Sterility Testing

Question 1

what is killing of microorganisms influenced by

Answer 1

a time dependent process influenced by duration of biocidal action and initial bioburden

Question 2

when can true sterility be achieved

Answer 2

can only be achieved after an infinite exposure period

Question 3

what does a higher bioburden result in

Answer 3

results in longer sterilisation times

Question 4

what is the sterility assurance level

Answer 4

probability of a single surviving organism remaining to contaminate a processed product

Question 5

what is the standard sterility assurance level for pharmaceutical products

Answer 5

10-6
- probability of a non sterile unit is 1 in 1 million units processed

Question 6

what equation can be used to establish the sterilisation process necessary to achieve the SAL

Answer 6

IF = No/N= 10^t/D

Question 7

what are the steps in sterility assurance

Answer 7

1. bioburden determinations
2. environmental monitoring
3. validation and monitoring of sterilisation procedures
4. sterility testing

Question 8

what is a bioburden

Answer 8

the concentration of organisms in a material

Question 9

what is SAL achieved dependant on

Answer 9

dependent on the pre-sterilisation bioburden

Question 10

outline the benefits of low pre-sterilisation bioburdens

Answer 10

1. reduced exposure of the product to high sterilisation temperatures, minimising degradation of actives and excipients
2. greater probability of the product passing the bacterial endotoxins test (BET)
   - autoclaving doesn’t destroy endotoxins
   - high bioburden means high endotoxin concentration
3. shorter autoclaving cycles use less energy

Question 11

how can the bioburden of pharmaceutical products be determined

Answer 11

1. 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

Question 12

what is environmental monitoring

Answer 12

where levels of microbial contamination in manufacturing areas are monitored
- microbial numbers should not exceed specified limits

Question 13

how can microbial load in atmosphere be determined

Answer 13

may be determined by settle plates or use of air samplers, which cause a volume of air to be passed over an agar surface

Question 14

how can contamination on surfaces be measured

Answer 14

may be measured using swabs or contact plates

Question 15

what is measured in environmental monitoring

Answer 15

microbial load and contamination on surfaces, including equipment

Question 16

what is involved in the validation and monitoring of sterilisation processes

Answer 16

1. calibration and testing of all the physical instruments used to monitor the process
2. evidence that the steam is of desired quality
3. conduction of leak tests and steam penetration tests
4. production of data to demonstrate repeatability
5. comprehensive documentation of all sterilisation aspects
6. use of biological indictors

Question 17

why are biological indicators used in the validation and monitoring of sterilisation processes

Answer 17

sterilisation cycle should be capable of producing an acceptable SAL under worst case conditions

Question 18

describe how biological indicators are used in the validation and monitoring of sterilisation processes

Answer 18

1. standardised bacterial spore preparations
2. after the sterilisation process, the suspensions or spores on carriers are aseptically transferred to an appropriate nutrient medium

Question 19

what does sterility testing assess

Answer 19

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

Question 20

outline the principles of sterility testing

Answer 20

1. placing the product into a suitable liquid culture medium
2. 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

Question 21

what are the 2 main methods of sterility testing

Answer 21

direct innoculation and membrane filtration

Question 22

describe how direct inoculation is used in sterility testing

Answer 22

1. samples are introduced into nutrient media
2. alternatively, concentrated culture medium may be added to the test fluid in its original container
   - more sensitive test

Question 23

describe how membrane filtration is used in sterility testing

Answer 23

1. recommended by most pharmacopoeias
2. involves filtration of fluids through a sterile filter
   - pore size of 0.45um
   - filter is transferred into culture liquid media
3. water soluble solids can be dissolved in a suitable diluent and processed the same way

Question 24

what must be ensured when using conducting sterility tests

Answer 24

1. testing must be performed under aseptic conditions
   - eg. using a laminar airflow cabinet
2. control tests are necessary to confirm the adequacy of facilities
3. necessary to use negative controls
   - samples known to be sterile
4. where an antimicrobial forms part of the product, it must be inactivated before sterility testing

Question 25

name the methods for antimicrobial inactivation in sterility testing

Answer 25

1. specific inactivation 2. dilution 3. membrane filtration

Question 26

how can specific inactivation be used for antimicrobial inactivation

Answer 26

an appropriate neutralising agent is incorporated into the culture media - eg. penicillins are inactivated by B-lactamases

Question 27

how can dilution be used for antimicrobial inactivation

Answer 27

the antimicrobial agent is diluted in the culture medium to a level it ceases to have any activity - eg. phenols and alcohol

Question 28

how can membrane filtration be used for antimicrobial inactivation

Answer 28

the product is filtered through a membrane filter - membrane retains any contaminating microorganisms, is washed and transferred to liquid media - used with antibiotics

Question 29

why are positive controls used

Answer 29

it is essential to show that microorganisms will grow under the conditions of the test

Question 30

what does the Pharmacopoeia suggest the use of as positive controls

Answer 30

suggests several designated strains as positive controls - staphylococcus aureas - pseudomonas aeruginosa

Question 31

outline the limitations of sterility testing

Answer 31

1. destructive test- problem when testing large, expensive or delicate products 2. procedure intended to demonstrate a negative - failure to detect growth could be consequence of using unsuitable media or incubation conditions 3. no attempt is made to detect viruses 4. expensive test - need clean rooms, equipment and qualified staff 5. low sensitivity- part of batch is randomly sampled and dependence of sample size

Question 32

give an equation to determine the probability of obtaining 'n' steriles in a sterility test involving a sample size of n containers

Answer 32

q^n = (1- p)^n

Question 33

when do sterility tests take place

Answer 33

take place at the end of the manufacturing process

Question 34

can sterility assurance failures occur

Answer 34

they are not uncommon and there are several product recalls each year