Handbook of Microbiological Quality Control Pharmaceuticals and Medical Devices
Microbiological concerns in product manufacture, regardless of whether it involves pharmaceuticals, cosmetics, toiletries or medical devices, continue to challenge the minds of those associated with their production. Whilst much is known about how contamination arises, and control methods are well understood, there is no room for complacency; every microbiologist knows that from time to time a contamination issue inevitably will arise and must be promptly addressed. Detailed practical guidance on appropriate microbiological methods is an absolute necessity if such problems are to be resolved speedily and to the satisfaction of all involved.
This handbook attempts to fulfil this role in the laboratory, bringing together a wealth of information from diverse sources into a single volume. The reader is guided throughout the book by the advice and recommendations of expert contributors with an in-depth knowledge and personal experience of the likely practical issues to be faced by those working in the microbiology laboratory. Beginning with safety concerns and the design of laboratory facilities, guidance is provided on the choice and requirements of culture media, followed by a detailed consideration of specialized microbiological methods, including chapters on sampling, enumeration and identification of micro-organisms, involving both traditional and rapid techniques, sterility testing, environmental monitoring, preservative efficacy testing, endotoxin testing and antibiotic bioassays. Whilst established reference methods are of necessity included here, there is also an abundance of information on how these tests are interpreted in common practice. Cleaning and disinfection considerations, frequently a much neglected subject, are also addressed within this volume. Hazard analysis and audit approaches complete the routine tasks facing the company microbiologist and the reader is directed in how this might be successfully achieved. Finally, a number of worked case examples have been included to assist those involved in such every day calculations.
Although the book is intended as a complete reference source in its own right, it is in fact a companion book to the Guide to Microbiological Control in Pharmaceuticals and Medical Devices, shortly to be published in its second edition by Taylor and Francis. The intention here was to provide in the Handbook detailed practical information on methodology of the techniques advocated and identified for microbiological quality control in the Guide. The Handbook is therefore extensively cross-referenced to the Guide, and it is hoped that the collected wisdom of these two books provides the laboratory microbiologist with a unique, valued and much consulted reference source.
Safe Microbiological Practices
This chapter sets out to offer practical advice on safe microbiological practices to all persons involved with microbiological quality assurance. It describes the current legislation relevant to handling micro-organisms and discusses the key elements of safe microbial practices, namely facility design, personnel and training, and validation and monitoring of procedures.
Culture Media Used in Pharmaceutical
Microbiologists working today have considerable choice in the range and type of culture media available to them. This has largely arisen from the expansion of microbiology from medicine in its early days to agriculture, food manufacturing, water production and pharmaceutical applications, with each discipline having its particular and individual requirements for culture media. The design and subsequent development of culture media have largely reflected these requirements, but have also been influenced by the introduction of selective and differential indicator media, containing an array of inhibitory substances in various quantities and combinations. In addition, culture media, initially developed for a particular requirement, have sometimes later been successfully adopted for use in other disciplines of microbiology. In the case of pharmaceutical microbiology, however, a rather more conservative approach has been taken over the introduction of new media; the range of media used has remained largely consistent over the years, undoubtedly reflecting in part pharmacopoeial recommendations.
Sampling: Principles and Practice
Sample selection is the initial stage of a process whereby data on the characteristics of a batch are collected for evaluation. By definition, only a fraction of the batch is sampled for testing; clearly, therefore, that fraction must be representative of the batch in question. Since the fate of the batch depends upon the results generated from that first sample, sample selection must be regarded as a critical process and an essential part of the quality assurance system. As with any sampling process, appropriate predetermined indicators of quality, known as attributes, must be identified which reflect the characteristics of the batch; these are assumed not only to be homogeneously distributed throughout the batch but also to be recovered during a random sampling scheme. In microbiological sampling, however, these characteristics may not necessarily be randomly distributed throughout the batch and the sampling scheme should therefore reflect this. Sampling schemes currently employed in the pharmaceutical industry are increasingly based on an analysis of vulnerable points in a system, otherwise known as hazard analysis and control of critical points (HACCP), as discussed in more detail below.
Sampling for microbiological contamination poses additional problems. Owing to the risk of contamination, it should precede other forms of sampling. Previously unopened containers should be sampled, and suitably marked so that they can be easily identified in the case of failure. Additionally, special handling precautions should be adopted to minimize the risk of accidental contamination. These precautions will vary according to the sample type: when sampling an aseptically made product as part of a sterility test, specialized environmental conditions will be required (i.e. a Class A laminar flow cabinet in a Class B clean room, or an isolator located within a Class D clean room environment); however, for other less critical products, a dedicated sampling area in an uncontrolled environment may well suffice. Furthermore, since the microbiological characteristics of a given sample may change over time, account should be taken of this if sampling is carried out on a second occasion: localized environmental conditions may allow a contaminant to flourish in one situation but result in its death in another.
Enumeration of Micro-organisms
Microbiologists from all disciplines are concerned with estimating the number of microbial contaminants in samples under examination. This estimate in turn can be used to assess the risk presented to the recipient by that population of contaminants. Risk assessment forms an important part of the microbiologist’s role.
Various techniques are available to the microbiologist seeking to count the number of micro-organisms in a given sample, the method of choice being determined by such considerations as cost, time-constraints, equipment required, sample specification, anticipated sample quality, the physical nature of the sample and, of course, personal preference. This chapter reviews the more commonly employed approaches to microbial enumeration that are generally available to the microbiologist working in pharmaceutical quality control. The practices described are ‘traditional’ procedures; recently developed ‘rapid methods’ are addressed only superficially here as the topic is more fully reviewed in Chapter 7.
Identification of Microorganisms
5.1 Circumstances in which microbial identification is undertaken
Micro-organisms isolated from the pharmaceutical manufacturing environment, or which occur as contaminants of inadequately preserved products, require identification. There may also be other circumstances when process contaminants require rapid and reliable identification if heavy financial losses are to be avoided; contamination of a starter (inoculum) culture in a fermentation process is one such example. This chapter, however, is intended primarily for persons responsible for ‘routine’ identification of environmental and spoilage isolates rather than those working in more specialized fermentation or biotechnology facilities.
The need for identification is not, of course, simply to satisfy curiosity or to demonstrate a conscientious approach to control of the microbial quality of the manufacturing environment. Fast, accurate identification affords a practical benefit too, because certain organisms often arise in the same situations or from the same sources, for example, from dust, water or other raw materials, or from personnel. Thus, a reliable identification is often the first step in locating the source of a contaminant and formulating a strategy for its avoidance in future manufacturing batches.
Pharmacopoeial Methods for the Detection of Specified Micro-organisms
Tests for the detection of four named bacteria (Escherichia coli, salmonellae, Pseudomonas aeruginosa and Staphylococcus aureus) in pharmaceutical raw materials and finished products are described in the United States Pharmacopoeia (USP) (1995), and in the European Pharmacopoeia (EP) (1997) and British Pharmacopoeia (BP) (1999) (where the tests are identical). The EP and BP additionally describe a test for the detection of Clostridium perfringens. These organisms are singled out for special attention in this way because they represent particular infection hazards to the patient, or because their presence is a criterion of the quality of raw materials.
The detection tests are typically applied to raw materials of natural or biological origin (starches, gums, gelatin, talc, etc.) where there may be a background of unrelated organisms which may grossly outnumber the species of interest. Consequently, specific procedures for sample preparation and preliminary enrichment cultures are used to permit the organism of interest to increase in concentration and so become more readily detectable. The final stages of the detection tests invariably involve the use of solid selective media which, after incubation, are examined for the presence of colonies conforming to the standard textbook descriptions of the bacteria in question; any such colonies are then subjected to confirmatory biochemical or immunological tests. It is these final stages of the testing procedures which are the most problematical, because the textbook (or pharmacopoeial) descriptions of the bacteria are often imprecise and several relatively harmless organisms can also conform to these descriptions—leading to the possibility of false-positive identifications. The problem is compounded by the paradox that the tests are often undertaken by personnel working for companies using high-quality raw materials in which the organisms are rarely, if ever, encountered.
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