Lecture 18 - Sterilisation Techniques Flashcards
What do medical or surgical procedures often breach?
the protective barriers of the host e.g. skin and mucosal surfaces or are topically applied to important body structures e.g. eyes, ears
Why is it critical that the pharmaceutical or surgical products being used are sterile?
to prevent the exposure of host tissue to potentially harmful microorganisms
Examples of pharmaceutical sterile products?
parenteral injections & infusions, ophthalmic preparations, ear preparations, wound and bladder irrigations
Examples of sterile surgical products?
wound dressings, artificial joints, cardiac pacemakers, surgical instruments, surgical gloves, hypodermic needles
What is the concept of sterility from an academic perspective?
complete absence of viable microorganisms from a product
What is sterility from a Pharmacopoeial perspective?
the tests to establish sterility are often limited due to the grounds of statistical probability due to limitations in the testing criteria available
What is sterilisation?
the process of removing or killing microorganisms from the product to render it sterile
What are examples of sterilisation methods?
heat (steam & dry heat)
radiation (g-rays or high energy electrons)
gaseous (ethylene oxide or formaldehyde)
filtration (with subsequent aseptic processing)
What does the sterilisation method applied depend on?
the physiochemical stability of the product to be sterilised
What is the most common type of sterilisation used?
steam sterilisation, accounts for 80% of sterilisation applied
What does sterility depend on?
microbial burdens of raw materials, equipment and facility
the operators
the use of validated sterilisation protocols
in process control of the process and the production environment
suitable storage conditions of the finished products
Why is storage of the finished product important?
to prevent recontamination
What should parenteral products be?
be pratically free from particles
be pyrogen free
be physiological compatible in terms of pH, tonicity
What should eye drops be?
buffered near physiological pH of tears
What do particles do if they are entered into a patient?
they have the ability to block capillary beds or travel somewhere more distance and block an important capillary bed, leading to reduced oxygen flow
What might pyrogens cause?
unwanted side effects e.g. pyrexia or disturb blood pressure
What are the heat sterilisation methods?
moist heat and dry heat
What does moist heat denature?
cell wall and cytoplasmic constituents and/or hydrolysis
How does dry heat denature?
denatures by oxidation
What is the destruction action of heat on microorganisms?
most pronounced in the presence of moisture
What does the enhanced sensitivity of microorganisms to heat in the presence of moisture cause?
reduced operating temperatures and times to effect sterilisation
Time and temperature of moist heat sterilisation?
115-118 degrees C 69kPa = 30mind
121-124, 103kPa = 15 mins
126-129, 138kPa = 10 mins
134-138, 209kPa = 3 mins
Time and temperature of dry heat sterilisation?
160 degrees C = 120 mins
170 degrees C = 60 mins
180 degrees C = 30 mins
At normal atmospheric pressure steam kills?
most but not all types of microorganisms
bacterial spores remain viable even if they are heated for prolonged periods of time
What are higher steam temperatures used for?
to ensure that all microorganisms are killed in a product
How do we obtain higher steam temperatures?
the water must be heated under pressure in an autoclave
What is steam?
water in the vapour (gas) phase
What is needed to transform water from the liquid to the vapour state?
energy must be added to the liquid
How can the energy bringing about this change be subdivided?
energy to raise the temp of the mass of liquid water to its boiling point
energy to transform the mass of liquid water at its boiling point to a vapour pressure
What is the energy to raise the mass of liquid to boiling point?
4.2kJ/kg/degrees C
What is the latent heat of vaporisation?
the energy to transform the liquid at its boiling point to a vapour pressure
2220kJ/kg
What is the exact boiling point of water determined by?
the pressure of the atmosphere in which the water is being heated (boiling point will increase with increasing pressure)
What is the boiling point of water at atmospheric pressure?
100 degrees C
When is steam referred to as being saturated?
when it is at a temperature corresponding to the liquid boiling point appropriate to its pressure
What is the effectiveness of saturated steam under pressure at destroying organisms related to?
the physical properties of the steam under pressure that allow the steam to efficiently transfer heat to the product requiring sterilisation
What happens when steam comes into contact with an object whose temperature is below the steams saturation temperature?
the steam condenses into liquid water (at 121 degrees C) on the object and transfers the latent heat of vaporisation (2220kJ/kg) to the object
What does the condensation cause?
a rapid contraction in volume (~785x decrease) and creates a localised region of low pressure that is filled by additional saturated steam
What does this contraction in volume ensure?
the rapid penetration of the load by the steam
When does this process continue until?
the object reaches the temperature surrounding the steam
When are increased pressures used?
only to elevate the temperature at which saturated steam is produced
the pressure itself has no antimicrobial action
Why is water quality used to produce saturated steam important?
contamination with chemical residues within water is possible
What does purity of the water effect?
the temp it can achieve at a particular pressure
chemicals in water can contaminate the product
What is superheated steam?
when the temperature increases above the saturation-pressure boundary or if the pressure is reduced below the saturation temperature-pressure boundary then the steam is referred to as being super heated
How does superheated steam behave?
like an ideal gal and is not as lethal to microorganisms as saturated steam?
What does superheated steam cause?
do not get transfer of latent heat of vaporisation and do not get lethal kill of microorganisms
What happens if you get a reduction of temperature at fixed pressure?
you get a puddle of water which means there is no transfer of latent heat of vaporisation, not as much antimicrobial action
What are the thermal stages within an autoclave?
A (rising) - heating up phase
B (flat) - holding phase, where most of microbial killing happens
C (descending) - cooling down phase
What products are sterilised by moist heat sterilisation?
only products that can withstand the process temperatures and that are not susceptible to moisture damage can
3 categories of products sterilised by moist heat?
aqueous products
non-pourous loads
porous loads
Examples of aqueous products sterilised by moist heat?
ophthalmic products
large/small volume parenterals
What are aqueous products normally contained in?
a non-porous container made of glass or thermostable plastic
Why does the saturated steam not have to come into direct contact with the product?
the water content in the product will itself ensure inactivation of microorganisms as long as it is maintained at the sterilisation temperature
How are products in containers sterilised?
the steam condenses onto the surface of the container and there is a massive transfer of the latent hear which is transferred inside the container, warm up through convection processes and sterilises the products
What non-porous products are sterilised using moist heat?
surgical instruments, rubber closures for vials or items used in the production of sterile products e.g. rubber tubing for transfer of sterile solutions or stainless steel mixing blades
What are non-porous loads usually wrapped in?
special containers that allow the penetration of saturated steam during the sterilisation process and prevent the ingress of microorganisms after the sterilisation process is complete
What are porous loads that would be sterilised?
wound dressings and filters
For porous loads what is done after sterilisation?
there will be a vacuum to pull of any residual moist steam present on the product so it is not soggy
What does the paper do for non-porous loads?
prevents introduction of any new microorganisms onto the product once it has been taken out of the autoclave
Where is dry heat sterilisation performed in?
a hot air oven
poor heat transfer capacity of air lead to prolonged sterilisation cycles at higher temperatures than moist heat
What is heat provided by in dry heat sterilisation?
electrical heating elements placed around the internal insulated chamber wall
What do fans in the oven do?
ensure that air is evenly distributed throughout the oven to prevent temperature gradients within the oven
How is heat transferred to products in dry heat sterilisation?
by conduction, convection and radiation
What can influence heat transfer and the performance of the oven?
size and distribution of the load
Why is dry heat sterilisation insufficient?
there is transfer from air onto the product so it takes a lot longer than moist heat
What is dry heat sterilisation used to sterilise?
a variety of thermostable products that are moisture sensitive
ophthalmic ointments
oil, wax, fat excipients that are used in the manufacture of depot injections, implants or ointments
What else is dry heat used to sterilise>
powders used in the manufacture of sterile suspensions or powders
depyrogenation of glassware
What temperatures are routinely used in dry heat sterilisation?
> 200 degrees C
this destroys bacterial endotoxins on glassware
What does the BP require for endotoxin?
3-log10 reduction in endotoxin
How is depyrogenation of glassware done?
putting it through a heated tunnel of >220 degrees C, by the time it comes out all of the toxins have been chemically denatures
What is used in radiation sterilisation?
only high energy gamma rays and high energy electron beams
When did the introduction of radiation sterilisation become widespread?
after the development of the nuclear industry
What did the development of the nuclear industry allow?
the main isotope Co-60 to become available in sufficient quantities
What does Co-60 do?
decays in a single step process to produce Ni-60
Co-60 -> Ni-60 + 2g-rays + b
What is the total energy of the gamma rays and electron released from Co-60?
2.81Mev, which is 4.4x10-13J
What does the energies of the two rays allow?
them to penetrate through most products
penetrate into a product with a density of water to a depth of 30cm
What does the BP recommend a dose received to achieve terminal sterilisation?
a dose of 25kGy (2.5x10^4 J/kg)
What is the energy from the gamma rays?
small compared to the energy input provided by moist heat or dry heat methods of sterilisation
Why is the energy delivered by an individual gamma ray highly localised?
it penetrates through the product
What does the gamma ray have sufficient energy to do?
break covalent bonds found between atoms in organic molecules
What is a carbon to carbon bond broken by?
an energy input of 5.77 x10^-19 J
What is the alteration in the chemical composition of important macromolecules induced by?
the gamma rays results in the death of the microorganisms (may also denature product)
What is gamma radiation used for?
the sterilisation of medical devices such as prosthetics, catheters, disposable plastic syringes and surgical clothing
What else has gamma radiation been used as ?
a cold sterilisation process for heat sensitive pharmaceuticals such as monoclonal antibodies, enzymes and peptides in addition to certain product containers
What chemicals have been used as gaseous sterilants?
ethylene oxide
formaldehyde
peracetic acid
hydrogen peroxide
What are the majority of gaseous sterilisation operations performed using?
ethylene oxide
What is ethylene oxide?
a colourless, practically odourless cyclic ether with a boiling point of 10.7 degrees C at atmospheric pressure
What is ethylene oxide in its pure form?
explosive and highly flammable with air
What is done to reduce the risk of explosion of ethylene oxide?
it is commonly mixed with an inert gas such as carbon dioxide or dichlorordifluoromethane
What does ethylene oxide do?
chemically alkylates a range of chemical functional groups present within the microorganisms
What impact upon effectiveness of sterilisation cycle?
gas concentration (500-800mg/L)
process temperature (40-60 degrees C, typically 55-60)
RH (40-80%, most important parameter)
What are the typical process times of gaseous sterilisation?
between 3 - 36 hours
What is ethylene oxide used to sterilise?
wound dressings, prothesis and intravenous sets
What can ethylene oxide not sterilise?
aqueous solutions or gas impermeable products
When should ethylene oxide sterilisation be used?
when other methods are not available or applicable
it is less reliable than other sterilisation methods
What is filtration?
the separation of an insoluble solid from a liquid or gas by means of a porous medium (filter) that retains the solid but allows the passage of liquid or gas
When does filtration result in a sterile liquid or gas?
when the insoluble material is a microorganism suspended either in a liquid or gas and is removed by filtration
Why is sterilisation by filtration not a terminal process?
the resulting sterile solution obtained must be filled and sealed into its final container without re-introduction of microorganisms
How can contamination prevented?
further processing of the sterile filtrate must be carefully performed under aseptic conditions
what does aspetic processing require?
greater manipulations and is more sensitive to the environmental conditions and depended on the capabilities of the operators
What type of sterilisation method should always be chosen?
a terminal sterilisation method when possible
What are modern filters made of?
polymers such as cellulose esters (acetate and nitrate)
PTFE
nylon
polysulfone
What does polymer selection depend on?
process conditions e.g. pH, presence of organic solvents, salts
What is retention?
capture particles that are too large to fit through the membrane apertures (holes or channels)
What is inertial impaction?
particle momentum leads to it embedding/lodging into membrane crevices and dead-ends
What is adsorptive sequestration?
ionic or hydrophobic interactions between particles and membrane polymer
What is adsorptive sequestration dependent on?
chemistry and process conditions (e.g. pH, salts, solvents)
Wha is the nominal pore size of sterilising grade filters?
0.2um
What does the pore size still allow?
passage of certain types of microorganism or metabolites/macromolecules through
Examples of things that can fit through pore size?
viruses (20-300nm)
mycoplasma (200-300nm)
endotoxin (~10^-10m)
What does the BP qualify filter performance in terms on?
microbial retention due to the absolute pore size distribution
What does the BP state a sterilising filter must retain?
a challenge of 10^7 CFU of brevundimonas diminuta per cm2 of filter
Why must you qualify every membrane with your product before using to filter sterilise product?
no reference is made to the test conditions under which the filter is challenged
What is filtration primarily used for?
to sterilise solutions that cannot withstand head treatment
What else is filter sterilisation used for?
to sterilise gases used in other processes e.g. dry heat sterilisation using hot air
What is filtration commonly used to filter?
solutions that are undergoing terminal sterilisation to enhance the microbial quality assurance further
