Sterilisation Flashcards
What are the two approaches to producing sterile products?
Produce under clean conditions, terminally sterilise in final container (limit number of microorganisms in environment)
Produce and assemble under conditions free of microorganisms and other particulates (aseptic processing)
Describe the microbial content of raw materials
Synthetic materials have a low microbial count, populations generally not diverse - most contamination comes from process
Natural materials have a large, diverse population of microbial cells - population is usually unique to the material
What is the issue surrounding water presence during manufacture?
Generally microbial growth where there is water - exclusion will prevent growth/kill existing organisms
Where can microbes come from in the manufacturing environment?
Air
Personnel and equipment
Facilities
Movement can cause spread
What is the advantage of knowing resident organisms?
Allows specific controls against them when using materials in manufacturing process
What resident organisms are present in soil?
Gram positive
Endospore forming
Fungi
What resident organisms are present in water?
Gram negative
Yeast
Moulds
What resident organisms are present on animals and humans?
Gram negative
Obligate anaerobes
Gram positive
What resident organisms are present in plants?
Yeasts
Moulds
What are transient organisms?
Carried to a place of rest, generally by water or air
What is the difference between sterile and sterilisation?
Sterile means free of all viable microorganisms whereas sterilisation refers to the process of killing/removing all viable microorganisms
What are the methods of killing microorganisms?
Heating with dry or moist heat
Chemical use - Ethylene oxide
Use of gamma radiation
What are the methods of removing microorganisms?
Filtration - removes cells without killing
What needs to be considered when choosing a sterilisation process?
Will microbes be removed by chosen process?
Will end product withstand process?
What are the purposes of sterilisation standards?
Control microorganism numbers in a manufacturing environment
Validate a sterilising agent/process
Monitor a sterilisation process
How do manufacturers test the compatibility of a sterilant and product?
Expose a culture of cells to sterilant for extended period of time, remove sample at time points and perform viable count
How is the best sterilant for a product determined?
Expose to a number of different ones and compare the different effects
What are inactivation kinetics?
First order kinetics affected by concentration of sterilant
Organism specific
Infinite probability of survival
What is the D-value?
Time taken (mins) at a fixed temp/conc to reduce population by 90% (1 log cycle)
What influences the D-value of an organism?
Species Vegetative vs endospore form Production method Nutrient environment Treatment dose
What is the Z-value?
Change in temperature (°C) required to produce a 90% reduction in the D-value
What is the purpose of the Z-value?
Measures thermal resistance and therefore efficacy of heat as a sterilant
When is a product considered sterile?
No 0 on log scale so min. level defined (10^-6)
How do we know when a product is sterile?
Only accurately measure to 10^1/10^0 microorganisms so plot graph and extrapolate to find exposure time - generally go beyond minimum limit
What would the log plot look like for organisms with the same D-value?
Lines would be parallel
Define bioburden
A population of viable microorganisms on or in a product and/or its packaging
Why is a bioburden estimate important?
Initial population numbers are required in order to specify sterilisation parameter and inactivation kinetics
Where would details of sample selection be found?
Pharmacopeia
Why is storage of items important?
Prevents growth/death which would give a false estimation for the bioburden
What is direct treatment for cell sampling?
Direct interaction between product and growth medium
What is indirect treatment for cell sampling?
Break up structure into individual components
Wash with eluent to remove free cells BUT must not affect viability of organisms
Physical treatment - swab, ultrasound, glass beads
What are the considerations when removing cells?
Ability to remove Effect on viability Type and location of microbe Nature of product Culture conditions
What should be taken into account when selecting culture conditions?
Type of microbe depends on nature of product, manufacture process and sources of contamination
A specific growth medium will not work for all microbes
What is the purpose of enumeration and characterisation?
Looking for low numbers of colonies, manufacturing process should limit amount to be removed by sterilisation
What is process validation?
Proves there is a reliable process that will consistently produce a product that meets the predetermined specifications
What are the steps of process validation?
Installation Qualification - Check equipment works
Performance Qualification - Measure ability to sterilise a product
Physical Qualification - Monitor conditions of the sterilisation process
Microbiological Qualification - Confirms physical qualification or used instead
What are biological indicators?
Inoculated carrier contained within primary pack ready for use
Provides a defined resistance to a specified sterilisation process
Why are biological indicators used?
Direct assessment of microbial lethality of a sterilisation process
Validation and monitoring of a process
How are BIs used for validation and monitoring?
Proportion of surviving test organisms measured and related to expected lethality
How are BIs characterised?
Strain of test organism Reference to culture collection Manufacturer's name Number of colony forming units per test piece D-value for radiation or Z-value for heat Recommended storage conditions Expiry date Disposal instructions
What should be considered when choosing a biological indicator?
Stability
Resistance - should be high in comparison to product bioburden
Should be non-pathogenic
Recoverability
What are the BI recommendations for each sterilisation process?
Filtration - Brevundimonas diminuta Moist Heat - Bacillus stearothermophilus Dry Heat - Bacillus subtilus Irradiation - Bacillus pumilus EtO - Bacillus subtilus
What is the general guidance for sterilisation?
Advantages of available methods have to be balanced with disadvantages
Method chosen at design/development
What is the specific guidance for sterilisation?
Ideal terminal sterilisation instead of aseptic processing
Sterilising agent has to be in contact with all parts of product
Process variables should be controlled and monitored
No hazards to environment or operators
No toxic residues within product
What is filtration sterilisation?
Passage of fluid across a filter, contaminating solutes removed
What can block the filter pores?
Irregular shaped particle
Simultaneous passage of particles
Surface interactions
What are filter voidages?
Empty spaces between the filters, particles can accumulate here
Describe depth filters
Variable pore size Particles collide with matrix High retentive capacity Robust Cheap No sterility
Describe screen filters
Uniform pore size Direct particle interception Easily blocked Fragile Expensive Sterility 0.22µm
What are the mechanisms of filter validation?
Bubble point pressure test - Add water and increase pressure until bubbles come through, shows relationship between pressure and porosity
Challenge filter with Brevundimonas diminuta (0.4µm). Put population in and look for minimum removal of 10-1/cm2. Working capacity: 10^9-10^10/cm2
How does moist heat sterilisation cause cell death?
Protein coagulation and hydrolysis
What is moist heat sterilisation used for?
Aqueous products, devices, dressings
What is an autoclave?
Self boiler, maintains steam
How autoclave operate?
Downward displacement of cold air or evacuation of air
Heating, holding period (15 mins at 121°C), cooling, drying
How is heat transferred in moist heat sterilisation?
Latent heat of vaporisation removes air from chamber and product
What are the critical aspects of moist heat sterilisation?
Air removal
Saturated steam (fixed moisture content)
Steam under pressure
What are the critical lethal parameters for moist heat sterilisation?
Dry and saturated steam, not wet or superheated
Maintain temp. within 5K either side
Exposure time sufficient to reach SAL 10-6
Bioburden level should consider nature, number and location of microorganisms
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
What are the different methods of validation for moist heat sterilisation?
Master Temp. Record - Drain coolest part of autoclave, use min. of 12 thermocouples in chamber
Temp. Record Chart - Drain probe temperature
How does dry heat sterilisation kill bacterial cells?
Oxidative processes
What is dry heat sterilisation used for?
Dry powder, oil preparation, glass, instruments
What equipment is used for dry heat sterilisation?
Dry heat ovens
Sterilising tunnels
What are the critical aspects of dry heat sterilisation?
Product size, loading pattern, air circulation
How is an even temperature achieved in dry heat sterilisation?
Use fan assisted circulation
What are the mechanisms of heat transfer in dry heat sterilisation?
Conduction, convection, radiation
Describe the dry heat cycle
Drive off moisture by drying
Heat to required sterilisation temperature
Keep product exposed for appropriate time
Cool
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
What is the F0 concept?
Alternative to compendial cycles which allows comparison of lethalities
Why was the F0 concept developed?
Compendial cycles overkill microorganisms so economically wasteful and may lead to degradation
How is the lethality measured?
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
What is the minimum F0 value?
8 - 8 mins at 121°C
Define F
D(logN0 - logN)
Define F0
Cumulative measure of total process lethality
(log-1 (T-121)/Z) x dt; where T is the heating temp., Z is 10 and dt is the heating time
What are the benefits of F0?
Can be used for heat labile products and offers flexibility for heat sterilisation
Define Fh
(log-1 (T-170)/Z) x dt; where T is the heating temp., Z is 20°C and dt is heating time
Describe the characteristics of radiation sterilisation
Suitable for thermolabile materials Continuous or batch process Safe, reliable and reproducible Single process parameter Ease of dose measurement Ease of process control
What are the uses for radiation sterilisation?
Single use medical devices Surgical devices Containers Wrapping materials Pharmaceutical preparations
How does radiation sterilisation work?
Product exposed to high energy radiation, inactivating microorganisms
Chemical change induced in vital components of cell, causes death
Chemical change shouldn’t impact product integrity
What is ionisation?
Sufficient energy input to eject one or more orbital electrons from atom or molecule
What are the radiation dose units?
1 gray (Gy) = absorption of 1J/kg of irradiated material 1 Gy = 6.242 x 10^15 eV/g
What is eV/g?
Electron volts per gram
What is a Posimeter?
Measures absorbed dose in given material
Reproducible, measurable response to radiation
How is the minimum radiation dose determined and what is the minimum?
Determined per product by number and resistance to radiation
Min. dose 25kGy
What are the uses of EtO sterilisation?
Disposable items
50% of all medical devices
How is the explosivity of EtO nullified?
Mixed with inert gas, usually CO2 or nitrogen
How does EtO kill cells?
Alkylation of sulphhydryl, amino, hydroxyl and carboxyl groups
What factors affect lethality of EtO sterilisation?
Concentration
Temperature - Increased activity at higher temperatures
Relative humidity
Which is the worst method sterilisation? Why?
EtO sterilisation, less sterility assurance
Toxic residues and operator safety issues - residues may be carcinogenic
Issues with distribution and penetration of EtO
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
What species is used for the validation and monitoring of EtO sterilisation?
Bacillus subtilus
Briefly describe the process of EtO sterilisation
Pre - Conditioning
Sterilisation Cycle - Evacuation, vacuum hold, conditioning, sterilant injection, exposure, sterilant removal, flushing
Aeration
What are the new sterilisation techniques? Describe them
X-ray radiation - Ionising radiation, expensive, low power
Pulsed light - Short pulses of broad spectrum white light, UV output
Microwaves - Intense heating for short cycle
Gas plasma - Mixture of ions, free radical, neutrons and electrons, alternative to EtO
What are the uses of pulsed light, microwaves and gas plasma?
PL: In-line sterilisation and intravascular medical devices
MW: Solutions in vials, contact lenses
GP: Medical devices
What are the problems with new sterilisation techniques?
Unknown lethal effects Different kill kinetics to traditional processes Validation compliance Monitoring No established regulatory requirements
