Microbiological Suitability Tests of Non-Sterile Preparations

   

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In numerous regulations and pharmacopoeias it can be read - mostly in similar words - that methods used for testing products or substances must be verified specifically as concerns their suitability. This is then called "performance of a product-specific suitability test". This approach is used inter alia when testing non-sterile preparations for sterility or for microbiological purity. In the following, this issue is described in greater detail with regard to the testing of non-sterile preparations according to the harmonised chapters 2.6.12 of the European Pharmacopoeia or to chapter <61> of the United States Pharmacopeia (USP).

Suitability tests can be carried out in the own laboratory or at specialised contract laboratories. The laboratory LS SE carries out hundreds of such suitability tests commissioned by the customer per year. The range of these suitability tests comprises all sorts of finished medicinal products, medical devices, raw materials, excipients, active ingredients, in-process controls, packaging materials, food supplements or consumer products. Some years ago, finished medicinal products played the main role but now the demand for tests on raw materials, excipients and active ingredients is constantly increasing. It often poses a particular challenge to test raw materials, excipients and active ingredients in their concentrated form for suitability of the method instead of testing them as one of several components in a finished medicinal product. Special properties of individual substances often are less apparent in the mixture of a finished medicinal product. Therefore, during testing these properties pose less often a problem than when these substances have to be tested isolated in a larger quantity. The special properties which can turn out to be a challenge during testing are inter alia:

1. extreme ph values of concentrated acids and lyes
2. particularly pronounced antimicrobial properties such as antibiotics present as API
3. insolubility of the matrix in aqueous media
4. matrices the nature of which impedes a microbiologically compatible treatment, such as insoluble crystalline structures, matrices with a melting point > 40 °C
5. poor homogenisability of sticky greasy matrices in aqueous media
6. adverse reactions with the solution medium such as a strong heat development or swelling of the matrix in the course of the solution process

For the treatment of special matrices it is often necessary to resort to a very individual processing and sample preparation. For this, it is also possible to apply different tools and methods such as:

1. usage of special solvents or
2. application of thermal processes
3. application of mechanical processes
4. usage of further tools such as prefilters etc.

Prior to their application the advantages and disadvantages of these potential preparation alternatives must be carefully balanced against each other. Not each procedure leading to a homogenous sample preparation can be recommended unreservedly from a microbiological perspective. But then again, they allow the actual testing of the matrix in the first place. A lumpy resinous sample matrix can be shredded to a mostly homogenous sample material by means of a high-performance mixer. This allows for a better sample preparation in contrast to the starting material which would only enable a wetting of the outer surface. But at the same time, the mechanical shredding causes the danger that vegetative microorganisms might be damaged or killed because of heat development or of shear forces. This means that the danger of false-negative results increases. If the sample preparation is heated for better homogenisation in a water bath having a maximum temperature of 40 °C this will have no influence on the original microorganism load of the matrix. But if the preparation is left for one hour in a water bath having a temperature of 40 °C this might already lead to a multiplication of micro-organisms such as Escherichia coli. Now, it is the other way around and the danger for falsepositive results increases.

The classical methods reach their limits more and more frequently, or they can only be used applying a risk assessment. One concrete example is an API for which a specification of TAMC (total aerobic microbial count) max. 100 CFU / g and TYMC (total combined yeasts / moulds count) max. 10 CFU / g has to be complied with when testing for microbial purity. In the course of the development of methods different solvents, various dilutions and the addition of different culture media, modifications of the ph value as well as the usage of the pour plate method and the membrane filtration method have been tried. In the end, it was demonstrated that only by using a special prefilter system, acceptable recoveries of the prescribed test microorganisms were possible. At the same time the results also showed, however, that the prefilter system itself had a negative influence on individual test microorganisms and that therefore, the pharmacopoeia requirement of the absence of toxicity of the test method used could not be complied with for all test microorganisms. Another example is the testing of a raw material which reacts with a strong heat development and a simultaneous sharp increase of the ph value when the aqueous medium is added for the sample preparation. The attempt to set the ph value to the prescribed range of ph 6-8 leads to a continuous chain reaction.

In cases like these, where the development of a valid method is possible only to a limited extent or not at all, it is mandatory to carry out a risk assessment as concerns the vulnerability of the matrix to microbial contamination. The determination of the water activity can be an important element in such a risk assessment. The water activity is a measure for the free water in the matrix which consequently is at disposal for metabolic processes. The water activity values can be between 0 and 0.99. Only pure water has a value of 1. The higher the value, the microbiologically more instable is the matrix. This value has been used in the food production as important parameter for the products' quality for a long time. Methods of food preservation used for centuries such as salting (salting of fish), sugaring (production of jam) or drying (dried fruit or dried meat) aim at reducing the water activity and consequently, at delaying the microbial deterioration of food.

These methods can easily be transferred to other industries. Little changes in the content of sodium chloride, sucrose or alcohol can reduce the water activity of a formulation substantially. Lower water activity values are accompanied by manifold microbiological effects such as an extended lag-phase, slower growth rates or a reduced production of toxins. But apart from the microbiological effects the water activity can also have an effect on other properties of the matrix, for instance on the content of proteins and vitamins, on the stability and shelf life, or solubility and texture.

In order to use the effects of a reduced water activity value best possible it is indispensable to know the water activity values of selected microorganisms and preparations.

Table 1: Water activity values of selected microorganisms

Extract from USP <1112>, table 1

Table 2: Water activity values of selected medicinal products and OTC products

Extract from USP <1112>, table 2

All this information can be found in chapter <1112> of the current version of the USP. This chapter also contains a practical overview over the potential for application of the water activity value in the pharmaceutical industry. Further information, especially as regards the verification and validation of the method can be found in the new chapter <922>. This chapter became applicable on 01. May 2021.

About the Author
Christine Weiß is head of QC and head of the department for testing of non-sterile products at Labor LS.