirements and testing to one may not cover the others. A few things can be considered in the formulation stage that should help reduce the proportion of failures in the preservative efficacy test, and they all revolve around the premise of knowing your preservative:

• At what level is the preservative expected to be effective? For example, benzoic acid is effective against E.coli at 120 ppm, but requires 2000 ppm to be effective against A.brasiliensis.

• How soluble is the preservative in the product? The preservative may be efficacious at 1%, but if the solubility in the preparation is only 0.1% then it probably won't work.

• Is the preservative stable? Or is it likely to degrade with time, or temperature or by reaction with the preparation? All of these will reduce or negate its efficacy.

Some of above can be estimated in advance, but in the end the only way to know is to try.

Greg Snowdon is a microbiology scientist at RSSL.