Sterilisation Flashcards
Define clean conditions:
microbiologically clean (reduced number of microorganisms) – favoured by manufacturers.
TWO General Approaches to sterile products:
- Produce under ‘CLEAN’ conditions, then TERMINALLY sterilise in the final container.
2) Produce and assemble under conditions ‘FREE’ of microorganisms and other particulates.
Examples of microbial Contaminants within the Manufacturing Environment
- Raw materials (synthetic/semi synthetic tend to have low counts of microorganisms) (natural materials will have their own intrinsic populations of microorganisms depending on nature of product)
- Water – primary requirement for microbial growth
- Environment – air (vector for particles), personnel, equipment
Resident organisms:
Soil –> gram positive, endospore forming, fungi
Water –> gram negative, yeast and moulds
Animals and humans –> gram -ve, obligate anaerobes, gram positive
Plants –> yeasts and moulds rather than bacteria
Transient organisms:
Carried by air and water (two main vectors). Humans as operators can also act as vectors
What does sterile mean?
Free of viable (living) microorganisms (sterile is an absolute term, no such thing as “quite sterile”)
Sterilisation = killing or removal of ALL viable microorganisms
Name some methods of killing organisms:
Moist heat steam (autoclaving), dry heat sterilisation (absence of moisture e.g. oils), radiation (cobalt 60), chemical (ethylene oxide)
Sterilisation standards:
Used to: - Control no. of microorganisms in manufacturing environment
- Validate sterilising agent – based on experimental data
- Validate sterilisation process – e.g. length of time for autoclaving
- Monitor sterilisation process – e.g. what controls have been put in place
- Regulated by EN (European), FDA (USA)
Methods of removing organisms?
Removal – Filtration (porosity taken into consideration),
Antibiotic sterilisation?
Filter sterilisation (heat sensitive, chemical could contaminate)
Vial sterilisation?
Steam sterilisation
Stopper sterilisation?
EtO sterilisation
Sterilisation standards are used to:
- Control no. of microorganisms in manufacturing environment
- Validate sterilising agent – based on experimental data
- Validate sterilisation process – e.g. length of time for autoclaving
- Monitor sterilisation process – e.g. what controls have been put in place
- Regulated by EN (European), FDA (US
What is a kill curve?
Curve which plots number of survivors against time
How is a kill curve made?
- Part of a development phase which occurs in a lab
- Take samples at regular intervals and dilute culture so you get colony forming units that you can count on a agar plate.
- Treat sample to sterilise it before you dilute and identify colonies.
- Serial dilutions so when you plate out on to agar you want 30-300 colony forming units
Why do you specifically want 30-300 colony forming units’?
Anything under 30 is not statistically liable and anything above 300 is too hard to count
What type of curve represents a working kill curve?
Decrease in numbers as a function of time, whatever we are doing we are getting a decrease in number – this means it is working
This is an ASYMPTOTE CURVE – regular intervals you get the same proportion of cells killed as per the unit of time. The blue line will never reach zero as It will lose the same number of cells each function of time.
How to make kill curve a straight line?
If you want to make this a straight line you take a log of the survivors, this is a semi-athymic graph. You can then use the gradient to work out the rate of survival at a given temperature. We can then repeat this at different temperatures so you can build up a portfolio of survival at different temperatures (you can change what it is you are measuring).
= First order kinetics
Why use a kill curve?
This is good to see any patterns and trends when you change what is affecting the colonies.
If you are doing different organisms you will also be able to see which organism is more sensitive to the change.
What is the D value?
The time taken, at a fixed temperature, to reduce the population by 90% (1-log). This gives a measuere you can compare at different temperatures. It doesn’t matter which number you have on the y axis as long as it is a full log cycle- it’s a straight-line relationship.
Whats is a thermal resistance curve?
The temperature change required to produce a 90% reduction (1-log cycle) in D-value. Can only be used for temperature changes not any other sterlisation agent
What is the Z value?
- A measure of thermal resistance
- Indicator of efficiency
- Reference (indicator) organisms
- Bacillus stearothermophilus under steam sterilisation has a Z value of 10 degrees
- Bacillus subtilus under dry sterilisation has a z value of 20 degrees.
When is a product deemed sterile?
- Inactivation on log scale, therefore no 0 on the log scale. Therefore, need a way to determine there is no organisms due to 0 not existing
- Measure via sterility assurance level (SAL)
- SAL= 10-6
- Minimum is the SAL value, even better if it is less then this value.
Sterility assurance:
- Start of with 102 and end with 106 you have an 8D reduction, if you know how long your D value is then you can see 8x1=8 minute reduction cycle. This allows you to see how long you need to carry it out for in order to reach you minimum SAL level.
- D values are always expressed in minutes.
our processing is effected by the resistance of the organisms and also the population size of the contaminating organism. - Therefore, even if they have the same d value but a different populations they will have a greater different reduction time so may take longer to get to SAL minimum.
D-Values
Influenced by:
o Bacterial species
o Vegetative vs spore form
o Production method
o Nutrient environment (suspension media, carrier materials, culture media)
o Treatment dose (temperature, radiation, dose)
Importance of bioburden estimation?
Initial population numbers required in order to specify sterilisation parameters and inactivation kinetics.
What is bioburden?
a population of viable microorganisms on or in a product and or package.
Describe the bioburden estimation process:
- Sample selection = choosing samples statistically
- Collection of items for test
- Transfer to test lab = variability depending where the lab is, may need to be transported long distances, so how do you prepare them for them for this?
- Treatment (if required) = to remove the cells. What techniques will you use to remove microorganisms from the product, some of which will have complex designs
- Transfer to culture medium
- Incubation
Bioburden estimation techniques:
Direct contact between product and culture medium (agar plates). For some products this is easier than others. But sometimes you may be unable to do this (indirect approach).
This means you may have to wash the product to wash off any microorganism as bacteria like to stick to surfaces – usually full of nutrients.
We need to check that the eluent we are using isn’t causing effect by osmosis. Sometimes very mild detergents are used to remove cells from surface of the product. But some mild detergents have very weak antibacterial activity so we will need to check that this won’t kill the cells – lots of background checks that need to be carried out.
Some are more difficult to remove, and this means you may have to use physical treatment – vortex, ultrasound (high frequency causes vibration on surface), shake with glass beads to knock off the bacteria – but if they are too big you will harm the organisms you are looking at.
Selection of a Removal Technique.
Considerations:
- Ability to remove microbial contamination
- Effect of removal method on microbial viability = don’t want to cause change to numbers and get a false representation
- Types and location of microorganisms
- Nature of product
- Culture conditions
Selection of Culture Conditions
Types of microorganisms likely to be encountered dependent upon:
- Nature of product = natural are more likely to have a bioburden population
- Method of manufacture = have we added any microorganisms to the product how we made it
- Potential sources of contamination (operator, packaging)
Selection of Culture conditions: process operation:
- Cycle development is the heat killing the microorganisms you want to remove in test tube studies then moving to lab based
- Cycle validation proving that your experiment works. Key to the entire process.
- Cycle monitoring when you have all the evidence monitor every time to make sure you get the deserved end result
What is process validation?
Process validation the 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. This applies to all sterilisation techniques.
Stages of process validation:
What is installation qualification?
when you build sterilisation unit is everything you want to use working properly
Stages of process validation:
What is performance qualification?
does it work and do the job we want it to do properly. You can assess this in two different ways. The better of the two is physical qualification it is not subject to change (taking a physical measurement i.e. temperature profile). Microbiological is used as a back up to support physical qualification, this is when we use microorganisms which have a high resistance to the sterilisation process – but they are more prone to error and variability.
Biological Indicators (BIs) definition:
‘An inoculated carrier contained within its primary pack ready for use and providing a defined resistance to the specified sterilisation process‘. Usually they are simple things like spores’ strips, as endospores are the most resistant to remove, so you stick a stick in and you will be able to kill the most resistant form.
BI Use?
‘To provide a means of assessing directly the microbial lethality of a sterilisation process‘
• Standardised preparations containing selected microorganisms having known stable high resistance to sterilising agents – can collect D vales for endospores
• Used for validation (steam, dry heat, radiation, EtO) and monitoring (EtO) of sterilisation process
• In use, proportion of test organisms surviving the process are measure and related to the expected lethality of the process
BI’s Characterised By:
- Strain of test organisms
- Reference to culture collection
- Manufacturers name, details of who produced
- Number (usually 106) CFU’s per test piece
- D-value
- Z-value
- Recommended storage conditions
- Expiry date – can last to 50-100 years
- Disposal instructions – how to get rid if you need to remove of them
Factors Governing choice of BI:
- Stability
- Resistance (high in comparison to natural bioburden)
- Non-pathogenic – think of the people manufacturing the product
- Recoverability – you want to be able to check 100% where killed and if some don’t die you want to be able to recover the surviving spores to dispose of them properly
Recommended Test BI’s?
Filtration - Brevundimonas diminuta Moist heat - Bacillus stearothermophilus Dry heat - bacillus subtilus Irradiation - bacillus pumilus EtO - bacillus subtilus
Selection of Sterilization Method
General Guidance:
- Balancing the advantages of available methods against their disadvantages
- No requirements to specify which method should be used
- Choice of method made at the design / development stage
Selection of Sterilization Method:
Specific Guidance
• Terminal sterilization of product in final container is preferred to aseptic processing
agent in contact with all parts of product
• Process variables are controlled and monitored
• Process does not present hazards to operators or environment
• Process does not leave toxic residues within the product
Selection of Sterilization Traditional Methods:
Removal:
Filtration – passage of a fluid across a filter, removing any contaminating solutes.
Destructive:
• Heat (moist and dry)
• Ethylene Oxide
• Radiation
Nature of filter is important (pore size). Could affect properties of solution being filtered
As long as pore size is smaller than solutes in liquid, particles should be removed.
Factors affecting sterile filtration:
Particles < Pore Diameter
- Irregular Shape - Angle that particle approaches pore is important
- Simultaneous Arrival – a sudden rush of particles
- Blocked Pore – pore is blocked therefore smaller particles can’t squeeze past.
- Surface Interactions – most bacterial cells are negatively so you could produce a filter that has a slightly positive charge leading to attraction of charges
What is filter voidage?
- no direct passage/channel from top to bottom. Convoluted.
- Open area of a filter is called a voidage which is the area where particles accumulate.
- Filter void age – accumulation of particles
Depth filter types:
Depth
- Non-fixed pore size (variable)
- Rely on Inertial impaction – (particle runs/collides into filter matrix
- High retentative capacity (can retain a large number of particles)
- Robust
- No sterility (no guarantee of producing a sterile product)
- Cheap
Screen filter types:
- Uniform pore size (0.8µm. 0.45µm…) – smaller pore size, higher chance of it being blocked
- Direct interception
- Easily blocked
- Fragile
- Expensive
- Sterility 0.22um – gurantees a sterile filtrate
Describe filter validation:
- Bubble point pressure test – gradually increases the amount of air that passes through the filter until you get bubbles.
- Challenge filter with Brevundimonas diminuta (0.4µm)
- Min. requirement 107 / cm2
- Working capacity 109 - 1010 / cm2
What is moist heat sterilisation?
Moist Heat:
- Death by protein coaggulation and hydrolysis (due to presence of moisture)
- Steam at elevated temps (exceeding 100 degrees)
- Used for aqueous products / devices/ dressings
How does dry heat sterilisation work?
- Death by oxidative processes (due to absence of moisture)
- Used for dry powders / oil preparations / glassware and instruments
Technology for dry heat sterilisation:
- Dry Heat Ovens
- Sterilising Tunnels (continuous process)
Mechanism of heat transfer in dry heat sterilisation:
- Conduction
- Radiation
- Convection (heating of air within chamber)
Critical aspects of dry heat sterilisation?
- Product Size (larger product, longer it takes)
- Loading Pattern
- Air Circulation (otherwise those at the bottom may not be sterilized properly)
What is the dry heat cycle?
- Drying – glassware in oven
- Heating
- Exposure – where sterilization takes place
- Cooling – longest part of sterilization cycle
16 hours start to finish
Dry heat sterilisation: pharmacopeial cycles=
Holding temp and time: 120degrees = 480 mins 160 degrees = 120 mins 170 degrees = 60 mins 180 degrees = 30 mins
Moist heat sterilisation: Technology:
Autoclave (pressure cooker)
o Self-boiler
o Mains steam
Mechanism of heat transfer in moist Heat Sterilisation:
- Latent heat of vaporization- continuous process until product is in equilibrium with steam.
Moist heat sterilisation: Critical aspects?
- Air Removal – if air is present, unlikely to get temps exceeding 100 degrees
- Saturated Steam – super heated steam = low moisture content, saturated steam has a known moisture content.
- Steam under pressure – targeting removal on endospores, need pressure to exceed temp
Moist heat sterilisation: Critical lethal parameters?
- Steam - Dry Saturated NOT wet or superheated
- Temperature - Maintained within +/- 5Kelvin of limit
- Time of Contact - Sufficient to give > 10-6 SAL
- Bioburden Level - Nature, number and location of M/Os
Autoclave Operation:
- Air Removal - downward displacement; evacuation to replace with steam so can heat up to app temperature.
- Heating
- Sterilisation / Holding Period (where you reach the agreed sterilization temp)
- Cooling
- Drying
Autoclave Cycles
Types:
- Fluid Cycle (most common) – that would be used for aqueous liquids (mostly)
- Porous Load Cycle (relatively quick) – however due to the fact they’re fabric, air can get trapped, air has to be replaced with steam.
- Air Ballasted Cycle
Moist Heat Sterilisation:
Cycle Validation & Monitoring
1) MTR - master temp record
- Test load
- Thermocouples
2) TRC - temp record chart
- Drain probe temp. drain in theory should always be the coldest part of the autoclave. As soon as they drain reaches the required temperature by default the chamber and the product should also be at the right temp
Determining the MTR
Key Points:
- Drain is coolest part of autoclave
- Minimum of 12 thermocouples used in chamber (all computerized)
- Run through cycle and check that every thermocouple reaches the minimum requirements giving a nice profile of the whole chamber. As long as everything has reached the conditions that you want.
What is compendial lethality?
- calculating lethality associated with the kill curve
- Compendial cycles based on holding period
- Need to be able to measure total lethality
What is the Fo Value?
- Alternative to compendial cycles
- Allows lethalities to be compared
- Defined as: The lethality expressed in terms of the equivalent time in minutes at a temperature of 121oC (standard refrence) delivered by the process to the product in its final container with reference to microorganisms possessing a z-value of 10.
- Should assure min. SAL of 10-6
- Min Fo = 8 (ie equivalent to 8 mins @ 1210C)
THE Fo CONCEPT:
Compendial Cycles – the Reality!
- (SAL = Sterility assurance level)
- Require SAL of min 10-6
- Typical cycle of eg 15 min @ 1210C
- Measured SALs o 10-15, 10-20 etc
- Gross Overkill !
- Problems of product degradation
- Economically wasteful & expensive
Fo Calculations
1. Biological Data
Relationship between F and D values:
F = D (logN0 - Log N)
Where:
- D is the D-value at the given temp.
- N0 is the initial number of microorganisms present (bioburden)
Fo Calculations
2. Thermal Data
- F0 = F1 + F2 + F3…. + FN
- Cumulative
- Measure of Total Process Lethality
F0 = Log-1 (T-121) x dt
Z
Where:
- 121 degrees is the reference temp (Bacillus
stearothermophilus)
- T is the temp. of heating
- Z is 10oC
- dt is the time of heating
FH value equation:
FH = [log-1(t-170)/z] x dt
- 170o is the reference temp. (Bacillus subtilus) - T is the temp. of heating - Z is 20oC - dt is the time of heating
Radiation sterilisation:
- Cold’ – suitable for thermolabile materials
- Continuous or batch process
- Safe
- Reliable
- Reproducible
- Single process parameter (dose)
- Ease of dose measurement
- Ease of process control
- Single-use medical devices
- Surgical Devices
- Containers
- Wrapping materials
- Pharmaceutical preparations (especially powders in bulk)
What dos RADIATION STERILISATION involve?
- Involves exposure of product to high energy radiation (ionizing radiation) to inactivate microorganisms that are present as contaminants
- Induced chemical change in vital components of the microbial cell is responsible for cell killing
- Induced chemical change in the ‘product’ has to be at an acceptable level and without effect on product integrity
ETHYLENE OXIDE STERILISATION formula?
(CH2)2O
What is ETHYLENE OXIDE STERILISATION used for?
Used for disposable Items; 50% of all medical devices]
How does EthO sterilisation work?
Blockage of reactive sites in the microorganism.
Alkylation of sulphhydryl, amino, hydroxyl and carboxyl groups on proteins and nucleic acids.
Lethality of EthO on micoorganisms affected by:
Conc. of EthO, Temp and RH (non-uniform) - so to be effective, need moisture content
Does ETHYLENE OXIDE STERILISATION use same degree of Sterility Assurance as other methods?
No - people strive to get SAL 10-6 but its unlikely to achieve.
Why does EthO sterilisation require standard product load containing suitable BI (biological indicator)?
Because there is no means to physical means measuring concentration.
Biological qualification
Issues with EthO?
- Toxic residues on product – have to be removed before product is released for patient use
- Operator safety (EtO in air is highly explosive therefore now has to be mixed with either CO2 or nitrogen
EtO Critical Lethal Parameters?
- Time - 1 – 24 hours
- Temperature 25 – 65OC
- Humidity 40 – 85% RH (helps kill bacteria as it enhances operation of EtO which kills bacteria)
- EtO Concentration - 250 – 1200mg/L EtO (difficult to monitor)
- EtO distribution & penetration issues
- B. subtilus (indicator organism) used for BOTH
- Validation and Monitoring
EtO process flow:
1) Pre-conditioning (room/chamber)
2) Steriliser
3) Aeration (room/chamber)
How many steps in sterilisation cycle?
7 steps: - Regardless of product, cycle tends to take on this shape
-What will vary is the time – depending on nature of the product i.e. what it is, volume etc.
Describe the EtO pre-conditioning stage:
Preconditioning (ensure that the product reaches the right humidity levels – moisture is needed with the EtO to increase effectiveness)
Describe the 7 step EThO sterilisation process:
- Evacuation (removal of air from chamber and product as EtO is explosive in air therefore nitrogen/CO2 are added)
- Vacuum hold (leak test) – to check air has been fully removed
- Conditioning – starting to heat the product
- Sterilant injection – where the EtO is injected via a canister and the EtO vaporises
- Exposure
- Sterilant removal (length process) Catalytic converters are used to convert the EtO into CO2 and water.
- Flushing – filtered sterile air introduced, vacuum used to remove air and any EtO
Selection of Sterilising Agent
General Guidance:
• Balancing the advantages of available methods against their disadvantages
• No requirements to specify which method should be used
- Choice of method made at the design / development stage
Selection of Sterilising Agent
Specific Guidance:
- Terminal sterilization of product in final container is preferred to aseptic processing
- Agent in contact with all parts of product
- Process variables are controlled and monitored
- Process does not present hazards to operators or environment
- Process does not leave toxic residues within the product and the product is adequate for use
New & Emerging Sterilisation Technologies
Examples:
- X-ray Irradiation - Ionising radiation; expensive; low power
- Pulsed light - Broad spectrum white light, short pulses; UV output;
Used for In-line sterilisation & intravascular Med Dev. - Microwaves - Intense heating; short cycle (sec);
- Used for solutions in
vials, contact lens - Gas plasma - Mixture of ions, free radicals, electrons & neutrons;
Alt to EtO; used for Med Dev.
Sterilisation agent characterisation forms part of the initial research and development: What is the process?
- Precise description of nature & quality of sterilization agent described
• Microbiocidal effectiveness needs to be demonstrated e.g. enzyme kinetics, environmental parameters etc.
• Material effects, i.e. product compatibility
• Safety and the environment
QUALITY CONTROL & QUALITY ASSURANCE:
Generic Tests?
- Bioburden estimation
- Test for Sterility
- Test of Sterility
- Test for Pyrogens (LAL Test)
Microbiological Methods
Test of Sterility:
1) Direct immersion in medium and incubate
2) Remove microorganisms by elution (either use medium as fluent or dilute fluent with equal volume of D/S medium and incubate)
- Filter to recover microorganisms
- Transfer filter to medium and incubate
Microbiological Methods: Precautions to minimise level of false positives?
– 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 train personnel
Microbiological Methods: What are false negatives?
- Inadequate culture conditions
- Presence of microbiostatic / cidal substance
- Interval between treatment and testing
Microbiological Methods: Define false negative defined in pharmacopoeia.
Defined in Pharmacopoeia
• Testing for a ‘negative’, ie absence of M/O’s
• Probability of rejecting a batch as a function of
o frequency of contamination
o number of times tested
- Probability of rejection = 1 - (1 - p)n
Where
p = proportion contaminated
n = number of items tested
How many further re tests allowed in failure sterility test?
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
Additional tests therefore increase chance of passing the test!
Two tests for sterility?
- Destructive Test
- Statistical test (imprecise, The greater number of samples tested, the greater the probability of rejection)
The batch passes the Test for Sterility, NOT that the batch is sterile!
What are pyrogens?
Pyrogens are endotoxins produced by the LPS from Gram-negative bacteria
Structure of pyrogen?
Lipid A –> core –> O antigen
What is the LAL test?
Bacterial Endotoxins Test
- LAL (Limulus Amebocyte Lysate) test is used for detecting endotoxin
Whta is limulus amebocyte lysate (LAL)?
an aqueous extract of blood cells (amoebocytes) from the blue blooded horseshoe crab, Limulus Polyphemus
What is LAL test based on?
clotting reaction of horseshoe crab lysate by endotoxin
LAL test process?
- Equal volumes of test solution and LAL reagent are mixed in glass tubes
- After incubation at 37oC for 1h, the tubes are observed for clot formation after inverting them
- Formation of a solid clot that withstands inversion of the tube constitutes a positive test
Three types of LAL test?
- Gel Clot
- Turbidometric (kinetic)
- Colorometric (chromogenic)
Methods of depylrogenation:
- Rinsing or dilution is a good way to eliminating pyrogenic activity
- Pyrogens in vials or glass components may be destroyed by dry heat sterilization at high temperatures (2500C for 45mins)
- Pyrogens removed from Water for Injections by distillation
