Environmental Monitoring is a GMP requirement to ensure drug products are made in a non-contaminating environment. Andrew Ramage and Harshad Joshi, Cherwell Laboratories, give a scientific and regulatory perspective on considerations when using neutralisers in growth media
Getting a true picture of environmental bioburden requires the use of settle plates with the correct growth media and neutraliser
When working as a microbiologist in an aseptic manufacturing facility, getting accurate results from the environmental monitoring regime in cleanrooms, specifically from contact plates and swabs, is always a concern. Similarly, the accuracy of sterility testing results is a concern, especially when the pharmaceutical product formulation could have anti-microbial properties. It can be both disastrous and extremely frustrating when a contamination event takes place, particularly one involving a sterile product, and one which cannot be linked to a specific reason or root cause.
As its name suggests growth media is designed to offer ideal conditions to encourage microbial growth; unfortunately, it does not take much to reduce the efficacy of the media. Generally, microbiological testing in the pharmaceutical industry consists of: environmental monitoring; sterility testing of final products, intermediates and active pharmaceutical ingredients (APIs), etc. and the testing for specific undesirable micro-organisms. In all of these situations, there will be chemicals present that inhibit microbial growth.
It is important to stress that some micro-organisms are extremely difficult to kill and can survive in extreme environments – the presence of a particular chemical may stop it growing, but the micro-organism is not dead. In the right conditions, when the inhibitory chemical has been neutralised, it will start to grow again.
The inhibitory chemical can be described as ‘bacteriostatic’1, an agent that inhibits bacterial growth and/or reproduction1 and/or ‘fungistatic’ (for fungi). How these chemicals inhibit growth depends on the context. When surface sampling with contact plates is performed, there may be a chemical residue on that surface from the disinfectant used. That residue will be transferred to the growth media on application. Surprisingly, micro-organisms do survive in the presence of residue and will then be inhibited from growing on the contact plate.
There is also a scenario when performing air monitoring as well as surface monitoring that chemicals released by the pharmaceutical product itself will inhibit growth, especially in the production of antibiotics. From the testing of products and intermediates, part of the formulation may contain an inhibitory substance, so there will need to be a neutraliser to be able to truly confirm the absence of micro-organisms.
There is an expectation for neutralisers to be used in the testing of sterile and non-sterile products according to the harmonised methods.2 Some of the inhibitory agents and suggested neutralisers are found in the harmonised method for microbial enumeration tests (see Table 1).
|Interfering substance||Potential neutralising method|
|Glutaraldehyde, mercurials||Sodium hydrogensulphite (sodium bisulphite)|
|Phenolics, alcohol, aldehydes, sorbate||Dilution|
|Quaternary ammonium compounds (QACs), parahydroxybenzoates (parabens), bis-biguanides||Lecithin|
|QACs, iodine, parabens||Polysorbate (Tween)|
|Mercurials, halogens, aldehydes||Thiosulphate|
|EDTA (edetate)||Mg2+ or Ca2+ ions|
A number of the interfering substances in Table 1 are commonly found in disinfectants. Most manufacturers of solid media will sell versions with combinations of a neutraliser that will neutralise the most commonly-used disinfectants. Bearing in mind that most users will be rotating their disinfectants, the media will need to have the capability to neutralise multiple agents. The concentration of neutraliser is usually reasonably standard between prepared media manufacturers. If uncertain on how to neutralise the disinfectant, contact the manufacturer of the disinfectant for advice.
In terms of inhibitory substances arising from the manufactured products being tested, the knowledge of the product has to come from the R&D department or development team. It would be extremely useful to have a microbiologist as part of the development team, as the microbiologist needs to ascertain and understand the formulation of the product and the influence of the product matrix, as well as the use of the neutralisers and media composition. The other way to neutralise an interfering substance is to dilute it (more on this later). If dealing with antibiotics manufacture, the presence of antibiotics in the air and surfaces needs to be considered when performing environmental monitoring. The medium can contain appropriate enzymes to neutralise the antibiotic.
If the active product itself is antimicrobial, then this is also recognised in the pharmacopoeias. To quote from the non-sterile products section: ‘If no suitable neutralising method can be found, it can be assumed that the failure to isolate the inoculated organism is attributable to the anti-microbial activity of the product. This information serves to indicate that the article is not likely to be contaminated with the given species of the micro-organism. However, it is possible that the product only inhibits some of the micro-organisms specified herein, but does not inhibit others not included amongst the test strains or for which the latter are not representative’.2
Knowing what is in the product and what microbial growth it can inhibit is of great importance.
It is important to take a closer look at the types of product testing to highlight the regulatory requirements. There are typically two types of products – filterable and non-filterable.
Filterable products are those where the bioburden of the product has to be determined by the filtration method. The regulatory expectation is simple in these harmonised methods, all requirements suggest that filtration is the method of choice when analysing bioburden or sterility testing of the intermediate or final products that are filterable.
Most organisations will use the filtration process including a rinse with an appropriate diluent, e.g. peptone mixtures, such as buffered sodium chloride peptone or phosphate buffered saline. The number of rinse steps can be validated to accommodate into the standard operating procedure (SOP) such that recovery in the presence of product is achieved within the specified time using the specific 10–100cfu of representative challenge micro-organisms. In essence, the rinsing steps dilute the antimicrobial/ preservative substance out of the product matrix, while retaining any viable isolates on the membrane for growth.
Non-filterable products can be dealt with through dilution. The testing of non-filterable products utilises direct inoculation methodologies, where subset aliquots of manufactured samples are placed into microbiological media to encourage the growth of contaminants or selected micro-organisms via enrichment/selective media.
The logic within the dilution effect is that the product matrix, which consists of the antimicrobial/preservative compound, is diluted to a concentration that enables growth and therefore recovery of spiked 10–100cfu of representative challenge micro-organisms.
There is excellent guidance in The Pharmaceutical Microbiology Manual (PMM)3 that covers in detail the possible methods and rationale but this needs to be applied to each validation method appropriately.
Method validation is the process of determining whether a method is suitable for its intended use. Validation can be divided into primary and secondary tasks. Primary validation is an exploratory process for establishing the operational limits and performance characteristics of the method. Secondary validation is the process of gathering evidence that a laboratory can meet the specifications of the primary validation.
There is plenty of information available in the literature about validation criteria, but here is a list of other questions to consider before pursuing a validation plan for a method:
One needs to reflect on the validation method – and follow this with both a thorough, systematic plan and preparation.The written plan should be prepared for each step of the validation process. Stakeholders should review the study plan prior to implementation and each laboratory involved within the validation process should have a QA programme, as well adequate QC activities.
There is another school of thought whereby the method used is a recognised pharmacopoeia method and therefore doesn’t need validation. Why not just perform a verification step to test for bacteriostatic and fungistatic effect? My reflection is that the method of choice is about placing confidence in the testing. You can gain an important insight into how the products are behaving in microbiological methods, so are therefore armed to think using a risk-based approach.
Does your growth media have the correct neutraliser? Contact plates commonly use them
It may be stating the obvious, but it can be overlooked – the neutraliser chosen should not have a detrimental effect on the growth potential of the growth medium. One way to ensure that a neutraliser is not detrimental to growth potential is to compare the growth potential of the medium with neutralisers against that of the equivalent without neutraliser. For example, in terms of solid media, certain neutralisers can assist in the desiccation of agar plates, but there are means to counteract this issue.
Some manufacturers, as standard, will add additional agar to strengthen the growth medium. In the case of 90mm plates, which are at greater risk of desiccation and cracking, deeper filled versions of those plates are offered. There are also versions containing a small amount of glycerol to help reduce the rate of desiccation. The version chosen depends on the environment to which the plates will be exposed.
It is important that the exposure time of the settle plate is assessed to ensure that the proportion of weight loss (through the loss of moisture) does not result in a loss of growth-promoting properties. A second important concern regards the avoidance of cracks in the agar, which might render reading sections of the exposed plate impossible4.
As microbiologists, we must be aware of the challenges involved when choosing the correct growth media, in the correct container, let alone what neutralisers (if any) to choose. The above advice should help in not only choosing the correct neutraliser but also in using the most appropriate method to meet regulatory requirements.
1. McGraw-Hill Concise Dictionary of Modern Medicine, McGraw-Hill, New York Written in English. 2002
2. European Pharmacopoeia 9th edition, Chapter 2.6.12 Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests
3. Pharmaceutical Microbiology Manual 2014, US FDA: http://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM397228.pdf
4. Sandle T., European Journal of Parenteral & Pharmaceutical Sciences 2015; 20(2): 45-50 Further reading http://www.cherwell-labs.co.uk/2016/01/08/ why-use-neutralisers-in-your-environmental-monitoring-plates/