Sterility Testing Flashcards
what is killing of microorganisms influenced by
a time dependent process influenced by duration of biocidal action and initial bioburden
when can true sterility be achieved
can only be achieved after an infinite exposure period
what does a higher bioburden result in
results in longer sterilisation times
what is the sterility assurance level
probability of a single surviving organism remaining to contaminate a processed product
what is the standard sterility assurance level for pharmaceutical products
10-6
- probability of a non sterile unit is 1 in 1 million units processed
what equation can be used to establish the sterilisation process necessary to achieve the SAL
IF = No/N= 10^t/D
what are the steps in sterility assurance
- bioburden determinations
- environmental monitoring
- validation and monitoring of sterilisation procedures
- sterility testing
what is a bioburden
the concentration of organisms in a material
what is SAL achieved dependant on
dependent on the pre-sterilisation bioburden
outline the benefits of low pre-sterilisation bioburdens
- reduced exposure of the product to high sterilisation temperatures, minimising degradation of actives and excipients
- greater probability of the product passing the bacterial endotoxins test (BET)
- autoclaving doesn’t destroy endotoxins
- high bioburden means high endotoxin concentration - shorter autoclaving cycles use less energy
how can the bioburden of pharmaceutical products be determined
- use of standard microbiological methods
- pour and spread plates
- membrane filtration
application of these methods to raw materials and manufactured medicines may need distinct adjustments
- due to presence of solid particles, viscosity
what is environmental monitoring
where levels of microbial contamination in manufacturing areas are monitored
- microbial numbers should not exceed specified limits
how can microbial load in atmosphere be determined
may be determined by settle plates or use of air samplers, which cause a volume of air to be passed over an agar surface
how can contamination on surfaces be measured
may be measured using swabs or contact plates
what is measured in environmental monitoring
microbial load and contamination on surfaces, including equipment
what is involved in the validation and monitoring of sterilisation processes
- calibration and testing of all the physical instruments used to monitor the process
- evidence that the steam is of desired quality
- conduction of leak tests and steam penetration tests
- production of data to demonstrate repeatability
- comprehensive documentation of all sterilisation aspects
- use of biological indictors
why are biological indicators used in the validation and monitoring of sterilisation processes
sterilisation cycle should be capable of producing an acceptable SAL under worst case conditions
describe how biological indicators are used in the validation and monitoring of sterilisation processes
- standardised bacterial spore preparations
- after the sterilisation process, the suspensions or spores on carriers are aseptically transferred to an appropriate nutrient medium
what does sterility testing assess
assesses whether a sterilised product is free from microorganisms
- can only show that a proportion of the products in a batch is sterile
- provide no guarantee about the sterility status of an entire batch
outline the principles of sterility testing
- placing the product into a suitable liquid culture medium
- if, after incubation, there are no signs of microbial growth (measured as turbidity), the item has passed the test
- counting of colonies is not involved
what are the 2 main methods of sterility testing
direct innoculation and membrane filtration
describe how direct inoculation is used in sterility testing
- samples are introduced into nutrient media
- alternatively, concentrated culture medium may be added to the test fluid in its original container
- more sensitive test
describe how membrane filtration is used in sterility testing
- recommended by most pharmacopoeias
- involves filtration of fluids through a sterile filter
- pore size of 0.45um
- filter is transferred into culture liquid media - water soluble solids can be dissolved in a suitable diluent and processed the same way
what must be ensured when using conducting sterility tests
- testing must be performed under aseptic conditions
- eg. using a laminar airflow cabinet - control tests are necessary to confirm the adequacy of facilities
- necessary to use negative controls
- samples known to be sterile - where an antimicrobial forms part of the product, it must be inactivated before sterility testing
name the methods for antimicrobial inactivation in sterility testing
- specific inactivation
- dilution
- membrane filtration
how can specific inactivation be used for antimicrobial inactivation
an appropriate neutralising agent is incorporated into the culture media
- eg. penicillins are inactivated by B-lactamases
how can dilution be used for antimicrobial inactivation
the antimicrobial agent is diluted in the culture medium to a level it ceases to have any activity
- eg. phenols and alcohol
how can membrane filtration be used for antimicrobial inactivation
the product is filtered through a membrane filter
- membrane retains any contaminating microorganisms, is washed and transferred to liquid media
- used with antibiotics
why are positive controls used
it is essential to show that microorganisms will grow under the conditions of the test
what does the Pharmacopoeia suggest the use of as positive controls
suggests several designated strains as positive controls
- staphylococcus aureas
- pseudomonas aeruginosa
outline the limitations of sterility testing
- destructive test- problem when testing large, expensive or delicate products
- procedure intended to demonstrate a negative
- failure to detect growth could be consequence of using unsuitable media or incubation conditions - no attempt is made to detect viruses
- expensive test
- need clean rooms, equipment and qualified staff - low sensitivity- part of batch is randomly sampled and dependence of sample size
give an equation to determine the probability of obtaining ‘n’ steriles in a sterility test involving a sample size of n containers
q^n = (1- p)^n
when do sterility tests take place
take place at the end of the manufacturing process
can sterility assurance failures occur
they are not uncommon and there are several product recalls each year
