1
Q
What are the 5 recognised methods for sterilisation of pharmaceuticals?
A
- Steam/moist heat (autoclave)
- Dry heat
- Ionising radiation
- Gas sterilisation
- Filtration
2
Q
Outline autoclave sterilisation
A
- Exposure to dry saturated steam under pressure in an autoclave
- 121 degrees for 15 minutes for thermostable aqueous preparations
- 100% water vapour
Consists of:
- Chamber + door
- Steam let in via baffles
- Thermocouples
- Temperature of drain valve should = autoclave temperature (otherwise there is a fault and procedure needs to be stopped)
- Some have a double walled jacket
3
Q
How does autoclaving sterilise?
A
- At high temperature, it kills microorganisms rapidly. Can transfer a large amount of heat quickly
- Kills microbes via hydrolysis of proteins and nucleic acids (denaturation)
- Convection
4
Q
Why is there a large amount of heat obtained from steam?
A
- Intrinsic heat as high pressures produce temperatures of 121 degrees
- Latent heat- contribution from condensation
5
Q
Why is there an issue if air enters the autoclave process?
A
- Equilibrium between temperature and pressure is related to partial pressure of the steam
- If there is a mixture of steam and air present - temperature and pressure is lowered (compared to pure steam in same conditions)
- Related to Dalton’s law- Total pressure of a mixture of gases= sum of individual partial pressures
6
Q
Outline the process of autoclaving sterilising bottled aqueous fluids
A
- Does not rely on direct contact between steam and the product
- Steam condenses on the surface of the bottle which is followed by heat transfer across the container walls
- Pressure within the bottle increases
- Heating up
- Sterilisation (holding time) temperature within the product will stabilise and then be held at a sterilising temperature (121 degrees for 15 minutes)
- Cooling stage- can be accelerated by spraying with STERILE water
Jacket preheating and pulling a vacuum is NOT needed
7
Q
Outline the process of autoclaving porous loads
A
- Most efficient sterilisation process
- Wrapped goods and porous materials
- Air removal via evacuation and steam injections and repeat
- Sterilisation holding time is low but temperature is high (134 degrees for 3 mins)
- Drying, steam is drawn off by pulling a partial vacuum
- Allows air admission via bacterial proof filter
Autoclave design must have:
- Jacket to preheat the chamber to lower condensation (will avoid too much condensation within the porous load, e.g. dressing)
- Bacteria proof filter over air entry
- Must be able to draw a vacuum
8
Q
In terms of autoclaving, what is heating affected by?
A
- Size of container
- Size of load
- Shape of container
- Porosity and entrapment of air
9
Q
What are advantages and disadvantages of autoclaving?
A
Advantages:
- Terminal process for aqueous solutions or suspensions
- Wide safety margin
- Kills bacteria and viruses
- Use for products given by IV route
Disadvantages:
- Only useful for thermostable solutions and suspensions
- Cannot be applied to bottled products with oily bases as water is essential for the process
10
Q
Outline the hot air oven (Dry heat Sterilisation) process and the criteria for it to occur
A
- There is no steam
- 170 degrees for 1 hour
- Convection fan to give forced air circulation
- Radiation from walls
- Silvered shiney layer to reflect heat within chamber
- Load should be same type and size, evenly spaces to reduce interference with air flow (heat transfer shouldn’t be obscured by neighbouring load)
- Containers should be sealed
11
Q
How does dry heat kill microorganisms?
What kind of products is it used for?
A
- Causes cell death by oxidative processes although
- Slower and less efficient proces
- Used for oils, powders, apparatus, glassware
- Not for samples vulnerable to high temp (Biologicals and liquids)
12
Q
What are some important points about hot air ovens?
A
- Maximum heat within the apparatus is at the walls, so contact with the walls must be avoided
- Avoid packing of many small items in large scaled tins
- Screw caps of containers should be unscrewed 1/2 a turn to prevent distortion of closure or bursting of containers
13
Q
What are the advantages and disadvantages of hot air ovens?
A
Advantages:
- Can be used for substances which can be harmed by moisture
- Suitable for assembled apparatus (glass syringes)
- Less damaging to glass and metal equipment compared to moist heat
Disadvantages:
- Drastic conditions are not tolerated by packaging material (plastics, rubber)
- Not suitable for surgical dressings
- Does not penetrate as well as moist air
- Non-terminal as you have to repackage materials
14
Q
Outline Gas sterilisation
A
- Ethylene oxide (volatile- boiling point is 10.8 degrees)
- 40 degrees
- 2 hours (lengthy process time)
- Gas tight chamber with vacuum pump
- Air is removed by drawing a vacuum
- Water vapour is admitted to the required RH (minimum 30% is optimum antimicrobial activity)
- Sterilisation at a pre set pressure and and a set time
- Gas is removed by drawing a vacuum. Air is admitted via bacterial filter
- Total air replacement allows air entry via a filter to atm pressure then chamber may be opened up
15
Q
What are the problems associated with ethylene oxide?
A
- Toxic gas so sterilisation must take place within enclosed chamber fitted with vacuum pump
- Forms an explosive mixture with air (overcome this by mixing it with inert gases such as carbon dioxide)
- 12% ethylene oxide with 88% dychlorodifluoromethane - under these conditions it is not explosive but does not interrupt the killing action
16
Q
What can gas sterilisation be used for?
A
- used for single use medical devices such as catheter, pacemaker wires
- Not for products which may absorb ethylene oxide such as porous products (toxicity may stay in it however it penetrates well)
17
Q
What are the advantages and disadvantages of gas sterilisation?
A
Advantages:
- Few materials damaged as minimal heat
- Good penetration into porous loads and plastics
- Effective against all organisms (bacterias, moulds, viruses)
- Effective at low humidity
Disadvantages:
- Expensive
- Need to control RH closely
- Toxic gas is being used and risk of explosion if mixed with air
- Resides of ethylene oxide must be allowed to disperse and be fully removed as it is dangerous if it stays in the product
- What do you do with the toxic gas after processing?
- Slower process (especially with plastics and nylon wraps which have to be left open and sealed aspetically)
18
Q
What is the MOA of ethylene oxide?
A
- Alkylation -CH2 addition
- Reacts with amino groups, OH
- Destroys function of essential proteins and nucleic acids preventing reproduction of contamination
19
Q
Name some other gases that can be used in sterilisation
A
- Hydrogen peroxide- cheap but reacts with many things
- Formaledehyde (CH2O)
- Pereacetic acid
- Ozone (hazardous)
- Chloride dioxide
20
Q
What are the ideal properties of a sterilant gas?
A
- Effective at low humidity and low temperature
- Non-toxic (safe to operator)
- Volatile, high penetration
- High activity against a broad range
- Ease of disposal
- cheap
- Compatibility with wide range of products and materials
- Non-explosive in air
21
Q
Outline ionising radiation as a method of sterilisation
A
Exposing product in final container to:
- Gamma radiation e.g. from Cobalt-60 OR electron beam e.g. linear acceleration with high energy electrons
- Needs minimum absorbed dose at 25KGy (kilograys)
- Terminal sterilisation
Aspects of the process:
- Irradiator containing the source
- Sample area (in a box)
- Conveyor belt passing samples through
- Measured time of exposure
- Dosimeter to measure exposure dose
22
Q
What is the MOA of ionising radiation?
A
- Targets site in microorganisms in DNA
- Ionising radiation induces structural damage in microbial DNA
- Ionising radiation causes the ejection of an electron that produces highly reactive radicals and positively charged ions
- Primary event
Interaction with gamma and water:
- Short lived hydroxyl radical that disrupts ability of cfu to reproduce as it interacts with DNA
23
Q
Outline ionising radiation as a method of sterilisation
A
Exposing product in final container to:
- Gamma radiation e.g. from Cobalt-60 OR electron beam e.g. linear acceleration with high energy electrons
- Needs minimum absorbed dose at 25KGy (kilograys)
- Terminal sterilisation
Aspects of the process:
- Irradiator containing the source
- Sample area (in a box)
- Conveyor belt passing samples through
- Measured time of exposure
- Dosimeter to measure exposure dose
24
Q
What is the energy of ionising radiation expressed in?
A
Electron volts = Energy gained by an electron moving through a potential difference of 1 volt
25
What is the absorbed dose of ionising radiation measured by?
```
Unit gray (Gy)
Defined as 1 joule of energy absorbed per kilogram of material irradiated
```
26
What are the advantages and disadvantages of ionising radiation?
Advantages:
- Effective against a large number of bacteria, yeast, moulds, some viruses
- Terminal
- Efficient
- Can be used on thermo-labile samples
Disadvantages:
- Cost of installation, training, disposal
- Not effective against some viruses and prions (bent proteins that cause disease)
- Dangerous
- Perceived s/e as people still think sample is radioactive afterwards
27
Outline filtration sterilisation
- Membrane with holes in it and you can sterilise fluids/gases
- Non-terminal
- Liquid is passed through a sterile bacteria retaining filter of suitable construction
- Probability of organism in filtrate < 1 in 1 million
- After filtration, the liquid has to be placed in pre-sterilised containers
28
What size is pseudomonas?
0.22-1 microns
29
What are the two types of filters?
Depth and screen
30
Outline a screen (membrane) filter
- Better type of filter
- e.g.) cellulose acetate
- Absolutely retentive
Integrity test must be applied:
- Before use- check for possible defects in the filter
- After use- reveals leaks which may develop during use
31
Outline a screen filter
- Better type of filter
- e.g.) cellulose acetate
- Absolutely retentive
Integrity test must be applied:
- Before use- check for possible defects in the filter
- After use- reveals leaks which may develop during use
32
What are the two integrity tests for filters?
1. Pressure retention test
| 2. Bubble pressure test
33
Outline the pressure retention test
- Method to test filters
- Push fluid through it and there should be a back pressure (if there is no pressure, liquid is going through filter too easily and there is a fault, such as too big holes)
34
Outline the bubble pressure test
- Allows you to calculate the pore diameter (D)
- Will change between liquids, but generally D is proportional to liquid (e.g. if it is viscous, it is harder to push through the pores and D is higher)
35
Outline criteria for an ideal aspetic sterilising filter
1. Unlimited flow rate
2. Inert
3. Thermal and strength stability (won't tear/puncture and can withstand pressures and steam sterilisation)
4. Low cost
5. Longevitity - with high particle contamination use a pre-filter such as Seitz
6. Target should be able to withold a certain number of organisms
36
What are the factors affecting efficiency of filtration?
1. Initial microbial count
2. Duration of filtration - prolonged filtration (> 1 day) must be avoided in case a contaminant grows through the filter medium
3. Types of organisms:
- Cannot filter viruses
- Cannot filter mycoplasms (a form of bacteria that has no cell wall, making it not rigid so can squeeze through pores)
37
What are the advantages and disadvantages of filtration?
Advantages:
- Used with thermolabile products (those unstable with heat)
- Removes bacterial bodies, so no remnants of dead cells are floating about the product (so no endotoxins and no LPS from gram -ve)
Disadvantages:
- Not for suspensions as active ingredient may be filtered out
- Non-terminal process (contamination risk and further sterility testing is needed)
- Sterility test required (7-14 day delay)
- Viruses and mycoplasms not removed
38
What is enumeration?
- Measurement of number of microbial cells in a sample
| - Total viable count/cfu
39
What is the simplest method for enumeration?
1. Dilute the sample with an appropriate media and then inoculate growth media with this sample.
2. It is then incubated for a set length of time
3. Number of CFUs is determined and the numbers are related back to the original sample
40
What are the methods of enumeration for viable counts?
- Pour plate (counting colonies in agar)
- Surface spread- counting colonies on agar surface
- Membrane filter methods- sample passed through a filter then the colonies are grown on the membrane on an agar surface
41
What are the methods of enumeration for total counts?
- Direct microscopic counting using a counting chamber
- Turbidity methods - measures opacity in suspensions/cultures
- Dry weight determinations
- Nitrogen, protein or nucleic acid determinations
42
What are the methods of enumeration for indirect viable counts (rapid methods)?
- Epifluorescence - dyes that have a characteristic fluorescence when interacting with living cells
- ATP methods- bioluminescence measures ATP production in living cells (firefly luciferase)
- Impedance - measures change in resistance on growing cultures
- Manometric methods - measures oxygen consumption/CO2 production by growing cultures
43
How do calculate log % survivors?
log % S= log (Nt/No x 100)
```
Nt= survivors after time t
No = original number of organisms
```
in cells/mL
44
What is the inactivation factor and how do you calculate it?
Degree of reduction in the viable population
IF= No/Nt
45
Death of microbial contamination is a probability function. What is it based on and what can assure all viable organisms are absent?
- It is based on the length of time of exposure to the lethal agent
- Only an infinite exposure to the lethal agent can absence of all viable organisms be assured with certainty
46
What does the BP state is an appropriate sterilisation assurance level?
1 viable microorganism in 1 million sterilised items of the final product
47
What is the decimal reduction time and what is it used for?
Time to achieve 90% kill
Time for reduction in number by one log 10
Used to:
- Characterize the resistance of spores under the influence of heat
- To measure the efficacy of sterilisation process
Each spore has a characteristic D value
48
1. Under what temperature is Lethality given the symbol Fo?
2. What does the BP recommend as an Fo value?
3. What is the equation for Fo?
1. 121 degrees
2. Fo=8
3. Fo = D x log(No/Nt)
Therefore, Fo= D X log(IF)
Fo means total thermal effect
49
1. Under what conditions if Lethality given the symbol Fo?
2. What does the BP recommend as an Fo value?
3. What is the equation for Fo?
1. 121 degrees
2. Fo=8
3. Fo = D x log(No/Nt)
50
Inactivation factor can be expressed in terms of D. How?
IF= 10^(t/D)
51
What is validation?
The process of demonstrating that a sterilisation method/protocol will consistently produce the results that it intended to
52
What is validation?
The process of
53
What 3 ways do you carry out validation and in-process monitoring?
1. Physical Indicators
2. Chemical Indicators
3. Biological Indicators
54
Outline physical indicators for validation and in-process monitoring.
For autoclaving and hot air ovens:
- Temperature recording chart as part of the batch record
For ionising radiation:
- Plastic dosimeter measures radiation dose absorbed
For filtration:
- Measures pressure retention by the filter
Gas Sterilisation:
- Temperature, relative humidity
55
Outline chemical indicators for validation and in-process monitoring
Autoclave and hot air oven:
- Browne's tubes and tape- tube will go from red to green if heat sterilisation conditions are adequate
Gas sterilisation:
- Chemical sachets that show whether the appropriate gas concentration has been present for the appropriate time
- Shows whether a product has been processed or not
56
What are biological indicators?
- Placed directly into the container
- Contain known number of microorganisms deposited on a carrier
- Often in the form of metal foil/paper strips/discs
- D value and z value known
- Uses various Bacillus strains depending on the sterilisation method
57
How do biologically monitor sterilisation?
1. Put the organism in the batch being sterilised
2. This introduces a risk and see if it is there at the end of sterilisation
Only in a few procedures is this essential
58
What is the general process for sterility testing?
1. Sterilise
2. Take sample e.g. from injection
3. Transfer to a suitable growth medium
4. Observe to see if growth has occurred
59
What is the membrane filtration technique for biological monitoring of validation?
What is the advantage of this?
1. Filter sample through 0.45 microns
2. Wash filter with suitable diluent such as peptone
3. Transfer filter to suitable liquid medium (in filter holder)
Advantage:
You can filter the whole contents of the container and any contamination will end up on the filter
60
What is the direct inoculation technique for biological monitoring of validation?
Inoculation into culture medium
Sample is diluted 10 times if it is a liquid and diluted 100 times if it is a solid
Disadvantage:
Can only take a small portion of the sample to be tested i.e if you had 1L you would need to dilute it to 10L which is a lot
61
What is the direct inoculation technique for biological monitoring of validation?
Inoculation into culture medium
Sample is diluted 10 times if it is a liquid and diluted 100 times if it is a solid
