Aseptic Production 2 Flashcards
Sterility
- Sterilisation processes must be specified to ensure that the probability of microorganisms surviving is low enough to ensure patient safety. The accepted low probability is that there should be not more than one chance in one million of viable micro‐organisms surviving.
- Called a sterility assurance level (SAL) of 10‐6
Sterility definition
• The total absence of viable microorganisms and an absolute state (Winfield and Richards)
What are we trying to destroy?
Anything that causes harm to patients
• All viable microorganisms
• Bacterial spores
• Pyrogens
– Produced by Gram‐negative bacteria on cell death
– Endotoxins ‐ lipopolysaccarides from cell walls
– Cause inflammatory response on injection; starts with a
fever, can result in cardiogenic shock (multi-organ failure)
– Distillation of aqueous products the most effective method of removal
What are bacterial spores
what bacteria are reproduced from, they are not living themselves but are able to reproduce
When are pyrogens released
released from bacteria upon cell death
Sterilisation – the main processes
• Heat – Moist heat – Dry heat • Gaseous – Ethylene oxide – Formaldehyde • Irradiation • Filtration
Which sterilisation process is commonly used
- used most commonly are moist heat, ethylene oxide and irradiation
- size of batch affects choice of methodology
D value
• Decimal reduction time
• Time taken at a fixed temperature to reduce the number of viable organisms by 90% (i.e. 1 log cycle reduction)
• Also applies to radiation
– D value is the radiation dose required to achieve the above (where time taken at a fixed temp to the time taken at a radiation dose to achieve a 1 log cycle reduction)
What is D value used for
D value is what we use to calculate the amount of time a particular product will need in the sterilisation process to achieve your sterility assurance level of 10-6
-D value = time taken to reduce at a fixed temp the amount of contaminant you have by 90%
Z value
• Only applies to heat sterilisation
• Assesses the influence of temperature changes on thermal resistance
• Increase in temperature required to reduce the D‐value by 90% (i.e. 1 log cycle)
-so its an additional value on top of D value. Allows you to reduce time exposure to heat if you have a product that can withstand higher temp
How to measure D value and Z value
D value - Time (horizontal axis)
Z value - Temp (vertical axis)
Bioburden
- Need to know at beginning of process
• Concentration of microorganisms in a material
• Normally given by the supplier of a raw material
• Specifications within pharmacopoeial entries, or from product development
• In order to assure sterility, the bioburden must be as small as possible at the start of the process (if bioburden is too large, product will be exposed to sterilising agent for too long which could denature product)
Inactivation factor (IF)
With a bioburden of 103 organisms prior to sterilisation, an inactivation factor of 109 would be required to achieve sterility (10‐6). I.e. the product would have to be exposed to 9 times the D value of a reference organism to achieve sterility.
Inactivation factor (IF) equation
IF = 10t/D
Where t = contact time or dose and D = the D‐value appropriate to the process being used.
IF is expressed as a log value for reference.
Heat Sterilisation
- Most reliable and most widely used method (but decreasing now)
- Destroys enzymes and other essential cell components
- Used with thermostable products
- Moisture sensitive products use dry heat
- Moisture resistant products use moist heat
When would moist heat or dry heat be used?
- Wouldn’t use moist heat for powder
- For glass and metal you would use moist heat to sterilise
- Sterilised piece of equipment that is susceptible to water ingress you would use dry heat
Heat sterilisation graph
Heating stage - holding stage - cooling stage
IF is applied from the time the autoclave reaches the required temp to the time the if is required to stop
Moist Heat Sterilisation
• Most commonly used method of terminal sterilisation – Dressings – Clothing – Equipment – Aqueous injections – Irrigation fluids – Opthalmic preparations
Moist Heat Sterilisation
- Steam under pressure (autoclave )to produce temperatures in the range of 121oC – 134oC
- Causes denaturation and coagulation of essential proteins
- 15 minutes at 121oC (standard for most products, very small D Values , 15 mins would knock out bioburden) or 3 minutes at 134oC (Z value used to caulcuate 3 mins at 134 degress)
- Shorter times cause less product damage
- Shorter times have higher IF value (so if you’ve got a high bioburden and will stand high heat you can put it in at higher temp and reduce the time )
The F‐value
-calculates damage done by heating and cooling phase
• Mathematical method of converting time in the
heating and cooling phases to time spent at 121oC
• Useful for products where the holding time of 15 minutes at 121oC are not possible
• EP (European Pharmacopia) states that “continuous and rigorous microbiological monitoring” should take place during manufacture to ensure consistency
Dry Heat Sterilisation
• Not as commonly used as moist heat • Most commonly used in hospitals – Surgical instruments sterilisation • Other products include – Non‐aqueous thermostable liquids (oily) – Thermostable powders – Glassware ‐ depyrogenation (pyrogens cling to surfaces - to sterilise glassware use dry heat)
Dry Heat Sterilisation
- good for solid or thermostable items
• Less effective than steam
– Lethal effects due to oxidative processes
• Hot air oven
• Therefore need higher temperatures and longer exposure times
• 160–180oC for up to 2 hours (exposure time is greater than moist heat)
• 250oC for 45 minutes for depyrogenation of glassware
Gaseous Sterilisation
- reserved for large batches or temp sensitive items
• Ethyleneoxide
or
• Formaldehyde
Gaseous Sterilisation
• Generally reserved for temperature sensitive items
– Medical, diagnostic and electrical equipment e.g. for use in operating theatres
– Re‐sterilisation of commonly used equipment
Gaseous sterilisation
- Cause alkylation of sylphydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic acids (destorys proteins and changes nature of cells and cell dies)
- Both gases potentially mutagenic and carcenogenic (used with extreme caution)
- Formaldehyde can be detected by smell, ethylene oxide cannot so its often mixed with an agent to get a smell
Gaseous Sterilisation – Ethylene Oxide
-have to be careful about what you mix it with
• Explosive when >3.6% v/v with air
• Supplied as 10% in carbon dioxide or 8.6% with HFC 124 (hydroflurocarbon)
• Good penetration of most materials - gas is atomic and will penetrate materials easily
• 800 – 1200mg/L at 45‐63oC
• Typically for up to 4 hours
• Products must be left to desorb toxins after sterilisation (products must be left to stand in a nongaseous environment after sterilisation)
Gaseous Sterilisation ‐ Formaldehyde
-isn’t most ideal - has to be moist heat at a higher temp
• Formalin (37% w/v aqueous solution of formaldehyde)
• Heated to 70 – 75oC with steam
• Cycle of up to 4 hours length
• Products must be allowed to desorb toxins following sterilisation
Radiation Sterilisation
-sterilise big batches at one time, gamma radiation passes through everything
• Accelerated electrons (particulate radiation)
• Gamma‐ray (electromagnetic radiation)
• UV light (electromagnetic radiation) - least efficient
• Damage microbial DNA – UV light is the least efficient at this
• Good for heat sensitive products
Radiation Sterilisation
• Most common method is gamma‐ray – industrial scale manufacture
• Can damage aqueous solutions and certain types of packaging
• Generally reserved for dry articles
– Surgical instruments
– Plastic syringes
– Dry powders
Gamma‐ray Sterilisation
• Cobalt‐60 (60C) source
• Half‐life of 5.25 years
• Dose of 25kGy (2.5Mrad)
• Dwell time of around 20 hours dependent on source strength
• Lethal to humans in minutes!
-you have a generator of gamma rays, at the start of its life it will have a lot of radiation coming out of it but as it gets older the radiation will be decreasing so for a old generator you need a longer dwell time
-biomarkers placed to know how effective it is
Biological Indicators
• Standardized bacterial spore preparations
• Suspensions or spores dried onto paper, aluminium or plastic
• Placed in with each load for sterilisation
• Cultured after cycle has ended (Culturing process tells you whether cycle has been effective in terms of sterilisation)
-Can’t release batch of terminally sterilised products until bacterial testing has been done. Takes at least 2 days
Biological Indicators examples
allow you to see how well your batch has worked
Moist heat
- BI organism: B. stearothermophilus
- D-value: 1.5 min
- Log IF: 10
Dry heat
- BI organism: B. subtilis var. niger
- D-value: Max. 10 min
- Log IF: Min. 12
Irradiation (25kGy)
- BI organism: B.pumilus
- D-value: 3kGy (quite a high D value)
- Log IF: 8.3
Disinfection
is not sterilisation. it is used in hospital manufacture to assure sterility • Process of removing microorganisms from the surface of inanimate objects – Alcohols: generally used (70% ethanol) – Aldehydes – Halogens – Peroxygens – Phenolics – Quarternary ammonium compounds
- dressing is sterile but package isn’t when it comes out of the sterile environment as its handled by different people
- have to disinfect package to avoid transfer of germs
STERILISATION INDICATOR
- ETO (ethylene oxide) Sterilisation indication 13122 Purple to green
- Steam sterilisation indication 13123 blue to pink
- Radiation (Gamma or E-beam) sterilisation indication 13124 yellow to red
