Sterilisation Flashcards
(138 cards)
1
Q
What are the two general approaches to producing sterile products?
A
- produce in clean conditions, then terminally sterilise in final container (the better one, less error)
- produce and assemble in conditions free of micro-organisms (aseptic processing)
2
Q
What are sources of microbial contamination in the manufacturing process?
A
Raw materials, water, the manufacturing environment
3
Q
How can raw materials contaminate manufacturing processes?
A
Synthetic materials: low and not diverse microbial count, come from the process as don’t carry an intrinsic population
Natural materials: large and diverse microbial population, unique to that product
4
Q
How can water contaminate the manufacturing process?
A
It’s the primary requirement for microbial growth - denying water prevents growth
5
Q
How can the manufactuing environment contaminate processes?
A
Air, personnel, equipment and facilities
spores can survive and move sites, humans carry micro-organisms. any moving machinery parts can spread microbes, may have organisms associated
6
Q
What are the resident organisms in soil?
A
gram positive
endospore forming
fungi
7
Q
What are the resident organisms in water?
A
gram negative
yeasts & moulds
8
Q
What are the resident organisms in humans and animals?
A
gram negative and positive
obligate anaerobes
9
Q
What are the resident organisms in plants?
A
Yeasts & moulds
10
Q
What are transient organisms?
A
spread from one source to another
carried by vectors (e.g. air and water, personnel)
11
Q
Definition of sterile?
A
Free of viable micro-organisms
12
Q
Defintion of sterilisation?
A
killing/removal of all viable micro-organisms
13
Q
Methods of killing viable micro-organisms?
A
heating, chemical (ethylene oxide), radiation
14
Q
Methods of removing viable micro-organisms?
A
filtration
15
Q
What is a kill curve?
A
looks at numbers of surviving colonies over a period of time
take samples from a dilution at intervals and plot number of survivors
16
Q
What shape is a kill curve?
A
asymptote
17
Q
Why is a logarithmic kill curve used?
A
gives linear relationship (log survivors vs time)
18
Q
What does the gradient mean on a logarithmic kill curve?
A
death rate
19
Q
What does the aymptote shaped curve mean?
A
there is an infinite probability of survivors
20
Q
What order are inactivation kinetics?
A
first
21
Q
What is the D-value?
A
time taken (at a given temperature) to reduce the population by 90%
90% reduction in population is 1 log cycle
22
Q
What is plotted on a thermal resistance curve?
A
Log D value vs temperature
23
Q
What is the Z-value?
A
the temperature change required to produve a 90% reduction (1 log cycle) in D-value
24
Q
What does the Z-value show?
A
a measure of thermal resistance of the organism
25
What are reference organisms?
something to compare other values to. usually the most resistant form to a given process
26
What is the Z-value of Bacillus stearothermophilus?
10 degrees
27
What is the Z-value of Bacillus subtilus?
20 degrees
28
What is the need for a sterility threshold?
Absolute zero is not possible
29
What is SAL?
Sterility assurance level
30
At what value can you guarantee a product is sterile?
10 ^-6
31
At what value does it become impossible to count the number of survivors?
10^0 (1) - must extrapolate from plots obtained
32
How can you use an extrapolated log kill curve to calculate the length of the process needed to reach SAL?
Calculate number of log cycles needed to reach 10-6 (from start count)
this many D cycles gives a duration of exposure required
33
What can affect how long an organism must be processed to reach SAL?
resistance the process, higher starting count, D-value
34
What are D-values influenced by?
the species, vegetative vs. spore form (vegetative more sensitive), production method, nutrient environment, treatment dose
35
What is the definition of bioburden?
The population of viable micro-organisms in a product/package
36
What is the importance of bioburden?
the initial numbers are required to determine sterilisation parameters and inactivation kinetics
37
Process of bioburden estimation?
- sample selection
- collection of items for test
- transfer to lab
- treatment if required
- transfer to culture medium
- incubation
38
At which stage does the greateset variability occur in bioburden estimation?
treatment - it's very product specific
39
stages in direct methods for bioburden estimation?
product --> contact with culture medium --> incubation
40
stages in indirect methods for bioburden estimation?
product --> contact with eluent --> physical treatment --> transfer to culture medium --> incubation
41
What eluents can be used in estimating bioburden ?
the correct one for expected organisms.
mild detergent can help remove from surfaces, but have mild antibiotic properties so some bacteria are sensitive
42
What are examples of physical treatments that might be required for bioburden estimation?
swabs, ultrasound
43
What considerations are necessary when selecting a removal technique for bioburden estimation?
- ability to remove microbial contamination
- effect of removal method on microbial viability
- types and location of organisms
- nature of product
- culture conditions
44
What are the types of micro-organisms likely to be encountered dependent on?
nature of the product, method of manufacture, potential sources of contamination
45
Why is selection of culture conditions so important?
there is no universal medium - no medium will support the growth of every single micro-organism
46
What are CFUs?
Colony forming units (aka microbial count)
47
What types of organisms can grow in Saraboud?
Yeast and fungi
48
What is cycle development in sterilisation processing?
Determining bioburden to define the process
49
What is cycle validation in sterilisation processing?
establishment of documentary evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications
50
Pooled raw milk at the processing plant has bacterial population of 4x10exp5/mL. It is to be processed at 79°C for 21 seconds. The average D value at 65°C for the mixed population is 7 min. The Z value is 7°C. How many organisms will be left after pasteurization? What time would be required at 65°C to accomplish the same degree of lethality?
At 79°C, the D value has been reduced by two log cycles from that at 65°C since the Z value is 7°C. Hence it is now 0.07 min. The milk is processed for 21/60=0.35 min, so that would accomplish 5 log cycle reductions to 4 organisms/mL. At 65°C, you would need 35 minutes to accomplish a 5D reduction.
51
What is performance qualification in process validation?
measure of ability to produce a sterile product
52
What are the two types of performance qualification in process validation?
phyisical and microbial qualification
physical is preferable, it is far more reliable. microbial qualification is a confirmation of your physical measure, or used if there are no physical parameters
53
Definition of a biological indicator?
An innoculated carrier contained within its primary pack ready for use, and providing a defined resistance to the specified sterilisation process
i.e. we already know their D values, so can test the effectiveness of a method using the theoretical conditions
54
What processes are biological indicators used for?
Validation of steam, dry heat, radiation and EtO
| Monitoring of EtO
55
What are biological indicators characterised by?
- strain of organism
- reference to culture collection
- manufacture details
- number (10^6) CFUs per piece
- D and Z values
- recommended storage conditions
- xpiry date
- disposal instructions
56
What factors determine choice of biological indicator?
- stability
- resistance to method (should be high compared to product bioburden)
- non-pathogenic (safety)
- recoverability
57
What biological indicator is recommended for filtration?
Brevundimonas diminute (the smallest)
58
What biological indicator is recommended for moist heat?
Bacillus stearothermophilus
59
What biological indicator is recommended for dry heat?
Bacillus subtilus
60
What biological indicator is recommended for irradiation?
Bacillus pumilus
61
What biological indicator is recommended for EtO?
Bacillus subtilus
62
What is the first choice according to the EMEA decision tree?
autoclave (moist heat) - 121 degrees for 15 mins
63
What is the second choice according to the EMEA decision tree?
moist heat with sufficient lethality >8 mins
64
What is the third choice according to the EMEA decision tree?
Filtration and aseptic processing
65
What is the final choice according to the EMEA decision tree?
Pre-sterilised individual components and asecptic filling
66
What factors determine choice of biological indicator?
- stability
- resistance to method (should be high compared to product bioburden)
- non-pathogenic (safety)
- recoverability
67
What biological indicator is recommended for filtration?
Brevundimonas diminute (the smallest)
68
What biological indicator is recommended for moist heat?
Bacillus stearothermophilus
69
What is filter voidage?
The empty space inside the filter. Actually a network of tunnels, not straight through pores, which allows for more trapping of microbes
70
What biological indicator is recommended for irradiation?
Bacillus pumilus
71
What biological indicator is recommended for EtO?
Bacillus subtilus
72
What is the first choice according to the EMEA decision tree?
autoclave (moist heat) - 121 degrees for 15 mins
73
What is common procedure for filtration sterilisation?
depth filter first to remove large particles, then screen filter to sterilise
74
What is the third choice according to the EMEA decision tree?
Filtration and aseptic processing
75
What is the final choice according to the EMEA decision tree?
Pre-sterilised individual components and asecptic filling
76
Important considerations when it comes to selection of a sterilisation process
- terminal sterilisation is preferred to asecptic processing
- agent must be in contact w all parts of product
- process variables must be controlled and monitored
- process must not present hazard to operators or environment
- cannot leave toxic residues on product
77
Definition of filtration?
“Passage of a fluid (liquid or gas) across a filter, removing any contaminating solutes”
78
Mechanisms through which filters remove bacteria?
irregular shapes, simultaneous arrival at a pore, blocking pores, surface interactions (most bacteria are negatively charged)
79
What is filter voidage?
The empty space inside the filter. Actually a network of tunnels, not straight through pores, which allows for more trapping of microbes
80
What are the two types of filters?
depth and screen
81
What are depth filters?
e.g. filter paper - variable pore size, intertial impaction, high retentive capacity, robust, cheap, no sterility
82
What are screen filters?
uniform pore size (fixed), direct interception, easily blocked, fragile, expensive, pore size less than 0.22 microns gives sterile product
83
What are the critical aspects of dry heat sterilisation?
Loading patterns, air circulation, product size
84
What are the two types of filters?
depth and screen
85
What are depth filters?
e.g. filter paper - variable pore size, intertial impaction, high retentive capacity, robust, cheap, no sterility
86
What are screen filters?
uniform pore size (fixed), direct interception, easily blocked, fragile, expensive, pore size less than 0.22 microns gives sterile product
87
What is common procedure for filtration sterilisation?
depth filter first to remove large particles, then screen filter to sterilise
88
What is the bubble point pressure test?
place filter in a closed system with water on top, apply pressure underneath. relationship between when bubbles appear and the porosity of the filter
89
What type of qualification measure is the bubble point pressure test?
physical
90
What is a method of microbial qualification of filters?
challenge filter with its biological indicator, minimum requirement of 10^7 / sqcm removal. working capacity should be 10^9-10^10
91
How does moist heat kill microbes?
protein coagulation and hydrolysis
92
What products is moist heat used for?
aq. products, dressings, swabs, devices
93
How does dry heat kill microbes?
oxidative processes
94
What products is dry heat used for?
dry powders, oil preparations, glassware and instruments
95
What are the two types of machinery used for dry heat sterilisation?
dry heat ovens and sterilising tunnels
batch vs continuous processing
96
When are sterilising tunnels used?
In industry, easier at scale. move at a set speed to expose product for long enough
97
What are the critical aspects of dry heat sterilisation?
Loading patterns, air circulation, product size
98
What are the four stages of a dry heat cycle?
drying, heating, exposure, cooling
99
what are the holding times accoring to pharmacopoeia?
480 mins at 120 degrees
120 mins at 160 degrees
60 mins at 170 degrees
30 mins at 180 degrees
100
what is used for moist heat sterilisation?
autoclave (essentially a pressure cooker)
101
What heat transfer mechanism occurs in moist heat sterilisation?
latent heat of vapourisation
102
What does the high pressure inside an autoclave enable?
temperatures to reach higher than 100 degrees
103
why must all air be removed from an autoclave?
this is the only way temperatures will reach higher than 100 degrees
104
How does steam cause the product to heat in moist heat sterilisation?
When heat transfers from steam to product, a slight vacuum forms around the product. this draws in more heat
105
What are the critical lethal parameters for moist heat sterilisation?
- steam (must be dry steam, not wet or superheated)
- temperature (maintain within 5k of limit)
- time of contact (sufficient to reach SAL)
- bioburden level (nature, number and location of microbes)
106
what are the conditions for moist heat cycles according to pharmacopoeia?
115-118 degrees - 30 min - 10psi
121-124 degrees - 15 min - 15psi
126-129 degrees - 10 min - 20psi
134-138 degrees - 3 min - 30psi
(psi = steam pressure unit)
107
What are the stages in an autoclave cycle?
1. air removal (by downward displacement or evacuation)
2. heating
3. sterilisation/holding period
4. cooling
5. drying
108
whats the difference between downward displacement and evacuation of air in moist heat sterilisation?
displacement - steam enters in top and forces air out the bottom
evacuation - air is suctioned out. used for products that may retain air e.g. porous dressings
109
what are the three types of cycle used in an autoclave, and what types of product are they used for?
1. fluid cycle - used for most products e.g. saline, containers, glass, heat stable aq products
2. porous load cycle - used for fabrics/dressings etc. introduce steam and actively withdraw a few times to ensure all air is removed. higher temp (134)
3. air ballasted cycle - used for sealed containers that would explode in a regular cycle
110
How are moist heat cycles monitored and validated?
Master temp record (uses a test load and thermocouples to profile the temperatures reached) - validation
Temp records throughout the cycle - monitor. drain should always have the lowest temperature
111
What is the minimum number of thermocouples to use in master temp records for moist heat sterilisation?
12
112
What is the problem with compendial cycles in moist heat sterilising?
achieve 10^-15 ish, gross overkill past SAL
risks of product degradation, economical waste etc
113
How does the gross overkill from compendial cycles arise?
Uses only the holding period lethality, where in reality death of organisms will start to occur from 50 degrees. need to determine total lethality
114
What is the F0 value?
The lethality expressed in terms of the equivalent time in minutes at a temperature of 121oC delivered by the process to the product in its final container with reference to microorganisms possessing a z-value of 10.
provides an alternative to compendial cycles, and allows lethalities to be compared, can choose lower temperatures
115
What is the minimum F0?
8 (i.e. 8 minutes at 121 degrees)
116
What is the relationship between F and D values using biological data?
F = D (logN0 - Log N)
```
N0 = initial bioburden
N = number of organisms surviving the process
```
117
What is the relationship between F and D values using thermal data for moist heat sterilisation?
F0 = ( Log-1 (T-121)/10) ) x dt
118
What is the relationship between F and D values using thermal data for dry heat sterilisation?
F0 = ( Log-1 (T-170)/20) ) x dt
119
What are the benefits of radiation sterilisation?
- 'cold' so suitable for thermolabile products
- continuous or batch
- safe, reliable, reproducible
- single parameter (dose)
- ease of dose measurement
- ease of process control
120
What types of products is radiation sterilisation used for?
single use medical devices, surgical devices, containers, wrappers etc - DRY products
121
How does radiation sterilisation work?
expose product to high energy radiation to inactivate any present micro-organisms
induces a chemical change in vital components of the microbial cell
induced chemical changes in the product must be at an acceptable level and not affect integrity
122
What is ionising radiation?
contains sufficient energy to eject one or more orbital electrons
Important characteristic is the localized release of large amounts of energy. The energy per ionizing event is 33eV, which is more than enough to break a chemical bond. For example, the energy associated with a C=C bond is 4.9eV.
123
What are the units for radiation dose?
Gray (Gy)
1 Gy = absorption of 1J/kg off irradiated material or = 6.242 x10^15 eV/g)
124
What is a dosimeter?
Device having reproducible measurable response to
radiation used to measure absorbed dose in a give
material.
125
How is the radiation dose determined for a product?
based on the innate population's resistance
minimum dose 25Gy
126
What source is used for ionising radiation
Cobalt 60
127
What types of devices is EtO used for?
Disposable, approx 50% of medical devices
128
By what mechanism does EtO kill bacteria?
alkylation of sulphhydryl, amino, hyroxyl and carboxyl groups on Protein and nucleic acids
129
Disadvantages of EtO sterilisation?
- lethality is not uniform, affected by conc, temp and RH
- not same degree of sterility assurance
- explosive
- toxic residues (can be carcinogenic)
- must be over processed to ensure each part of the product is sterile, higher risk to operators
130
What are the critical lethal parameters for EtO sterilisation?
- time (1-24 hours)
- temperature (25-65 degrees)
- humidity (40-85%)
- EtO concentration (250-1200mg/L)
131
Which organisms are used for validation and monitoring of EtO sterilisation?
B. subtilus for both
132
What are the three stages of EtO sterilisation?
1. pre-conditioning
2. sterliser
3. aeration (expose to sterile air to remove residuals)
133
what are the 7 stages within the sterilisation cycle?
1. evacuation
2. vacuum hold (leak test)
3. conditioning
4. sterilant injection
5. exposure
6. sterilant removal
7. flushing
stages 1-3 done at low pressure, increases for sterilant injection
flushing involves cycling pressure to flush product with sterile air
134
how long does the aeration phase last?
12 hours to 7 days depending on product
135
How is sterilant removed from the chamber?
catalyst
136
How is choice of sterilising method determined?
initial product research and development
- microbiocidal effectiveness should be demonstrated (e.g. enzyme kinetics, environmental parameters)
- product compatibility should be demonstrated
- safety and environment
137
What are some examples of new/emerging sterilising technologies?
- X-ray irradiation (low power, expensive)
- Pulsed light (short pulses broad spectrum white light, UV output, used for in-line sterilisation and intravascular medicines development)
- microwaves (intense heating for short cycles, used for solutions in vials, contact lenses etc)
- gas plasma (mix of ions, free radicals, electrons/neutrons, alternative to EtO)
138
Problems with new and emerging sterilisation techniques?
unknown lethal effects, kill kinetics are different to existing processes, validation compliance, monitoring, no established regulatory requirements
