Microbiology and Good Laboratory Practice in GMP

Defining micro-organisms Micro-organisms are defined as living organisms that cannot be seen with the naked eye. The presence of micro-organisms not only has the potential to cause product degradation, but also to cause disease as the result of administration of the product. Families:

• Bacteria
• Fungi (yeasls and moulds)
• Algae
• Protozoa

Characteristics of eukaryotes and prokaryotes Eukaryotes:

• Cell has a nucleus
• Examples: yeasts, moulds, algae

Prokaryotes:

• Cell does not have nucleus
• Examples: bacteria, archaebacteria

How to classify bacteria Bacteria can be classified according to:

• Size
• Shape (e.g. rod, coccoid)
• Arrangement
• Cell wall structure (e.g. gram-positive, gram-negative)
• Oxygen requirements (e.g. aerobes, anaerobes, facultative anaerobes)
• Temperature requirements (e.g. mesophile, thermophile)
• Nutrient source (e.g. carbon, sugars)
• Biochemical reactions (e.g. ferment sugars to produce acid and gas)

Difficulties in quantifying micro-organisms Micro-organisms are constantly multiplying and dying. They multiply through binary fission, and can produce up to 17 generations overnight. To get a true count, multiplication must be slowed or stopped altogether. Since micro-organisms rarely occur alone, estimates must be made. Estimates can be made through Total Viable Aerobic Count (TVAC) by counting the number of colony forming units (CFU). CFU counting assumes one colony has originated on the media from one cell; this introduces significant error into the count. Some micro-organisms do not grow together due to toxicity, in the same temperature, moisture, oxygen, nor nutrient conditions. Colony Forming Unit When a plate is being read, it is the visual colonies on the plate that are quantified. Each colony on the plate originated from a single bacterial unit called a colony forming unit. Objectionable Organisms Concern of objectionable organisms “Objectionable” organisms are organisms that should not be present in a product, because:

• They can be potentially pathogenic to users, especially to immunocompromised patients

They may cause degradation of the product, thus compromising therapeutic effectiveness GLP Requirements Role of the laboratory The role of the microbiological quality control laboratory is to find, and sometimes enumerate and identify, micro-organisms present in samples. Samples can be from:

• Sterile, pre-sterilisation, or non-sterile products
• Environment
• Raw materials, e.g. water
• Ancillary materials, e.g. lubricating fluids

Tests can include:

• Preservative efficacy or effectiveness test (PET)
• Microbial limit testing
• Sterility
• Anti-microbial
• Bioburden program
• Growth promotion quality control (fertility)
• Bacteriostasis/fungistasis (stasis)
• Identification

Risks posed by the laboratory If there is not sufficient hygiene nor cleaning practices, there is a risk of organisms entering the manufacturing areas via staff, media, or materials utilised in sampling.  Sampling requirements

• The sample needs to be representative of the batch. Problems can include micro-organisms multiplying and dying.
• The sample must accurately reflect the product, surface, or raw material.
• Check the sampling plan, technique, technique validation, and operator training and validation.
• Microbial contamination may not be evenly distributed within a container.
• The sample must be transported and stored so that the micro-organisms neither die nor multiply.
• Check the time the sample was taken, time the test commenced, the transport medias, and all temperatures.

Overcoming sampling problems There should be systems in place to:

• Log in and track the sample and storage conditions
• Correctly define and validate storage systems and requirements
• Assign the sample to a trained technician
• Training records should show test method competence, preferably validation
• Assign a test method correctly
• The method must be validated
• Monitor and document readings for validated incubators and refrigerators where samples are stored
• Assure that monitoring equipment is calibrated

Laboratory system elements

• Equipment qualification/validation
• Thermal mapping of incubators
• Calibration and maintenance system
• Calibration of temperature monitoring devices for incubators
• Annual maintenance on heating and cooling system for incubators
• Test method validation and change control
• Documentation
• Training of staff
• Sample handling and storage

Key requirements for media In order for media to be used in testing, it must be deemed competent by conforming to the following:

• Media batch records must be present
• Storage of media must be controlled
• Expiry date and date of manufacture must be recorded
• It must be QC tested
• pH, fertility, and sterility
• Media sterilisation must be validated and monitored for every batch

Maintaining culture collections A culture collection consists of a variety of micro-organisms, and maintained under clearly defined procedures. These organisms must be well-characterised by:

• Supporting documentation
• Traceability to the source (internal and external)
• Confirmatory testing supporting identification and purity

Sub-cultured organisms should be traceable to the original organisms. Sub-culturing should be done weekly. All access to the culture (and sub-culture) collection should be restricted. Periodic identification of organisms should be performed. Biological indicators (BIs) BIs provide a microbiological challenge to the process being evaluated, e.g., 106 spores on a Bacillus stearothermophillus spore strip for use in validating steam sterilisation processes. For quality control:

• Treat BIs as a raw material: apply QC to lots.
• Treat BIs as biological “contaminated”‘ material: uncontrolled dispersion may compromise manufacturing or QC testing.
• QC the BI count and identity.
• Control, issue, and reconcile usage, use, and storage.
• Require an SOP on BI handling and record system supporting use.

Requirements for reporting results Microbial results have an inherent variability. The accepted variability is usually ± 0.5 log. Another way of understanding variability is to use the 95% confidence interval approach. For example, if the limit for the microbial test is 100 cfu/g, and the result is 80 cfu/g, then it is likely that the true result could be between 62 cfu/g and 98 cfu/g. This is calculated by the formula: Confidence Interval = c ± 1.96 x c^0.5 Reporting of zero detected results in a 10g sample should not be slated as absent or zero, but rather <10 cfu/g Positive and negative controls Testing using positive controls involve deliberately introducing organisms into the sample. This is done in order to demonstrate that should the organism have been present, it would have been detected. Positive controls are used in the microbial limit test and media QC tests. Testing using negative controls involves terminally sterilising a sample so that it does not contain any organisms. Negative controls are used in bioburden, sterility testing, and endotoxin tests. Issues that affect validity The growth of organisms can be affected by:

• Incubation time
• Temperature
• Medium used
• Disinfectants
• Bactericidal and bacteristatic agents

Additionally, the sampling procedure, handling procedure, and transport of the media may also affect test results. Housekeeping and Waste Disposal  Risks to product quality and safety The laboratory may be a source of introduction of micro-organisms into the manufacturing Possible sources of contamination:

• Staff
• Staff garments
• Sampling equipment
• Sampling materials (e.g. media)

Entering and exiting the laboratory When entering:

• Change outer garments
• Wash hands with disinfectant

When exiting:

• Change outer garments
• Wash hands with disinfectant
• Sanitise or sterilise any materials being transferred from the laboratory

Risks of transferring media Media may be contaminated with a significant number of micro-organisms in the process of forming a colony. Therefore, they are not visible. These micro-organisms may be dispersed or transferred into the manufacturing environment when exposed. Transfer containers that hold these media must also be sterilised, and the media should be transferred into the container within the confines of a laminar flow hood. Similarly, external surfaces of the media plate should also be maintained sterile.  Contaminated samples left in laboratory Contaminated processed samples left in the laboratory can result in the dispersion of micro-organisms. This could pose a health risk to staff, contaminate staff when they enter manufacturing, and contaminate other samples and materials used in testing. All biological wastes should be placed into “Biological Hazard” labelled bags and steam sterilised. These bags should then be securely transferred to a specialised waste disposal company. Materials Flow  Importance of materials flow Materials flow describes the physical movement that a material takes within a defined area. Materials are processed through a series of steps. Materials at different stages of processing should be maintained in a different flow to prevent sample mix-up. It is important to ensure that contaminated samples, media, or product do not overlap in an uncontrolled manner with non-contaminated materials. This may result in “false positives”. Key attributes for effective materials flow

• Well-defined flow that may be represented schematically
• Procedure control
• Strict labelling and storage requirements indicating material status, identification, and storage conditions
• Ensure storage is designated and segregated
• Contaminated materials and materials of unknown state or media do not cross the non-contaminated materials flow
• Contaminated materials and equipment must not contact items or documents that are non-contaminated or destined for outside the laboratory area

Stages of materials flow Typical sample progression:

1. registration and tracking
2. storage
3. processing
4. incubation
5. reading and recording
6. results verification
7. destruction

Materials other than samples should be handled in dedicated areas, segregated from sample processing areas. Conclusion This module reviewed the basic concepts and requirements of GLPs:

• Issues focused on by laboratory auditors
• Specific pharmaceutical regulatory requirements
• Sterility test features

By now, you should be able to:

• Identify different classes of micro-organisms
• Describe and list typical objectionable organisms for different products
• Follow correct procedures for safe disposal of cultures
• List the key attributes of the flow of samples and materials through the laboratory