Handbook of Microbiological Quality Control in Pharmaceuticals and Medical Devices

Rosamund M. Baird, Norman A. Hodges, Stephen P. Denyer, Rosamund M. Baird, Norman A. Hodges, Stephen P. Denyer

• Publisher: CRC Press
• Year: 2000
• ISBN:
  9781135739980
• Edition: 1
• Topic: Medicina
• Subtopic: Farmacología

Microbiologists working in both the pharmaceutical and medical device industries, face considerable challenges in keeping abreast of the myriad microbiological references available to them, and the continuously evolving regulatory requirements. The Handbook of Microbiological Quality Control provides a unique distillation of such material, by provi

1
Safe Microbiological Practices

ANTHONY W.SMITH

Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom

1.1 Scope and aims

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.

1.2 Overview of current legislation

The key feature of current legislation is risk assessment, recognizing that only after all the component elements of a process have been fully analysed, can effective and safe procedures be put in place. The three major pieces of legislation affecting handling of micro-organisms are the:

• Health and Safety at Work Act 1974
• Control of Substances Hazardous to Health (COSHH) 1994
• Genetically Modified Organisms (Contained Use) Regulations 1992 (amended in 1996)

A major change in legislation arose in COSHH 1994 which, unlike COSHH 1988, stipulated that control measures minimizing risk of illness resulting from occupational exposure should be extended to biological agents as well as chemical agents. Resulting from the Biological Agents Directive 90/679/EEC, the definition of a biological agent used in COSHH 1994 is:

‘any micro-organism, cell culture or human endoparasite, including any which have been genetically modified, which may cause infection, allergy, toxicity or otherwise create a hazard to human health’

Therefore, for the first time COSHH 1994 specifically stipulated the need to carry out an assessment of risks and to select and maintain measures where exposure to any microorganism might present a hazard to the health of any person. Clearly, all micro-organisms vary in their ability to cause infection in humans. Indeed, those micro-organisms used in pharmaceutical quality assurance procedures range from organisms not associated with any human disease to those which have significant pathogenic potential.

1.3 The Advisory Committee on Dangerous Pathogens (ACDP)

1.3.1 Background

The ACDP was set up in 1981 and was the successor to the Dangerous Pathogens Advisory Group. Its remit is to advise the Health and Safety Commission, the Health and Safety Executive (HSE) and Health and Agriculture Ministers on all aspects of hazards and risks resulting from exposure to pathogens.

1.3.2 Classification

ACDP has categorized biological agents into four Hazard Groups based on the pathogenic potential to humans, hazard to employees, probability of transmission to the community, and availability of effective prophylaxis or treatment. These Hazard Groups are classified as follows:

• Hazard Group 1: A biological agent unlikely to cause human disease.
  
• Hazard Group 2: A biological agent that can cause human disease and may be a hazard to employees; it is unlikely to spread to the community, and there is usually effective prophylaxis or effective treatment available.
• Hazard Group 3: A biological agent that can cause severe human disease and presents a serious hazard to employees; it may present a risk of spreading to the community, but there is usually effective prophylaxis or treatment available.
• Hazard Group 4: A biological agent that causes severe human disease and is a serious hazard to employees; it is likely to spread to the community and there is usually no effective prophylaxis or treatment available.

The Approved List includes bacteria, viruses, fungi and parasites in Groups 2, 3 and 4. However, the guidance notes emphasize that it should not be assumed that absence from the list means that the agent is a Group 1 organism. Cell lines, which may become increasingly important in pharmaceutical processes, are not included in the Approved List; however, it is expected that they will be handled in laboratories equipped to conform to the containment requirements for Hazard Group 2 organisms. None of the organisms used in common pharmacopoeial tests is in Hazard Groups 3 and 4; however, a number of species are in Hazard Group 2 (Tables 1.1–1.3). This has important legal implications since notification of use and containment conditions are mandatory.

Table 1.1 Micro-organisms used in European Pharmacopoeia (1997) Test for the efficacy of antimicrobial preservation

Table 1.2 Bacteria recommended in the European Pharmacopoeia (1997) as biological monitors for sterilization processes

The COSHH regulations call for notification to HSE 30 days in advance of first use of organisms in Groups 2, 3 or 4 where it is the intention to culture, propagate and store the organisms. For a new pharmaceutical Quality Assurance (QA) laboratory the range of organisms used need not be restricted to named organisms in Hazard Group 2, rather it is sufficient to notify use of all organisms in Hazard Group 2.

Table 1.3 Test organisms for antibiotic assays in the United States Pharmacopoeia (XXIII, 1995) and European Pharmacopoeia (1997)

Tables 1.1–1.3 list the commonly used organisms in pharmaceutical QA processes, together with their ACDP classification.

1.3.3 Containment Level 1

Each of the four Hazard Groups is associated with a degree of laboratory containment relevant to the risk of occupational and community exposure from organisms within the Group. Laboratory Containment Level 1 is suitable for work with agents in Group 1 and should conform to Good Laboratory Practice. The main features of a Containment Level 1 facility are described in full since there are elements pertinent to laboratory design and to establishing Local Rules.

1. The laboratory should be easy to clean. Bench surfaces should be impervious to water and resistant to acids, alkalis, solvents and disinfectants.
2. Effective disinfectants should be available for immediate use in the event of a spillage.
3. If the laboratory is mechanically ventilated, it is preferable to maintain an inward flow of air while work is in progress by extracting room air to atmosphere.
4. All procedures should be performed so as to minimize the production of aerosols.
5. The laboratory door should be closed when work is in progress.
6. Laboratory coats or gowns should be worn in the laboratory and removed when leaving the laboratory area.
7. Personal protective equipment, including protective clothing, must be stored in a welldefined place, checked and cleaned at suitable intervals and repaired or replaced if found to be detective.
8. Personal protective equipment which may be contaminated must be removed on leaving the working area, kept apart from uncontaminated clothing and decontaminated and cleaned or, if necessary, destroyed.
9. Eating, chewing, drinking, taking medication, smoking, storing food and applying cosmetics should be forbidden.
10. Mouth pipetting should be forbidden.
11. The laboratory should contain a basin or sink that can be used for hand washing.
12. Hands should be decontaminated immediately when contamination is suspected and before leaving the laboratory.
13. Bench tops should be cleaned after use.
14. Used glassware and other materials awaiting disinfection should be stored in a safe manner. Pipettes, for example, if placed in disinfectant should be totally immersed.
15. Contaminated materials whether for recycling or disposal, should be stored and transported in robust and leak-proof containers without spillage.
16. All waste material, if not to be incinerated, should be disposed off safely by other appropriate means.
17. Accidents and incidents should be immediately reported to and recorded by the person responsible for the work or other designated person.

1.3.4 Containment Level 2

Laboratory Containment Level 2 must be used for work with agents in Hazard Group 2. Since some organisms used in pharmaceutical QA procedures are in Hazard Group 2, this Containment Level will represent the minimum standard for safe handling of microorganisms. There are some additional points which are statutory requirements to operate a laboratory at this level, over and above those described for Laboratory Containment Level 1, and in addition some control measures change from ‘should’—indicating the ACDP’s strong recommendation—to ‘must’—indicating a requirement defined in legislation. The additional features required for a laboratory to operate at Containment Level 2 are as follows:

1. Access to the laboratory is restricted to authorized persons.
2. There must be specified disinfection procedures.
3. If the laboratory is mechanically ventilated, it must be maintained at an air pressure negative to atmosphere while work is in progress.
4. Bench surfaces must be impervious to water, easy to clean and resistant to acids, alkalis, solvents and disinfectants.
5. There must be safe storage of biological agents.
6. Laboratory procedures that give rise to infectious aerosols must be conducted in a microbiological safety cabinet or isolator, or be otherwise suitably contained.
7. The laboratory should contain a washbasin near the exit. The taps should be of a design that can be operated without being touched by the hands.
8. Hands should be decontaminated immediately when contamination is suspected, after handling infective agents, and before leaving the laboratory.
9. An autoclave for the sterilization of waste materials should be readily accessible in the same building as the laboratory, preferably in the laboratory suite.
10. Materials for autoclaving should be transported to the autoclave in robust containers without spillage.
11. There should be a means for the safe collection, storage and disposal of contaminated waste.

1.4 Risk assessment and Control of Substances Hazardous to Health Regulations 1994

In essence, these regulations require that suitable and sufficient assessment of risks to health be made by employers before work with chemicals or biological agents is started. Moreover, they require that appropriate measures to prevent or control exposure are in place, and that procedures exist to assure that measures are always in operation.

1.4.1 Assessment of risk

Assessment of risk requires a pragmatic approach to protocol design and a thorough and detailed understanding of the process under consideration. In the context of handling micro-organisms in the laboratory, the two ‘worst-case’ scenarios are loss and escape of containment and infection of the laboratory worker. The excellent book by Collins (1993) reviews in detail the history of laboratory-acquired infections, and the many important lessons that have been learned and applied to safe microbial handling.

1.4.2 Eight steps to risk assessment

• Step 1: Define the work activity.
• Step 2: Identify the hazards.
• Step 3: Identify the control measures.
• Step 4: Evaluate the risks.
• Step 5: Maintain the control measures.
• Step 6: Arrive at a conclusion.
• Step 7: Record the assessment.
• Step 8: Review the assessment.

For a pharmaceutical QA process, Step 2 is easily satisfied by reference to the tables in this chapter and to the ACDP Categorization of biological agents according to hazard and categories of containment (4th edition, 1995). Steps 3 and 4 are somewhat interdependent in that effective control measures cannot be considered independently of the risks involved. Step 3 covers the issue of control safeguards such as biological cabinets, personal protective equipment and personnel instruction, training and supervision (discussed later), whereas Step 4 requires evaluation of the risks, such as transmission routes and potential for exposure, which remain despite the control measures put in place.

It is interesting to note that many surveys, reviewed by Collins (1993), indicate that an infection is more likely to arise simply as a consequence of working with a microorganism than through an accident, although clearly the risk assessment should consider both possibilities. Assessment of risk will result in prohibiting some activities and acknowledging that in some procedures a degree of risk is unavoidable. In such circumstances, the challenge of effective risk assessment is to ensure risk minimization, for example through process and facility design and monitoring and personnel training. It should be implicit in risk assessment that the process is not a ‘once-only’ event and that it should be modified and up-dated as new information becomes available. Much of what we may take for granted for safe practices in the microbiology laboratory, although perhaps previously developed on an ad hoc basis, result from risk minimization based on an awareness of the likely routes of self-contamination and infection. Examples of development of safe practices based on knowledge of the principal routes of infection are given below:

1. Through the mouth. Therefore eating, drinking, smoking and application of cosmetics in the laboratory must be prohibited as prescribed in the conditions for operating a Containment Level 1 facility. Mouth pipetting is also a significant hazard and is prohibited. Transfer of micro-organisms to the mouth (or hair) by contaminated hands is a personnel training issue.
2. Through the skin. Laboratory personnel applying cosmetics or handling microorganisms with cuts and abrasions are clearly risks, and again represent training issues. Accidental inoculation with a ‘sharp’ is always a risk and so consideration should be given to substitution wherever possible. Accidental inoculation with a ‘sharp’ is always a risk and so consideration should be given to substitution wherever possible.
3. Through the eye. There is evidence that some organisms, perhaps hepatitis B, could be transmitted via the eye. While this is not directly relevant to pharmaceutical QA procedures, it should always be considered when handling any unscreened blood products, such as serum samples for drug level monitoring and pharmacokinetic analysis. Of more direct concern are organisms such as Staphylococcus aureus and Pseudomonas aeruginosa, which are well documented to cause eye infections. These may be transmitted by direct transfer by contaminated fingers or perhaps from aerosols. There are a number of issues here, notably aerosol minimization and personnel training. It is also prudent to recommend that safety spectacles are worn at all times in the microbiology laboratory.
4. Through the lungs. Again, steps to minimize aerosol generation and use and maintenance of biological safety cabinets are important here.

In the spirit of developing risk assessment based on previous experience it is worth noting the types of accidents which precede infection (Collins, 1993). These are spillage and splashes, needles and syringes, sharps and broken glass, and aspiration through pipettes. It is therefore essential to the whole risk assessment process that procedures are in place such that the potential for these accidents to occur is either removed or, at the very least, minimized.

The remaining steps of risk assessment centre largely on the maintenance of records and procedures to ensure the continued safety of the operator. These measures will include issues such as maintenance and testing of biological safety cabinets, environmental monitoring and personnel training before commencing a procedure and ‘in-service’. The record of the assessment is clearly an important document and must be cross-referenced to Standard Operating Procedures (SOPs; see section 1.1.10). In the context of employee safety, it is important not to assume simply that the presence of SOPs guarantees safe practices. ‘SOP fatigue’ is well-recognized, and periodic checks should be carried out to ensure that procedures are followed. Finally, and perhaps most importantly, it must be recognized that risk assessment is a continuous process. It is not acceptable to continue a procedure over months or years without reviewing the risk assessment. A review process should be carried out at least once every five years, or more frequently if new information becomes available. This might include additional information on the pathogenic potential of the micro-organisms being used, or perhaps new innovations in laboratory safety equipment or changes in experimental methods.

1.4.3 Risk ranking

Clearly, not all risks are of the same importance and this is reflected in the rigour of the control measures to be adopted. In the case of handling micro-organisms this can be readily appreciated since the likelihood of exposure to organisms such as the harmless Bacillus stearothermophilus and the Category 2 pathogen Pseudomonas aeruginosa is the same, and yet the consequence of exposure to the latter is greater. In the event that it is not possible to deal with all risks equally, risk ranking is an approach to managing risks in the most important order of priority.

Risk level=Probability of occurrence×Severity of occurrence

Probability levels

5 Certain or will occur at some time

4 Probably or very likely to occur

3 Possible or likely to occur

2 Remote or unlikely to occur

1 Improbable or very unlikely to occur

Severity levels

5 Fatality

4 Serious injury (hospitalization)

3 Moderate injury (>3-day absence)

2 Minor injury (first aid only)

1 No injury

Combining the two risk elements giv...