Sterilisation Flashcards
(137 cards)
1
Q
What are the two approaches to producing sterile products?
A
1) Produce under ‘clean’ conditions (reduced no. of microorganisms) and terminally sterilise in final container
2) Aseptic technique - produce and assemble under conditions free of microorganisms
2
Q
Describe the microbial content of raw and synthetic materials
A
Synthetic and semi-synthetic materials:
- Low microbial count
- Populations generally not diverse
- Most contamination comes from process and operator handling
Natural materials:
- Large, diverse population of microbial cells
- Population is usually unique to the material (intrinsic flora)
3
Q
What is the issue surrounding water presence during manufacture?
A
Generally microbial growth where there is water - exclusion will prevent growth/kill existing organisms
4
Q
What are the potential sources of microbial contamination within a manufacturing environment?
A
Raw material:
- Synthetic and natural
- Synthetics and semi-synthetics tend to have low counts of microorganisms
- Natural products will have their own intrinsic flora – type and amount varies depending on product
Water:
- Essential for microbial growth so if used to wash/cool products will increase growth
Manufacturing environment:
- Air/equipment/personnel
- Any moving parts will displace microorganisms into the environment
5
Q
What is the advantage of knowing resident organisms?
A
Allows specific controls against them when using materials in manufacturing process
6
Q
What resident organisms are present in soil?
A
Gram positive (80%) Endospore forming Fungi
7
Q
What resident organisms are present in water?
A
Gram negative
Yeast
Moulds
8
Q
What resident organisms are present on animals and humans?
A
Dependent on area of the body
Gram negative Obligate anaerobes (gut bacteria) Gram positive (strep and staph)
9
Q
What resident organisms are present in plants?
A
Yeasts
Moulds
10
Q
What are transient organisms?
A
Transferred from one place to another
Carried by water and air
Harder to control
11
Q
What is the difference between sterile and sterilisation?
A
Sterile means free of all viable microorganisms.
It is an absolute term - if there is one organism on a surface, it is contaminated.
Sterilisation refers to the process of killing/removing all viable microorganisms
12
Q
What are the methods of killing microorganisms?
A
Heating (Dry or moist)
Chemical (Ethylene oxide)
Radiation (Cobalt 60)
13
Q
What is the method of removing microorganisms?
A
Filtration - removes cells without killing
Efficacy dependent on the pore size
14
Q
What needs to be considered when choosing a sterilisation process?
A
Will microbes be removed by chosen process?
Will end product withstand process?
15
Q
What are the purposes of sterilisation standards?
A
- Control microorganism numbers in a manufacturing environment
- Validate a sterilising agent/process
- Monitor a sterilisation process
16
Q
How do manufacturers test the compatibility of a sterilant and product?
A
Culture of cells taken and exposed to sterilant for increasing number of time
At different time points, remove a sample and perform a viable count
Plot the number of survivors against time to produce an asymptote curve (kill curve)
17
Q
How is the best sterilant for a product determined?
A
Expose to a number of different ones and compare the different effects
18
Q
What are the four key points about inactivation kinetics?
A
Infinite probability of survival
First order kinetics
Organism specific
Affected by concentration of sterilant
19
Q
What is the D-value?
A
The time taken at fixed temp/conc/radiation to reduce the population of microorganisms by 90% (1 log cycle)
Always in minutes
20
Q
What influences the D-value of an organism?
A
Population size Bacterial species (vegetative vs endospore forming) Production method Nutrient environment (culture media) Treatment dose
21
Q
What is the Z-value?
A
Change in temperature (°C) required to produce a 90% reduction (1 log cycle) in the D-value
Units = Degrees C
22
Q
What is the purpose of the Z-value?
A
Measures thermal resistance and therefore efficacy of heat as a sterilant
23
Q
When is a product considered sterile?
A
There is no zero on a log scale so when it is below the SAL (10^-6)
24
Q
How long should a product be sterilised to reach the SAL level?
A
10^0 = 1 bacterial cell. Anything less than this is a probability measure and is not accurate.
Instead plot a kill curve (logSurvivors vs time). Extrapolate to line to SAL (logSurvivors = 10-6).
Find the D-values for each line.
Time to reach SAL = D-value x Log cycles until SAL is reached (e.g. from 10^2 to 10^-6 = 8)
Units = Minutes
25
What would the log plot look like for organisms with the same D-value?
Lines would be parallel
26
Define bioburden
A population of viable microorganisms on or in a product and/or its packaging
27
Why is a bioburden estimate important?
Initial population numbers are required in order to specify sterilisation parameters and inactivation kinetics
28
Where would details of sample selection be found?
Pharmacopeia
29
Why is storage of items important?
Prevents growth/death which would give a false estimation for the bioburden
30
What is direct treatment for cell sampling?
Direct contact between product and growth medium
31
What is indirect treatment for cell sampling?
Break up structure into individual components
Physical treatment - swab, ultrasound, glass beads
1) Contact with Eluent
- Wash product with an eluent (e.g. buffered saline) to remove free cells then can perform serial dilution to find the number of organisms in the solution
- Need to ensure the eluent does not affect viability of microorganisms (promote growth or kill)
2) Physical treatment
- E.g. vortex the product submerged within an eluent
- Shaken for a period of time and set speed
- Ultrasound is also commonly used but over processing can lead to the cell membrane perforating and cell lysis
- Can also use glass beads to physically remove the cells – need to be the correct size
3) Transfer to culture medium
32
What are the considerations when selecting a removal technique?
```
Ability to remove microbial contaminants
The effect of removal on viability
Types and location of microorganisms
Nature of product
Culture conditions
```
33
What should be taken into account when selecting culture conditions?
- Type of microorganism
| - No universal growth medium (different bacteria have different dietary needs)
34
What is the purpose of enumeration and characterisation?
Looking for low numbers of colonies, manufacturing process should limit amount to be removed by sterilisation
35
What is process validation?
The establishment of *documentary evidence* that provides assurance that a specific process will *consistently* produce a product that *meets the predetermined specifications*
36
What are the steps of process validation?
1) Installation Qualification
Checks the sterilisation equipment works
2) Performance Qualification
Measures efficacy
Split into two forms:
- Physical Qualification
Physically measuring the conditions of the sterilisation process
Consistent and accurate
- Microbiological Qualification
Can be used in addition to physical qualification or if physical qualification cannot be used
- Use microorganisms which are known to be highly resistant and virulent
- Prone to variability due to the use of biological microorganisms
37
What are biological indicators?
An inoculated carrier contained within its primary pack ready for use. It provides a defined resistance to a specified sterilisation process.
38
Why are biological indicators used?
Direct assessment of microbial lethality of a sterilisation process.
Part of the microbiological qualification for the validation and monitoring of a sterilisation process.
39
How are BIs used for validation and monitoring?
Proportion of surviving test organisms measured and related to expected lethality - are you achieving the expected D value reduction and ASL levels?
40
How are BIs characterised?
```
Strain of test organism
Reference to culture collection
Manufacturer's name
Number of CFUs per test piece (1 x 10^6)
D-value (radiation)/Z-value (heat)
Recommended storage conditions
Expiry date
Disposal instructions
```
41
What should be considered when choosing a biological indicator?
Stability
Resistance - should be high in comparison to product bioburden
Should be non-pathogenic (low risk)
Recoverability - If there are surviving spores, these need to be recovered and cultured so they can be tested
42
What are the BI recommendations for each sterilisation process?
Dry Heat - Bacillus subtilus
EtO - Bacillus subtilus
Filtration - Brevundimonas diminuta
Moist Heat - Bacillus stearothermophilus
Radiation - Bacillus pumilus
43
What is the *general* guidance for selecting a sterilisation method?
Balance advantages and disadvantages (efficacy, safety etc.)
No requirements to specify which method should be used
Method chosen at design/development stage
*Use EMEA Decision Tree*
44
What is the *specific* guidance for choosing a sterilisation method?
Terminal sterilization of product in final container is preferred to aseptic processing
Default is heat sterilisation
Ensure sterilising agent is in contact with all parts of product
Process variables should be controlled and monitored (moisture level, heat etc.)
No hazards to environment or operator
Process does not leave toxic residues within product
45
What is filtration sterilisation?
Passage of fluid (or gas) across a filter, removing any contaminating solutes
46
What can block the filter pores?
Filters remove particles which are bigger than the pore diameter. However, some filters can remove particles which are slightly smaller than the pore size due to:
- Irregular shaped particle (rod cells)
- Simultaenous arrival of two or more cells
- Blocked pore (large particle preventing small particle from being filtered through)
- Surface interactions
Most bacterial cells are negatively charged, a filter with a positive charge will form interactions with the cells and prevent them going through the pore
47
What are filter voidages?
Empty spaces between the filters, particles can accumulate here.
Accumulation of filtrate at the top of the filter is an indication that the voidage is full. This will indicate the filter capacity (dependent on particle size).
48
Describe depth filters
Variable pore size
Particles collide with matrix and are retained (inertial impaction)
High retentive capacity
Robust
Cheap
No sterility (cannot guarantee they'll produce a sterile product)
Example: Paper filter
49
Describe screen (absolute) filters
Uniform pore size (0.8µm - 0.45µm)
Direct particle interception with pore (larger particle cannot pass through)
Easily blocked
Fragile
Expensive (5x cost of depth filter)
Sterility (0.22µm)
- The smallest vegetative cell is 0.45 µm
50
What are the mechanisms of filter validation?
- Bubble point pressure test
Add water and gradually increasing amount of air that passes through the filter until bubbles form
Direct correlation between the pressure of air applied and the porosity of the filter membrane
- Challenge filter with Brevundimonas diminuta (0.4µm)
Min. requirement is that a filter will remove/retain 1 x 10^7 cells/cm^2
Working capacity: 1 x 10^9 - 1 x 10^10/cm^2
51
How does moist heat sterilisation cause cell death?
Protein coagulation and hydrolysis
52
What is moist heat sterilisation used for?
Aqueous products, devices and dressings
53
What is an autoclave?
Self boiler which maintains steam to produce high pressures.
Stainless steel to withstand pressure.
Steam supplied externally.
Temperature in excess of 100 degrees.
54
How do autoclaves operate?
Downward displacement of cold air or evacuation of air
Heating, holding period (15 mins at 121°C), cooling, drying
55
How is heat transferred in moist heat sterilisation?
Latent heat of vaporization.
Steam condenses around the object which creates a small vacuum pull around the product. This draws more steam and the heat from the steam is transferred to the product.
This processes continues until the product is in equilibrium with the steam (same temperature).
Product should be resistant to moisture as steam condenses.
56
What are the critical aspects of moist heat sterilisation?
Air removal
- Presence of air will limit the temperature (will unlikely get past 100C)
- Air needs to be removed and replaced with steam
Saturated steam
- Steam has different moisture content
- Saturated steam desired
Steam under pressure
- Removal of air will increase the pressure due to a formation of a vacuum
57
What are the critical lethal parameters for moist heat sterilisation?
- Dry saturated steam (not wet or superheated)
- Maintain temp. within +/- 5 Kelvin of limit
- Exposure time sufficient to reach SAL 10-6
- Bioburden level should consider nature, number and location of microorganisms
58
Describe the moist heat cycle
1) Air removal
- Air doesn't heat above 100C therefore needs to be replaced by steam
2) Heating
3) Sterilisation/holding period
- Pressure dictates temperature reached
4) Cooling
- Steam stops entering chamber
- Can be either natural decay (gradual cooling of product) or active (pumping in cold air)
5) Drying
59
What are the different methods of validation for moist heat sterilisation?
M.T.R. - Master Temp. Record
- Always used for validation
- Test run with sample products
- Thermocouples = probes which monitor temperature
T.R.C. - Temp. Record Chart
- Use a temperature probe to monitor temp of the Drain
- Drain should be the coldest part of the autoclave (exit of cold air)
60
How does dry heat sterilisation kill bacterial cells?
Oxidative processes
| Longer process than moist heat
61
What is dry heat sterilisation used for?
Dry powder, oil preparation, glassware and instruments
62
What equipment is used for dry heat sterilisation?
Dry heat ovens
- Batch processing
- Similar to domestic ovens
Sterilising tunnels
- Continuous hear
- Conveyor belt
63
What are the critical aspects of dry heat sterilisation?
```
Product size
Loading pattern (should allow free circulation around the product)
Air circulation (dependent on loading pattern, need to ensure even distribution)
```
64
How is an even temperature distribution achieved in dry heat sterilisation?
Use fan assisted circulation
65
What are the mechanisms of heat transfer in dry heat sterilisation?
Conduction
Radiation
Convection
Layout (Heating elements inside the walls of the oven with fan assisted air circulation, no moisture)
66
Describe the dry heat cycle
Drying - remove moisture
Heat to required sterilisation temperature
Keep product exposed for appropriate time (holding period)
- This is when sterilisation takes place
Cooling
- Longest time of the cycle
- Sterile air can be pumped into the oven to quicken the cooling process
Dry heat cycle can take up to 16 hours
67
What are the pharmacopeial heating temps. and exposure times? Which are the most common?
120°C for 480 mins
160°C for 120 mins
170°C for 60 mins
180°C for 30 mins
160°C and 170°C most common
68
What is the F0 concept?
Alternative to compendial cycles
| Allows comparison of lethalities
69
Why was the F0 concept developed?
Compendial cycles overkill microorganisms so economically wasteful and may lead to degradation
70
What is the definition of the F0 value?
Lethality expressed in terms of the equivalent time in mins at 121°C delivered by process to product in final container, with reference to microorganisms with a Z-value of 10°C
E.g. an F0 of 3 means that the lethality is equivalent to heating at 121°C for 3 minutes
71
What is the minimum F0 value?
8 (8 mins at 121°C)
| Should guarantee a min. SAL of 10^-6
72
How is F0 calculated for biological data?
F0 = D(logN0 - logN)
D is the D-value at the given temp.
N0 is the initial number of microorganisms present (bioburden)
N is the number of microorganisms surviving the process (actual or expected)
• Likely N = 10^-6 (SAL)
73
How is F0 calculated for thermal data?
F0 = [log^-1 (T-121)/Z] x dt
121°C is the reference temp. (Bacillus stearothermophilus)
T is the temp. of heating
Z is 10°C
dt is the heating time (for a range, take the range)
Note: FOR HOLDING TIMES ONLY!
74
What are the benefits of F0?
Can be used for heat labile products and offers flexibility for heat sterilisation
75
How do you calculate Fh?
Same as F0 but for dry heat sterilisation.
Fh = [log-1 (T-170)/Z] x dt
170°C is the reference temp. (Bacillus subtilus)
T is the heating temp.
Z is 20°C
dt is heating time
76
What are the uses of EtO sterilisation?
Disposable items
| 50% of all medical devices
77
How is the explosivity of EtO nullified?
Mixed with inert gas, usually CO2 or Nitrogen
78
How does EtO kill cells?
Alkylation of sulphhydryl, amino, hydroxyl and carboxyl groups
79
How does temperature affect the lethality of EtO sterilisation?
Increased activity at higher temperatures
80
Which is the worst method sterilisation? Why?
EtO sterilisation
Difficult to achieve min. SAL of 10^-6 due to a range of variables that affect EtO.
- Toxic residues and operator safety issues. Residues may be carcinogenic.
- Issues with distribution and penetration of EtO
- Lethality affected by Conc., Temp. and RH (non-uniform)
Therefore less sterility assurance.
81
What are the critical lethal parameters for EtO sterilisation?
Time: 1-24 hours
Temp: 25-65°C
Humidity: 40-85% RH
EtO Conc: 250-1200mg/L (no physical means of monitoring concentration)
82
What species is used for the validation and monitoring of EtO sterilisation?
Bacillus subtilus
83
Briefly describe the process of EtO sterilisation
1) Pre-conditioning
Ensures product reaches right humidity levels
2) Sterilisation Cycle Evacuation, vacuum hold, conditioning, sterilant (EtO) injection, exposure (holding period), sterilant removal (catalytic converters used to convert EtO to CO2 and H2O), flushing (input of filtered, sterile air into the chamber)
3) Aeration
- Vacuum cooling
- Safeguarding to ensure that no EtO residues remain on the product
All three processes can be in the same chamber or, for larger processing, in separate rooms where the product will have to be transferred to each one
84
What are the new sterilisation techniques? Describe them
X-ray radiation - Ionising radiation, expensive on mass scale, low power
Pulsed light - Short pulses of broad spectrum white light, poor penetration, non-ionising
Microwaves - Intense heating, short cycle (seconds), batch-like process,
Gas plasma - Mixture of ions, free radical, neutrons and electrons. Alternative to EtO, no residues
85
What are the uses of the new sterilisation techniques?
X-ray: Simple solutions
Pulsed light: In-line sterilisation and intravascular medical devices (catheters, tubing)
Microwave: Solutions in vials, contact lenses
Gas plasma: Medical devices
86
What are the problems with new sterilisation techniques?
- Unknown lethal effects
- Different kill kinetics to traditional processes
- Difficult to achieve validation
- Monitoring (What is the best way to monitor it? Best BI?)
- No established regulatory requirements
However, may be cheaper/more reliable methods
87
What are the sources of resident microorganisms?
Water
Soil
Plants
Animals and humans
88
How would an antibiotic contained in a vial with a stopper be sterilised?
Antibiotic sterilised using filtration technique
Vial - Steam sterilisation as can't pass through filter
Stopper - EtO
Antibiotic then filled in vial and sealed aseptically before it is packaged
89
What organisations regulate sterilisation?
European National Standards (EN)
FDA
AAMI (Japan)
The product must adhere to the standards of where it will be used (i.e. if produced in the UK but sold in America, it has to adhere to FDA standards)
90
What colony count is needed to produce a kill curve?
Perform serial dilutions to produce 30 – 300 colonies
< 30 = not statistically significant
> 300 = too many colonies to count
91
What is meant by an asymptote curve?
Rapid then gradual decrease in colonies
Decreases by the same proportion each time interval
Never reaches zero
First order
92
How can a kill curve be plotted to allow the calculation of a thermal death rate i.e. y and x axis?
Plot log of number of survivors against time to give a semi-logarithmic graph
This will produce a negative straight line.
Gradient = thermal death rate
(thermal if temp used)
Thermal death rate = how quickly an organism dies at a particular temperature
93
An asymptote curve can be used to compare results when organism exposed to different temps and when different organisms exposed to the same temp. True or false?
False - use a semi-logarithmic graph for this
94
A 90% reduction in population of microorganisms is how many log cycles?
1 log cycle
95
What are the units of the D-value?
Minutes
96
Z value is only for which type of sterilisation?
Heat sterilisation
97
How would you find the D-value on a logarithmic kill curve?
# Choose a value on the y axis (logSurvivors) then go across to the straight line. Note the time (mins) on the x axis. Then go down one log cycle and repeat process.
The difference in time between the two values is the D-value.
Doesn’t matter which two numbers you choose as it is a directly proportional relationship – just has to have a reduction of one log cycle
E.g. 100 to 10, 10 to 0 all have the same D-value.
98
How do you produce a thermal resistance curve?
Calculate 4 D-values and plot a graph of the log of D-values against the temperature which the D-values where produced.
Gradient = Z-value
Z-value can also be found by repeating the same process which D-values are found (difference in temperatures between two logD-values)
99
What is the relationship between Z-value and D-value?
For every increase in the Z-value, you get a decrease of one log cycle (90%) in the D-value
E.g. if Z = 10C then an increase of 10C will decrease the D-value by 90%
100
What can be used to make sense of data produced by Z-value?
Biological indicator used as a standard to compare values against
Bacillus used as it produces endospores therefore it is highly resistant
101
Which BI is used for dry heat sterilisation?
Bacillus subtilus
| Z-value: 20C
102
What is the Sterility Assurance Level (SAL)?
SAL = 1 x 10-6
| Out of a million products, only one can be contaminated
103
What are the steps in bioburden estimation?
1) Sample selection
- Statistical sampling
2) Collection of items for test
3) Transfer to test lab
- Preparation of the sample and their packaging for transport
- Keep at a controlled temperature in order to maintain the viability of the cells – reduces false representations
- Transfer includes the time it takes for the samples to start being processed
4) Treatment (if required)
- Treatment to remove cells
- Dependent on the product
5) Transfer to culture medium
6) Incubation
7) Enumeration
104
There is a standard universal bioburden level. True or False?
No standard universal bioburden level.
Each manufacturing company will set their own bioburden level standard. The lower the better.
105
What is an example of an Eluent than can be used for bioburden estimation? What are the problems with this eluent?
Sometimes mild detergents are used (breaking covalent bonds between bacteria and the surface of the product)
But some mild detergents have weak antibacterial activity.
Some organisms such as Pseudomonas like detergent and grow which is an inaccurate representation of the actual number present on the product.
106
What does the type of microorganism dependent on?
Nature of the product (a natural product likely has a higher bioburden)
Method of manufacture
Potential sources of microbial contamination (Operator/Packaging etc.)
107
How do you choose the best culture medium?
Conditions required assessed during validation of a technique.
- Using samples from the product, these samples are cultured on different medias.
- Each growth media chosen to grow specific bacteria – if you suspect that there are fungi on your product, can grow on Yeast extract agar to test.
- Can vary the temperature and length of time
- CFU (colony forming units) = Total number of cells
- Colony types will show the different types of microorganisms growing on the product
*Best culture condition has the most number of CFUs and the greatest variety of colony types present*
Must be performed for every new product and if you change your supplier.
108
What is process operation?
For any sterilization process, there are 3 stages:
1)Cycle Development
Testing the effect of the sterilization process on the product
2) Cycle validation
Proof that the sterilization process is working
3) Cycle monitoring
After the sterilization process, monitoring of all products
109
How is the monitoring process for chemical sterilisation different from other methods?
No physical means of monitoring EtO sterilisation therefore Microbial qualification is used (BIs)
110
Most bioburden cells found on products are what type of cells?
Vegetative
111
Filters have a direct passage from top to bottom?
False. Convoluted pathways with voidages.
112
Why is moist heat sterilisation faster than dry heat sterilisation and which one is used more commonly?
Moist heat is faster as microorganisms are killed faster by oxidative processes than hydrolysis. Moist heat is more common.
113
Why is dry saturated steam preferred to wet or superheated steam?
Wet steam will sterilise product but will make it extremely wet – may damage the product
Superheated steam has a much lower moisture content, similar to dry heat sterilisation More towards oxidative killing which is much slower process and may result in the SAL level not being reached as it may not have killed all the cells in time
114
What are the cycle types for moist heat sterilisation?
Fluid cycle
Porous Load Cycle - Air removed from products with matrix (e.g. fabrics or dressings)
Air ballasted cycle - Used for hermetically sealed plastic units
115
What are the methods of air removal in moist heat sterilisation?
Downward displacement
- Cold air pushed downwards and out, steam pumped in from the top
- Easiest
Evacuation
- Air pulled out
- More expensive
116
What are the temperatures, time and pressures used for moist heat sterilisation?
Holding Temp.(range) - Time - Steam Pressure
115 - 118C 30 mins 10 psi
121 - 124C 15 mins 15 psi
126 - 129C 10 mins 20 psi
134 - 138C 3 mins 30 psi
117
What are the 3 autoclave cycle types for moist heat sterilisation?
Fluid Cycle
- Most common
- Used for fluids and heat-resistant solid objects (glassware)
- Usually 2 hours
Porous Load Cycle
- For fabrics and dressings
- Quick (30 minutes)
- Fabrics trap air therefore air removal most important step
Air Ballasted Cycle
- For products which are ballasted sealed products (plastics)
- Delicate pressure balance
118
How do you determine the Master Temperature Record (M.T.R) to validate moist heat sterilisation?
Minimum of 12 thermocouples at different locations in the autoclave chamber.
Run through cycle and check that every thermocouple reaches their required temperature.
MTR specific for one type of load ONLY - different products require revalidation.
Even if the same product but at a different size e.g. if you increase from 500ml bottle to 1L bottle, revalidation is required.
119
What are the F0, holding temp and time values for pharmacopoeial cycles?
Holding temp(range)/Time(min)/F0 value (min)
115 - 118°C 30 min 7.5 - 15 min
121 - 124°C 15 min 15 - 30 min
126 - 129°C 10 min 32 - 63 min
134 - 138°C 3 min 60 - 150 min
Note: Holding times only
An F0 of 7.5 is acceptable because, even though it is below 8, by the time the process heats and cools down, the product would be sterile
120
Why are high humidity levels needed for EtO sterilisation?
High levels help kills microorganisms as EtO more lethal in the presence of moisture and bacteria more susceptible
Higher resistance in drier conditions
121
Why is EtO dangerous to work with?
Acts by blocking active sites. These target sites are not unique to bacteria – toxic to humans.
Need to ensure adequate removal of toxic residuals following sterilization of product.
EtO is also highly explosive in air.
122
What is the test of sterility?
Defined in the Pharmacopoeia.
Testing for a ‘negative’ i.e. absence of microorganisms.
Imprecise statistical test - the greater number of samples tested, the greater probability of rejection.
Performed on devices/products exposed to a fraction of the specified sterilization process i.e. part of Process Development.
Then perform a cell count and scale up.
123
Why is the test of sterility used?
For the validation of a sterilisation process
124
How is test of sterility different from bioburden estimate?
Different from bioburden estimation as performed on product in the sterilisation chamber
125
How is test for sterility similar to bioburden estimate?
Similar process.
Product unit (sample) is either:
1) Directly immersed in medium and immersed
or
2) Microorganisms removed by elution and:
- Filter to recover cells then transfer filter to medium and incubate
OR
- Use medium as eluent or dilute eluent with equal volume of medium and incubate
126
How do false positives happen in the test for sterility and how can they be controlled?
Important to record the frequency of occurrence
• Is there a pattern to their presence?
Perform simulated test of “sterile” samples
• Positive control
Precautions to minimise level of contamination:
• train personnel
• use environmentally controlled area/room
• use aseptic techniques
• avoid introducing contamination
• decontaminate test surfaces
• sterilize test equipment and materials
• minimise manipulations
• monitor and control incubator environment
• minimise aerosol production
127
How do false negatives happen in the test for sterility and how can they be controlled?
Inadequate culture conditions
• E.g. overprocessing of culture media which can lead to breakdown of nutrients → limited growth
• Can check using pure cultures and inoculating them
Presence of microbiostatic/cidal substance
• Positively charged cells may adhere to negatively charged substances which may not kill the cells but stop them from growing
Interval between treatment and testing
• A long gap between treatment and testing
128
What are the limitations of the test for sterility?
Destructive test.
Sample of products = 1-2% of product load. Presumption is that the sample tested represents the batch.
Additional tests increase chance of passing the test. Therefore just because the batch passes the Test for Sterility doesn't mean that it is sterile.
129
What is the probability of rejecting a batch during the test for sterility affected by?
Frequency of contamination
| Number of times tested
130
What do failures in the Test for Sterility mean?
Failure = presence of a microorganism
Up to 2 further re-tests allowed.
Reject batch on 2nd test if same m/o found.
Retest if 2nd fail due to a different m/o.
131
What are Pyrogens?
Pyrogens are endotoxins produced by the LPS from Gram-negative bacteria (Lipid A component of LPS).
Found in gram negative bacteria only.
132
Why are Pyrogens dangerous?
Lots of pyrogen produced during the killing of cells.
Can be toxic – fatal fever.
Any product that has to be injected into the body has to be pyrogen free.
133
How can you test for Pyrogens?
LAL (Limulus Amebocyte Lysate) test.
Aqueous extract of blood cells from a horseshoe crab (Limulus polyphemus) forms LAL reagent.
Equal volumes of test solution and LAL reagent are mixed in glass tubes.
After incubation at 37C for 1h, the tubes are observed for clot formation after inverting them.
Formation of a solid clot that withstands inversion of the tube = positive test (Pyrogen present).
134
What are the different types of LAL test?
Gel Clot (Horseshoe crab)
Turbidometric (kinetic)
- Rate of clotting
- Inaccurate
Colorometric (chromogenic)
- Addition of chromagen to the LAL
- Increase in colouration with the presence of pyrogen
- Concentration vs colouration curve to find amount of pyrogen present
135
What is depyrogenation?
Better to prevent endotoxin accumulation than remove from product once present.
Rinsing or dilution of materials is a good way to eliminating pyrogenic activity.
Pyrogens in vials or glass destroyed by dry heat sterilization at high temperatures (250C for 45mins)
Pyrogens removed from Water for Injections by distillation
136
Describe the relationship between lethality and F0 value
The higher the F0 value, the greater the lethality
137
Which BI is used for moist heat sterilisation?
Bacillus stearothermophilus
| Z-vale: 10C
