sterilisation Flashcards
There are two general approaches to produce sterile products. what are they?
- produce under clean conditions then terminally sterilise in the final container
(limit the no. of MO’s during the manufacturing process - best way) - produce and assemble under conditions free of MO’s and other particulates. (ASEPTIC)
Why is Aseptic processing the best way to produce a sterile product?
you are sterilising a product in one container and only using one container.
Which type of material out of synthetic and natural have a greater microbial count?
Natural
Synthetic have a low count - usually MO’s on synthetic materials are derived from the manufacturing process (the machines they are put under.)
Does the population count and type of MO’s present depend on the natural product itself?
YES
what are the three factors associated with the manufacturing process that can cause MO’s to appear on a product?
raw materials, water and the manufacturing environment
what does the manufacturing environment include?
AIR (spores and endospores)
PERSONNEL (ourselves, we carry a host of MO’s)
EQUIPMENT AND FACILITIES (moving parts)
What are the sources of MO’s?
organisms can either be resident or transient
Give examples of where resident organisms come from
soil (gram+, endospore forming, fungi)
water (gram-, yeasts and moulds)
Animals and humans (gram-, obligate anaerobes, gram+)
Plants (yeasts and moulds)
Give examples of where transient organisms come from
they are carried by water and air.
they are harder to control
may already packaged up with the product inside
what does sterile mean?
free of viable micro-organisms; ZERO MO’s present
There are different sterilisation methods: killing or removal of all viable MO’s
KILLING: heating - dry heat and moist heat chemical - EtO radiation - gamma REMOVAL: filtration (sieves the cells out, doesn't damage them)
AN antibiotic solution will be placed in a vial with a stopper - how are these three components sterilised?
solution: filter
vial: steam sterilisation
stopper: EtO sterilisation
then bring the 3 components into the clean room to be assembled together to form a packaged sterile product
Is water destroyed by high temperatures?
NO
What are sterilisation standards used for?
- to control the number of M/O’s in the manufacturing environment
- to validate the sterilising agent
- to validate the sterilisation process
- to monitor the sterilisation process
who are the sterilisation standards regulated by? Are they consistent across all countries?
European Networks
FDA
no, they can differ between continents/countries, therefore, if a drug wants to be sold in America BUT was created and tested against the sterilisation standards in England, it needs to be repeated in America, against their standards before being sold.
Inactivation kinetics summary (4 points)
- first order kinetics
- affected by temperature
- infinite probability of survival
- organism specific
Why is bioburden estimation important?
inital number required to specify sterilisation parameters - to know what the pop. count is or what the pop. looks like in order to kill them
What is the bioburden estimation?
A population of viable micro-organisms on or in a product and/or packaging - the packaging is part of the overall product
At what stage would you conduct indirect treatment to the product?
after transfer to the test laboratory
What does indirect treatment include to estimate the bioburden on a product?
- contact with eluent
- physical treatment
- Then transfer to culture medium
In which cases would you use indirect treatment?
a multi-component product: indirect route would break the product apart
what do you have to be sure to do before using an eluent on a product?
look in the pharmacopoeia to see if the eluent you are using does not affect the vialbility of the cells you are handling
What are the physical treatment measures you can undergo on your product to extract the MO’s?
after using a eluent
add a mild detergent to identify them and help remove them from the surface
treat them with a swab/ultrasound - need to consider frequency
extract via glass beads - shake the product with the glass beads, the smaller the size of the beads the better.
What is the direct technique of estimating the bioburden count?
direct transfer to culture medium (also need to consider which culture medium will be used, as there are so many different types.)
selection of a removal technique considerations (indirect method)
- ability to remove microbial contamination
- effect of removal method on microbial viability
- types and location of MO’s present on product
- nature of product
culture conditions (in which the MO’s will be in contact with after)
is there a universal growth medium?
NO
the type of MO’s likely to be encountered upon depend on what?
the nature of the product
method of manufacture
potential sources of microbial contamination (operator, packaging)
what are the stages involved in bioburden estimation?
sample selection collection of items for test transfer to test lab treatment (if required) transfer to culture medium incubation enumeration and characterisation interpretation of data
process operation - what are the three factors involved in this?
- cycle development
- cycle validation
- cycle monitoring
what is the definition of process validation?
high degree of assurance provided for a specific process consistently producing a sterile product every time (the process meets its pre-determined specifications)
performance qualification can be assessed in two different ways - what are these?
physical qualification
micro-biological qualification
are physical or micro-biological measures more reliable in assessing performance qualification?
physical measures, they can be measured/monitored e.g. the amount of radiation the product is being exposed to
microbiological measures are open to change. biological indicators are used to often validate physical qualification
Are physical and microbiological qualification methods, means of assessing the physical validation of a sterilisation process?
YES
what is the definition of biological indicators?
An inoculated carrier contained within its primary pack ready for use and providing a defined resistance to the specified sterilisation process
what does it mean if the sterilisation process can kill endospores?
the sterilisation process is good
when are biological indicators used?
used for validation of steam, dry heat, radiation EtO. only used routinely to monitor EtO.
What is the sterility assurance level?
10-6. this is used when using BI’s as a means of measure for performance validation. if the process can reduce the no.i of BI’s to this level or more then it is good.
What factors govern the choice of BI’s used?
stability
resistance (high in comparison to product bioburden.)
non-pathogenic (endospores should be non-pathogenic)
recoverability
What are the recommended BI’s to be used for the different sterilisation processes, to assess whether they meet the sterility assurance level at least?
Filtration - Brevundimonas Diminuta Moist - Bacillus Stearothermophilus Dry - Bacillus Subtilus EtO - Bacillus Subtilus Radiation - Bacillus pumilus
When is the choice of sterilisation method made?
often at the design/developmental stage of the process
Which is better - terminally sterilising the product in the final container or aseptic processing?
terminally sterilisation (product in its container with its label) - but this is not always possible, in which case you would use aseptic processing!
What are factors of the sterilisation method that should be followed?
shouldn’t leave any toxic residues;
agent should be in contact with all parts of the product
process variables are controlled and monitored
process does not present hazards to the operators or the environment
is filtration sterilisation interested in big solutes?
No, only small solutes
What is filtration sterilisation?
when a passage of fluid (liquid or gas moves across a filter, removing any contaminated solutes
A sterile filter (screen filter) should the particle size be less than the pore diameter of the filter?
yes
what are the four different sizes of the particles that could come into contact with a sterile filter?
irregular shape simultaneous arrival (two particles try to go through the same pore) blocked pore (fat) surface interactions
What is filter voidage?
the empty space of the filter- particles accumulate in the voidage and then you will notice a small layer of cells on top of the filter.
What two types of filters are there? (removal sterilisation, not killing!)
Depth and screen
which type of filter achieves sterility?
Screen
do depth filters have a fixed pore size?
No - it is variable
name other properties of depth filters that are associated with them.
robust
cheap
intertial impaction
high retentative capacity
what is the uniform pore size for a screen filter?
0.8um (EU) or 0.45um (America)
What is the benefit of using a depth filter and a screen filter in combo?
As the uniform pore size of a screen filter is quite low, if you initially tip the product straight over, alot of the pores may get blocked. in this case it is better to use a depth filter first to filter out all the larger particles and then having a screen filter to allow the small particles to hit the surface of the filter.
name other properties of screen filters that are associated with them.
fragile (very thin)
expensive
will produce a sterile product if the correct porosity is present
direct interception
What are the two different methods to validate a filter?
physical measurement: bubble point pressure test
biological measurement: Brevundimonas Diminuta
explain how the bubble point pressure test works?
use tap water
increase the pressure till a few bubbles arise
the relationship between the pressure and the porosity - to make sure the correct porosity is present in the filter.
how do you use brevundimonas diminuta to validate the filter?
a fully functional filter should be able to remove the organism at a size of 10^7 per cm2 at least! BUT working capacity is usually around 10^9-10^10/cm2 anyways.
how does death occur via dry heat sterilisation?
oxidative process
what is dry heat sterilisation used to sterilise?
glasswares, oil preps, instruments
why are dry heat ovens efficient?
they have stainless steel sides, therefore improving heat transfer. also withold sterilising tunnels (conveyer belts) which is a continuous process
what are the critical aspects associated with dry heat sterilisation?
Product size (larger products harder to heat up due to low SA:V ratio) loading pattern air circulation (fan assisted oven to achieve an even temp all throughout the oven)
what are the four phases of dry heat sterilisation?
- drying (moisture intitially driven off just by placing the product in the oven and heat starts to rise)
- heating (heating to the temp required for the sterilisation)
- exposure (exposure of the product to heat for the required amount of time)
- cooling (product needs to cool down)
which temps are the most commonly used from the pharmacopoeial cycles and what are the holding times associated with these temps?
170 degrees C: 60 mins
160 degrees C : 120 mins
How long will a dry heat cycle overall take?
12-15 hours
what technology is used for moist heat sterilisation?
autoclave (self boiler or mains steam) very similar to a pressure cooker
what products undergo moist heat sterilisation?
aqueous products/devices/ dressings e.g. swabs/dressings
how does death occur by moist heat sterilisation?
death by protein co-agulation and hydrolysis
in a autoclave, what temps can it reach?
in excess of around 100 degrees celcius
what is the mechanism of heat transfer in moist heat S.?
steam is generated by boiling of water
temps can reach in excess of around 100oC
steam rises
BUT only occurs if there is no air already present in the chamber of the autoclave
how is air removal achieved when you switch the autoclave on?
steam comes in from the top, pushes it downwards and pushes air out of the chamber for it to be drained (there is a drain connected at the bottom of the chamber for air to come out - steam pushes the excess air out)
how is the product heated in the autoclave?
after air removal, saturated steam will wrap itself around the product and some of the energy from the steam is transferred to the product. this causes a slight vaccum to be generated around the product - this cools additional heat and steam. Eventually the chamber and the product will reach the required temp (the chamber first)
what are the critical aspects of MHS?
air removal
saturated steam
steam under pressure to achieve temps in excess of 100oC
what are the critical lethal parameters associated with MHS? these need to be achieved!!!!
steam (dry saturated, not WET OR SUPER SATURATED)
temp (+/- 5K of the required temp)
time of contact (sufficient to given SAL of >10^-6)
bioburden level (nature, no. and location)
what are the 5 different procsses involved overall in the autoclave?
air removal heating sterilisation/holding period cooling drying
why are the temps very specific in the pharmacopoeial cycles?
they correlate with the steam pressures - it is easier to measure the steam pressure rather than the temp (Psi) therefore the temps are very specific
what is the most commonly used temp and holding time
121-124 oC for 15 mins
how long will the process take overall?
2-2.5 hours (much less than dry heat sterilisation)
What are the three different types of autoclave cycles?
fluid cycle (MHS)
porous load cycle
air- ballasted cycle
what is a porous load cycle usually used for?
fabrics and dressings, all the air needs to be removed then heat is introduced (injecting and withdrawing 6/7 times)
heated alot higher compared to a fluid cycle therefore cycle usually complete within around 30 mins
when would you use an air-ballasted cycle?
to sterilise sealed plastic units e.g. saline containing plastic bags in hospitals
they have specific pressures associated with them therefore you can’t put them under a fluid cycle
Which two ways can you validate and monitor MHS?
MTR and TRC
Master Temp Record - what does this include?
test load
thermocouples placed around the autoclave (minimum of 12 must be placed!) computer generated diagrams help to visualise/localise where the TC’s are in the autoclave
Temp Record Chart - what does this include?
drain probe temp
once the drain reaches the required temp, the everything else in the chamber should have already reached the required temp
which part is the coolest part of the autoclave?
the drain
What is important about MTR?
it is specific for one type of load only!!
if you increase/decrease the volume or size of product then you have to validate the product again - can’t mix and match
Why don’t we want gross overkill? (which some companies do by over achieving the SAL)
could cause product degradation
this is economically wasteful and expensive - we then make up for it because it pushes the price up of products
What is the Fo value?
the lethality of a process expressed in terms of the equivalent time in minutes at a temp of 121 degrees delivered by the process to the product in its final container - with reference to micro-organisms possessing a Z-value of 10.
what is the minimum value Fo should be?
8 mins @ 121 degrees celsius
The GREATER the Fo value the GREATER the lethality of the process. Corresponds equivalently to the holding times and temps.
the greater the lethality for the greater the Fo value!
e.g. 3 mins @ 134 oC >
8 mins @ 121 oC
what are the overall summary points for the Fo value?
alternative to compendial (dry heat/moist heat, Eto, radiaiton) cycles
allows lethalities to be compared
can be used for heat labile products
offers great flexibility for heat sterilisation
when is EtO sterilisation used?
for dispensing items and 50%| of all medical devices
what does EtO sterilisation require?
standard product load containing suitable biological indicators
What needs to be mixed with EtO to calm the explosivity down?
needs to be mixed with an inert gas e.g. nitrogen or mainly CO2
what is the measure of sterility assurance with EtO - does this differ from other sterilisation processes?
there is no accurate, physical measure for EtO, manufacturer’s rely on biological indicators (looking for a certain level of kill on endospores)
what are the critical lethal parameters for EtO?
time: 1-24 hours
temp: 25-65 oC
humidity: 40-85% RH
EtO conc: 250-1200mg/L EtO
B. Subtilus: used for BOTH validation and monitoring
what is the problem with EtO
has distribution and penetration issues
what equipment is needed for EtO sterilisation?
pre-conditioning
steriliser
aeration room (residuals are not explosive to sterile air, the air helps them be removed)
how does aeration help? (last step after pre-conditioning and sterilisation cycle|)
product is exposed to several pulses of air to make sure any residuals (which could be carcinogenic) are gotten rid of.
manufacturer’s will often quarantine with the product to make sure the product has no EtO left on it.
Specific guidance rules with alternative technologies
- terminal sterilisation preferred to aspetic processing
- agent should be exposed to all parts of product (including the packaging)
- process does not present hazards to the operators or the environment
- process variables are controlled and monitored
- process does not leave toxic residues
What other factors are associated with alternative technologies, other than specific guidance?
general guidance
sterilising agent characteristics
what are the sterilising agent characteristics for an alternative technology?
- precise description of nature and quality of sterilisation agent described
- microbial effectiveness needs to be demonstrated e.g. enzyme kinetics, environmental parameters
- material effects (product compatibility)
- safety and the environment
Give examples of new and emerging sterilisation techniques
x-ray irradiation
pulsed lights (specific wavelength of light)
microwaves
gas plasma - made the most progress in terms of being used as a sterilisation process
what can gas plasma be used to sterilise?
medical devices (alternative to EtO)
what are microwaves used to sterilise?
contact lens’, solutions in vials
what is the difference between x-ray irradiation and pulsed lights?
X ray: ionising radiation
pulsed lights: specific wave length of broad spectrum light used and it is non-ionising
what is pulsed light used to sterilise?
in-line sterilisation and intra-vascular medical devices
what are the current problems associated with new technologies?
- unknown lethal effects (should really know how the MO’s are killed)
- kill kinetics are different to the traditional processes
validation compliance: can the new techniques consistently show the end points? - monitoring: physical methods used
- no established regulatory requirements - therefore no standards!
