Sterilisation Flashcards
Spores are not killed using steam sterilisation but only through filtration
T/F?
false: Spores are not killed by filtration
1) filtration removes micro-organisms
2) spores requires temperature greater than 100 degree Celsius .
Top-to-bottom gowing up in the cleanroom is the only way in wearing the protective clothing
False, three types- top-bottom, bottom-top, inside-out
What are the main methods of sterilisation?
steam sterilisation
dry heat
filtration
You can always choose a rubber closure regardless of the active substance, vehicle and pH of the product
T/F?
False:
Rubber closure need to be compatible with the active substance, vehicle and pH of product - Go back to lecture 1
In the case of a injection controlled by Misuse of Drugs Act 1973, the label should state the total amount
T/F?
False:
Total VOLUME should be stated - Go back to first lecture and check the labelling requirements.
Viruses are only killed using filtration sterilisation?
T/F?
False;
Viruses cannot be killed by filtration:
1) filtration removes microorganisms but
2) the size of the pore 0.22um is bigger so viruses can pass through filters as they are small.
Viruses are killed using heat sterilisation techniques - Go back to final lecture about filtration.
The D value of an organism exposed to a temperature 121°C for 20 min is 5 min. What is the inactivation factor and calculate it?
Waiting for grade IF= 10^t/D = 10^20/5 = 10,000 ??
European cleanroom specification are more stringent (strict) than the US ones?
T/F?
True
because they look at in rest and in operation as well as 5 and 0.5 microns ( go over your second lecture of sterile products)
A constant number of organisms is killed during a sterilisation process
T/F?
False:
A constant proportion , not a constant number of organisms is killed per unit time
Moist heat sterilisation is unsuitable for oils and powders
T/F?
True
The preparation and sterilisation of a product must happen in the same area in the cleanroom
T/F?
False:
Should be separate from other manufacturing areas
Different types of operation must be effectively separated on from another
o Component preparation
o Solution preparation
o Filling
o Sterilisation
Filtration is a terminal sterilisation process
T/f?
False:
filtration is a non-terminal sterilisation method
Latent heat is heat required to raise the temperature of water to its boiling point ?
True/False?
False:
that is sensible heat.
Latent heat is additional heat absorbed when boiling water is converted into steam
Inside the cleanroom, the air supply should be at a positive pressure and of a suitable quality
T/F?
True
High pressure inside the clean room
Single- and multi-dose preparations require bactericides
T/F?
False:
only multi-dose containers require bactericides
What is the BP sterilisation method of choice ?
Sterilisation by saturated steam under pressure is preferred, wherever
applicable, especially for aqueous preparations
moist heat
Using steam sterilisation, aqueous preparation requires a minimum heating at 250°C for 15 min
T/F?
False
Using steam sterilisation, aqueous preparation requires a minimum heating at [121°C] for 15 min
Why do some products need to be sterilised?
- avoid infection
- medical equipment/products bypass innate immune system e.g. parentally administered products
What is meant by sterilisation?
Process that kills or remove all types of viable (living) microorganisms including bacterial spores
what products must be sterile?
why?
- Certain classes of medicinal products + equipment
- Parenterally administered products: IV/IM
- any other formulation in contact with eyes, broken skin, internal organs or tissues, want to keep blood sterile
microorganisms present in those items – bypass bodys first line of defence – give rise to severe infection. Don’t want to introduce anything that may cause infection
What is meant by disinfection?
A process that !reduces! the number of viable vegetative microorganisms but does not necessarily kill spores
What is meant by antisepsis?
how do antiseptics compare to disinfectants?
- Prevention of infection by applying antimicrobial agents to tissues
- Antiseptics are less toxic and less effective than disinfectants
What is meant by a viable cell?
One cell capable of division to form a visible colony on solid nutrient medium or visible turbidity in liquid medium (petri dish with agar to see area) can look to see grow/ do turbidity test
What is meant by a dead cell?
A cell that is not capable of division to form a visible colony on solid nutrient medium or visible turbidity in liquid medium
What is the best-case outcome of a product supposed to be sterile that is non-sterile?
- it’s identified before preparation is issued
- microorganisms induce spoilage of product (physic/chem degradation)
- product removed from use + destroyed
What is the worst-case outcome of a product supposed to be sterile that is non-sterile?
example scenario
- microbial survival not identified BEFORE USE
- infection occurs
Devonport incident (1971-1972)
- 5 patients died from acute endotoxic shock
- dextrose 5% infusion bottles were sterilised w a faulty autoclave
whats needed in place to ensure fatal infection will not occur from worst-case outcome of a product that is non-sterile?
need good quality control !!
whats Bioburden ?
Expression of the number of viable organisms a batch contains
define batch (in terms of contamination…)
Homogeneous collection of sealed packages/ containers, prepared in manner that the risk of contamination is the same for all the items – all contacted if problem
define pyrogens
and whats the most important type in sterile production. what may it lead to?
Substances that when administered to humans/animals result in an increase in temperature (bad- infection).
In sterile production, the most important pyrogen is the “endotoxin” originating in the cell wall of gram negative bacteria. Can lead to death
define endotoxins
High MW lipo-polysaccharides which are water soluble, heat stable (for method of sterilisation), can pass through bacteria proof filters.
how can endotoxins be inactivated?
They can ONLY? be inactivated with dry heat at high temperatures (170-350°C)
8 types of Sterile product formulations, medical products, and devices
- Injections
- Non-injectable sterile fluids
- Opthalmic preparations
- Dressings
- Implants
- Absorbable haemostats
- Surgical materials (e.g. sutures) on opening of skin
- Instruments and equipment ( Syringes, metal instruments, medical devices- endoscopes)
State 3 types of injections which must be sterile
o IV infusions
o Total parenteral nutrition (TPN) fluids
o Small volume injections
State 3 types of Non-injectable sterile fluids which must be sterile
o Non-injectable water,
o Peritoneal dialysis,
o Haemodialysis solutions
State Ophthalmic preparations which must be sterile
Eye drops , lotions and ointments ( Link to SOM1)
what are Absorbable haemostats - products that must be sterile
“Absorbable knitted fabric that is used to control bleeding in surgical procedures”- for blood tests etc
what are 4 general requirements for containers for sterile products
- Chemically compatible with the product: don’t want product to leak and destroy
- Withstand sterilisation – dry heat, don’t want product to break etc
- Maintain sterility of the product
- Permit safe withdrawal of the product
what is The purpose of packaging?
contain and protect the product
EP deifinition of container [ for sterile products ]?
“Article which contains/ is intended to contain a product and is, or may be, in direct contact with it.
The closure is part of the container”
Types of containers for Large volume parenterals (LVP) sterile products?
Rigid and flexible, glass and plastic
Types of containers for Small volume parenterals (SVP) sterile products?
Ampoules, vials, prefilled syringes, novel devices
types of containers for irrigations sterile products?
Glass and plastics, sachets
types of containers for eye drops sterile products?
Glass and plastics
types of containers for eye ointments sterile products?
plastics
main differences in the 2 types of containers for sterile products
Single-dose
- Hold enough for total/ partial use as single administration
- no bactericide
Multi-dose containers e.g. eyedrops
- hold enough for 2 or more doses
- Must contain an excessive number of doses and period between 1st and last doses should not be excessively prolonged
- Multidose formulations require a bactericide – as opened and closed may introduce bacteria
why must Intraspinal injections and intravenous injections greater than 15 ml be single use only?
As they must contain no bactericides
- Thus must be single use only
Materials used for sterile fluid containers
glass:
- ampoules: small containers
- vials: small bottles
plastics
plastic polymers
advantages of using Glass materials for sterile fluid containers
- Good chemical resistance (depends on glass type)
- Don’t absorb/ elute organic ingredients
- Impermeable. With proper closures.
- Easily cleaned
- Transparent – Easy inspection of contents and spoilage
- Rigid & Strong
- Resists puncture
why can glass containers be used to undergo dry heat sterilisation/ be subjected to it?
Can be autoclaved at 121°C or subject to dry heat sterilisation
Disadvantages of using Glass materials for sterile fluid containers
- Breakage during sterilisation (esp. soda glass) like in heat sterilisation
- Attack by alkaline solution
- Get hair-line cracks in transit, allowing entry of moulds
- Much heavier than plastics
- Require venting during administration
- Require inspection & washing prior to use- alcohol swab where needle goes in
- All except ampoules require sealing by closures of a different material, giving problems during autoclaving and risk of interaction between product and closure
describe glass (as an inorganic product of…)
Glass is an inorganic product of fusion that cools to a rigid condition without crystallising
advantages of using plastics for sterile fluid containers
- Relatively unbreakable
- Light ( > 1/10 glass weight)
- Easily fabricated
- Cheap
- Single use
- Small filling ports (less chance of contamination)
- Possible to completely seal by fusion
Disadvantages of using plastics for sterile fluid containers
i.e. what should the used polymers be carefully chosen to avoid:
- Inferior clarity compared to glass -> but can achieve translucency & transparency
- Cannot match the barriers properties of glass to moisture and oxygen
- Additives in plastic: potential leaching in the product
- Sterilisation of fluids in plastic packs -> container must be protected from deformation & bursting
when are plastics used in sterilosation packaging i.e. what did they replace? and problems with plastic ampoules?
became alternatives to glass in LVP packaging but also used on a more limited scale for PVS
Plastic ampoules- easily cracked
2 types of plastic polymers sued as materials for sterile lfuid containers
PVC: polyvinyl chloride
Polyethylene (Polythene)
describe the type of plastic polymer Most widely used in medical industry in containers
- Polymer repeat unit is (CH2-CHCl)n
- Medical used of flexible PVC
IV Tubing
Catheters
Blood bags
LVP containers
describe Polyethylene (Polythene) as plastic polymer sterile product container
melting temp?
o Polymer repeat unit is (CH2-CH2)n
o Fabrication of parenteral containers (provided relatively high-density material is used)
o Withstand sterilisation -> high melting temperature
o Loss of flexibility
o Increase in opacity
closures: how are glass ampoules vs glass vials and bottle closed?
ampoules: by fusion
vials and bottles: need a closure e.g. Elastomeric stopper (rubbers) coupled with a screw
how are Plastic containers closed?
o E.g Boots Polyfusor brand
o Most use elastomeric components in ports
Desirable properties of elastomeric closures: for plastics/ glass vials + bottles
o Compressibility
o Resealability
Factors affecting the selection of a rubber closure
Active drug substance • Vehicle • Preservative, if any • pH of the product • Buffer system • Moisture/gas protection required- could change conc • Colour
Factors affecting the selection of a rubber closure
- Active drug substance
- Vehicle
- Preservative, if any
- pH of the product
- Buffer system
- Moisture/gas protection required- could change conc
- Colour
what do you need to protect in terms of sterile products?
i.e. types of contamination (4)
From contamination & degradation -> protecting the patients • Chemical contamination • Particulate contamination • Microbiological contamination • Pyrogenic contamination
- Physical damage during sterilisation and subsequent handling – transport
- A parenteral container should be completely sealed- Leaks are most commonly due to thermal or mechanical cracks or faulty seals- Tests used to detect ampoule leakers
BP labelling requirements (not dispensing) for sterile products:
For all preparations
- Name of product/ preparation or an approved synonym
- Except in the case of a fixed recipe , the names and proportions of the medicaments
- Names and proportions of any added preservatives additional to, or alternative to any included in the recipe
- Batch number- should any problems be faced
BP labelling requirements (not dispensing) for sterile products:
For injections
- Amount of API in suitable dose volume
- Name + proportion of any added bactericide/ preservative
- Name of any added buffering/ stabilising agent
- Storage conditions- temperature
- Expiry date- 2 years or less
In the case of a product controlled by Misuse of Drugs
Act 1973, what should be stated on general label for injections?
total volume of injection should be stated:
1.1ml
MORPHINE SULPHATE INJECTION BP
PLUS
Containing Morphine Sulphate 10mg in 1ml
example of General label for injections on ampoule
1.1ml
MORPHINE SULPHATE INJECTION BP
Containing Morphine Sulphate 10mg in 1ml
For subcutaneous or intramuscular injection
Expiry date: 1.10.2022
BN:1234
Keep out of Reach of Children
School of Pharmacy, ….
how does packaging label req for IV infusion label differ? state specifics
different in stating ingredient concs (mMol [mEq]) of each ion, then e.g. Sodium Chloride %w/v
additional warnings needed:
- do not use if any solid particles are present
- do not use if seal/cap is opened/ rmeoved
- to be used on one occasion only and any remaining soln discarded
Label on the package of any medicinal product
The label on any container having a capacity of more than 10 ml must have :
(18)
a) Name of product
b) Description of pharmaceutical form
c) Strength
d) Official name –if applicable
e) International non-proprietary name –if applicable
f) Quantity of product
g) Directions for use-if applicable
h) Contra-indications- if any
i) Special handling & storage conditions
j) Expiry date- if product must be used within 3 years
k) Name & address of (a)holder of product license (b) retailer
l) Product license number
m) Batch reference
n) Manufacturer’s licence number- if applicable
o) Any other details required by licence
p) Keep out of reach of children
q) Any warnings or other special requirements
r) If prescription only medicine- the letters
summary:
Containers for sterile products should be: (4)
- Compatible with the product
- Withstand sterilisation
- Maintain Sterility of the product
- Permit safe withdrawal of the products
Why are microorganisms problematic to pharmacists? (size, amount, growth, can cause…, can form… that are…)
- small
- abundant
- can grow in unlikely situations
- can cause disease (pathogenic)
- can form spores resistant to killing by heat, chemicals, radiation or drying
What are 4 different types of microorganims?
- bacteria
- bacterial spores
- fungi
- viruses
What are the characteristics of bacteria?
size, multi/unicellular, shape, gram… dependent on… nutrients, O2 survival
- 0.7-4um
- unicellular
- rod-shaped (bacillus) or spherical in form
- gram +/- dependent on cell wall
- nutrients: C, H, N, O, S, P, H2O
- aerobic or anaerobic
How are bacterial spores formed?
and how killed?
bacteria undergo a profound biochemical change -> formation of a spore (white spots) - highly resistant to adverse environment
kill by sterilisation processes
What are the characteristics of viruses?
size, bacteria proof filters, destroyed by…
- very small (20-250nm)
- pass through bacteria proof filters
- destroyed by heat
- most inactivated at 60degrees C for 30min
- killed by boiling water
What is the level of chemical disinfectants’ viricidal activity?
minimal
What are the characteristics of fungi?
- single or multicellular
- individual cells are 5-10um in diameter
- commonly form spores/yeast/mould/mushrooms
Sterile medicinal products should be prepared where?
in specially designed and constructed manufacturing departments
Separate from other manufacturing areas
Cleanroom & Aseptic areas
what different types of aseptic operation are effectively separated? (4 rooms)
o Component preparation
o Solution preparation
o Filling
o Sterilisation
General requirement for cleanrooms and aseptic areas exist
how are aseptic rooms connected?
and how do the rooms prevent contamination?
via transfer hatch
air pressure controlled, prevent contamination
what are airbourne organisms usually associated with?
- Air is not a nutrient source but it is usually contaminated
- Airborne organisms are usually associated with matter e.g. streptococci with saliva
- Bacteria carrying particles (4-20 µm)
- Particle sediment at a rate dependent on size. Can be re-suspended when air is distributed
Airborne bacterial spores remain viable for a long time- what does this depend on?
the humidity (controlled in cleanroom) and availability of nutrients
how do cleanroom areas have to do to fulfill the requirements of specific grade?
areas have to be supplied and continuously flushed (filtered) with air of suitable quality and at + pressure
Cleanroom areas: where is air of highest and lowest pressure?
o Highest pressure inside aseptic room
o Through High Efficiency Particulate Air (HEPA) filters of appropriate efficiency
o Outside air (unfiltered and dirty) – low pressure
EU vs US cleanroom specs, what are the 4 grades/classes?
EU - US A - 100 - critical B - 1000 - clean C - 10,000 - controlled D - 100,000 - pharmaceutical
what are the most sterile grades/ classes in aseptics?
A and B = 0 small particles allowed at rest.
revisit table?
Many parenteral products are manufactured where?
at one site for global distribution
What req must air quality standards in aseptic processing areas meet?
Air quality standards in aseptic processing areas must meet both US and European requirements
What is the difference between the US and EU classification of cleanrooms? Which one is stricter and why? (4)
European standards:
• Use Grades A, B, C, and D classifications not Class X (100, 1 000, etc)
• Use particle and microbial limits per cubic meter, rather than per cubic foot
• Require particle measurements at 5 microns in addition to 0.5 microns in Grade A and B areas
• Differentiate area cleanliness dynamically “in operation” and ‘at rest’
Measurement of aerial contamination (2)
Settle plates
Slit Samplers
what are settle plates?
Measurement of aerial contamination
- Nutrient plates- collect aerial contamination > 100µm which settle by gravity
- Important hazard in aseptic work
- Small particles not represented
- Unaware of the volume of air introduced- good for learning but not practical
what are Slit Samplers?
what will deposition of particles depend on?
(Measurement of aerial contamination)
- Air drawn through slit/ small holes
- Beneath which rotates a “nutrient” agar plate
- Deposition of particles depends on the rate of air flow, slit width or dimensions of holes and distance between slit/holes and plate
- 99% efficiency of particle ~ 1µm
- Can count number of organisms associated with particles in a known volume of air drawn through the device
What quality control measures should be taking place while working in a cleanroom ( in other terms what do we need to monitor?) (3)
- Particulate contamination
- Temperature & humidity
- Air velocity and pressure
What are the requirement for staff working in aseptic cleanrooms?
• High standards of personal hygiene and cleanliness
• Report any medical condition (colds, skin infections)
• Minimum number of personnel should be present when work is in progress
Activities restricted to avoid excessive shedding of microorganisms
• Adequate training to be received
3 types of gowning up
- Top-down
- Bottom-up
- Inside-out
Sterilisation processes:
Dynamics of Sterilisation…
a single microbial cell is either viable or dead depending on what?
Depending on its ability to grow
However, after exposure to a lethal agent e.g. heat:
- Population of viable cells will not all die at the same time
dynamics of sterilisation: how is
Reduction in cell viability during exposure to a lethal agent plotted?
Reduction in cell viability during exposure to a lethal agent (e.g high temperature) is plotted as:
A log number or % of viable cells / time
I\
I \
I \
I\_\_\_\_\_\\_\_\_\_
time (min)
reduction in cell viability graoh: how to describe the organisms killed per unit time?
A constant proportion , not a constant number of organisms is killed per unit time
• Not all viable living cells die at once!!
Example reduction in cell viability:
• Consider 1 Litre of an intravenous infusion fluid
• Contaminated with one million (10^6) organisms per ml
• Assuming that the fluid is exposed to sterilising conditions which kills 90% of the population in each minute
• So after each minute of exposure, the number of viable cells per ml would be
Time of exp(min) number survivors per ml!! 0 1,000,000 1 100,000 2 10,000 3 1,000 4 100 5 10 ...
i.e. 1million x 90/100 then what you are left with (killed 900,000 on 1 minute, left with 100,000)
then killed 90,000 etc.
what does Reduction in cell viability during exposure to a lethal agent not take into account?
If a batch has 1000 units !
• If 1000 units are to be sterilised
• Probability = 100 of them would be contaminated with 1 organism
• Log survivor/time plot are non-linear?
• The distribution of resistance to sterilisation is unknown. -> bacteria around, resistant
- Allowance for this factors is usually made by using sterilising conditions that give:
- A probability level of one in one million units being non-sterile (based on linear death kinetics)
what is sterility assurance level (SAL)?
Sterility assurance level (SAL) of 10-6
• Degree of assurance with which the process in question renders a population of items sterile
• SAL of 10-6 = probability of not more than one viable organism in 1 x 10^6 sterilised items of the final product
• Cant get SAL of 0. Have to be small as possible
why are D values and Inactivation factors used?
Expression of rate at which micro-organisms are inactivated by a sterilisation process is needed
what are D (decimal reduction) values?
- The value of a parameter of sterilisation (duration/absorbed dose) required to reduce number of viable organisms by 90% (i.e. D= 1 minute from previous table example as 1m -> 100K survivors per ml)
o i.e. To 10% of the original number
o i.e.by 1 log cycle
what is Inactivation factor (IF)?
and equation
calculate it for: E.g. D value of an organism exposed to a temperature of 121ºC for 15 min was 2 min.. IF =?
reduction in the N of viable organisms produced by sterilising process
• 10^ t/D –> t= exposure time , D= D value
E.g. D value of an organism exposed to a temperature of 121ºC for 15 min was 2 min -> IF= 10^15/2 = 10^7.5
2 approaches to Sterilisation are?
Overkill method
Bioburden method
describe the sterilisationn approach most commonly used
basis of traditional BP methods of heat sterilisation
Overkill method
- Sterilisation cycles based on inactivation of a greater N of organisms of greater resistance(high number) than the natural bioburden of the product
- A minimum SAL is set for the cycle and the process defined
- With recommended combinations of time & temperature
what does bioburden method of sterilisation require knowledge of? (3)
- Bioburden
- D value
- Of the most resistant organism in the bioburden
in bioburden method, needs to be done when?
and 3 benefits?
- Prior to defining the sterilisation cycle
- Allows sterilisation cycles to be run with
- Acceptable SALs
- Minimal product deterioration
what is bioburden and whats it always expressed as?
Expression of the number of viable organisms a batch contains.
Always probability, cant have 0
how are sterilisation products classified? (2)
based on their method of production:
- Terminally sterilised
- Non-terminally sterilised
describe Terminally sterilised products class
give examples
where manufactured? (3)
• Those sterilised in their final containers
e.g. IV in glass vial, sterilised at end
- Manufactured in cleanroom
- Pyrogen free product
- Low microbial and particulate counts prior sterilisation
what is the sterilisation classification method of choice where possible?
Terminally sterilised
describe Non-terminally sterilised products class (1)
Those prepared under aseptic conditions from previously sterilised materials
where are non-terminally sterilised products processed? (3)
- Should be processed in cleanrooms
- Until they have been sterilised
- Must be filled into the final container in aseptic areas
what determines classification of products… terminally sterilised/ non?
Stability of drugs
• can determine which one used. If drug not stable with heat (sensitive)- dry/wet covered later
• filtration = long term. Based on product and drug
Methods of sterilisation: Official methods described in BP
what are the 2 heat based methods of sterilisation?
steam/moist sterilisation (heat in autoclave)
dry heat sterilisation
what are the 3 methods of sterilisation for terminally sterilised products?
- steam/moist sterilisation (heat in autoclave)
- dry heat sterilisation
- ionising radiation sterilisation (rare)
what method of sterilisation is used for non-terminally sterilised products?
filtration
what is the 5th method of sterilisation in BP (not used for terminally or non-terminally sterilised products)?
sterlisation by gases (ethylene oxide)
not common but does exist
what method of sterilisation is most reliable?
Heat. used for 2/3 methods for terminally sterilised products
= most reliable, versatile and readily available and economic method of sterilisation
heat sterilisation requires what materials to be stable?
Materials (drug, excipients) must be stable
o Heat decomposition of drug, interaction between drug and additives, modification of the pharmacological activity of the drug
o Stability of the container and closures
Safer than to chemical sterilisation processes- personnel etc (Ethylene oxide)
how does heat sterilisation acheieve effects?
Heat increases the rate of chemical reactions within bacteria cells= increased growth rate
Heat has a destructive effect on the more heat sensitive components of cells
NET RESULT DEPENDS ON A BALANCE OF THESE EFFECTS
net result in outcome of heat sterilisation depends on balance of what 2 effects?
Heat increases the rate of chemical reactions within bacteria cells= increased GROWTH RATE
Heat has a DESTRUCTIVE effect on the more heat sensitive components of cells
what is the effect of increasing heat on Bacteria? (2)
As temp increases:…
- Point is reached where organisms die at the same rate as they reproduce
= bacteriostasis : preventing growth
•A further increase in temperature -> a destructive bactericidal effect: kill
..
how does increasing heat affect death rate and time to sterilise bacteria?
what does heat sterilisation depend on?
death rate increases and time to sterilise decreases
• Sterilisation depends on time/temperature relationship
• Clear difference in the sterilising effect of moist and dry heat
name of and describe some relatively heat sensitive/resistant microorganisms?
- how are they killed?
Vegetative bacteria
• Killed rapidly in hot water at 60-100°C- with heat ster/autoclave above 100 anyway= good
• Spores usually req temperatures greater than 100°C
what does resistance to heat (sterilisation) depend on? microorganisms
Resistance to heat depends on the species
• E.g. Clostridium tetani is heat resistant pathogen
• Bacillus stearothermophilus is very heat resistant non-pathogen
why is Bacillus stearothermophilus used as a biological indicator to test the efficiency of sterilisation processes?
= very heat resistant non-pathogen, use in heat ster
Used as a biological indicator to test the efficiency of sterilisation processes
describe moist heat sterilisation
mechanism of destruction?
- In the presence of H2O. Hot air with H2O Vapour
- Mechanism of destruction is by protein denaturation, uncoiling DNA, enzyme inactivation – very high temp
- Unfolding protein = denaturation
- Changes to the cell membrane -> leakage of cell contents -> death
Method of choice (if possible)
how do temperatures used in moist/dry heat ster compare?
moist heat: relatively lower temperature compared than those causing death in dry heat
describe dry heat sterilisation
mechanism of destruction?
- In the absence of H2O
- Proteins much more stable to dry rather than moist heat
- More energy required for unfolding
what 2 things may death occur by in dry heat sterilisation?
Death occurs by
• Oxidation of cell components
• Requiring high temperatures
whats a subcategory in moist heat sterilisation for terminally sterilised products?
and what equipment used?
steam sterilisation
heating in an autoclave
Sterilisation by saturated steam under pressure is preferred wherever possible- BP
Moist: Steam sterilisation requires what conditions for aq preparations?
heating at a minimum of 121°C for 15min
whats saturated steam (moist heat ster)?
water vapour at a temp corresponding to the boiling point of water appropriate to its pressure
how does steam compare with water/air at same temp? (steam/moist heat ster)
Steam has greater heat content than water /air at the same temperature
define
a) sensible heat
b) latent heat
a) Sensible heat: heat required to raise the temperature of water to its boiling point
b) Latent heat: additional heat, absorbed when boiling water is converted to steam at the same temperature
advantages of moist heat sterilisation?
- Terminal sterilisation
- Any dose volume
- Good safety margin
- Viruses killed
- Relatively short process (15 min)
- Suitable for dressings, rubber and some plastics
- Suitable for solutions, suspensions, surgical materials
disadvantages of moist heat sterilisation?
whats it not suitable for…
- Unsuitable for thermo-labile materials (temp sensitive)
- Unsuitable for anhydrous materials (oils & powders) as penetrates
- Organisms killed but not removed
- Skilled operators required
- May damage glass and metal (stainless steel)
- Batch process
disadvantages of dry sterilisation?
- Drastic heat treatment- high temps
- Organisms killed but not removed
- Unsuitable for dressings, rubber, plastics
- Suspensions may give a different crystal form on cooling
advantages of dry sterilisation?
- Terminal sterilisation
- Any dose volume
- Viruses killed
- Used for anhydrous materials
- Suitable for solutions & suspensions
- Less damage to glass and metal than moist heat
- Suitable for some assembled equipment
process of Dry heat sterilisation (3)
- Preparation to be sterilised is distributed in its final container
- Then, sealed to exclude microorganisms
- Heated
dry heat sterilisation- why is preparation heated and in what conditions carried out?
To ensure the whole of the contents of each container is maintained for an effective combination of time and temperature
e.g. 30 min @180°C / 120min @160°C
what type of items is dry heat ster used for? (5)
- Used for thermostable items
Sensitive to moisture/ impermeable to steam (oils or powders)
Very high temps ( in excess of 250°C) used to sterilise and depyrogenate glassware - Anhydrous materials e.g. oils, fats, non-aqueous suspensions
- Glass and metal materials
- Powders
- Some rubbers e.g. silicone rubber
Sterilisation by Filtration- why do some preps require this?
- Active ingredients and preparations not sufficiently heat stable to allow autoclaving (THERMOLABILE)- sensitive
process of Sterilisation by Filtration?
- and how doe sit differ to other sterilisation methods?
Solutions/ liquids sterilised by passage through a sterile membrane filter of nominal pore size 0.22µm or less
organisms are removed rather than killed
what materials are NOT sterilised by filtration and WHY not?
- Suspensions
- Medicaments highly adsorbed by filters
- Readily oxidised solutions
- Products unstable in solution
Because: it eliminates bacteria by separating the microorganisms from the sterilized medium (but doesn’t kill or stop the bacteria’s ability to reproduce)
2 Advantages of Sterilisation by Filtration
- No thermal effects
* Remove dead and viable organisms (not killed)
Disadvantages of Sterilisation by Filtration
- Difficult technique
- Require sterility testing prior to release (delay)
- Will not remove/ inactivate viruses as viruses smaller than 0.22 microM
- Defects in filters not visible
- Possibility of adsorption of medicament, liberation of particles or alteration of pH by the filter
- Unsuitable for suspensions
main difference in Sterilisation by Filtration compared to other ster?
organisms are removed rather than killed
What type of viruses will sterilisation by filtration not work on?
Will not remove or inactivate viruses as viruses smaller than 0.22 microM
describe 3rd method of ster for terminally sterilised products:
ionising radiation sterilisation
- whats product exposed to and units?
• Exposure of the product in its final container to ionising radiation
• Minimum absorbed dose of 25 kGy
Gray (Gy) is the SI unit of dose defined as 1 joule of energy absorbed per kg of material irradiated
type of radiation that may be used in sterilisation by filtration
Gamma-radiation
- Very penetrating – stand behind shield, dangerous
Beta radiation ( high speed electron)
- Poor penetration
- Irradiation from a Num of sides/ rotate items in beam
-
Physical & biochemical effects in microorganisms (DNA)
Applications of ionising radiation sterilsation method (3)?
- Heat sensitive materials (disposables e.g. syringes, needles, surgical blades, gowns, masks)
- Dressings
- Organic compounds e.g. antibiotics, hormones, vitamins
advantages of Ionising radiation sterilisation (terminal)?
- Operates at ambient temperature- no heat
- Terminal sterilisation
- Continuous process
- Reliable and can be accurately controlled
- Treatment time short ( ~ few seconds)
disadvantages of Ionising radiation sterilisation (terminal)? (3)
- Expensive
- Damaging effects on some materials (some glass, plastics e.g PVC)
- Protective precautions and clothing
Principles of good manufacturing practice (GMP) must be observed in the design of the process including the use of …?
- Qualified Personal
- Adequate premises
- Suitable production equipment
- Validated procedures for all critical production steps
- Environmental monitoring and in process testing procedures – 2nd lec, petri dishes etc
- Precautions necessary to minimise the pre-sterilisation bioburden
Suitable production equipment included in GMP, what does this specify/ include?
Suitable production equipment, designed for easy cleaning and sterilisation
in sterilisation, Precautions necessary to minimise the pre-sterilisation bioburden means… (2)
- Use of components with an acceptable low degree of microbial contamination
- Start with this as want lowest possible bioburden
After steam sterilisation of intravenous injection ampoules, the D value of microorganisms exposed to a temperature of 121°C in an autoclave for 20 min is 5 min.
type of sterilisation?
Steam, wet sterilisation = Terminal sterilisation, BP recommended method where possible.
What other processes can be used if the drug is heat sensitive?
other than steam, wet ster
- Sterilisation by filtration
- ionising radiation/ gas/ filtration
Advantages of Ionising radiation sterilisation:
and what to consider?
and filtration?
done by exposure to gamma rays/etc.
- consider safety and how penetrate.
Filtration not done with suspensions? - Cost Gas/gamma radiation more expensive and not widely available. Can ur the filters easily.
Filtration: non-terminal process. Implications of ampoule and sterilisation of container used
Autoclave: time
Other two: more about dose administered
Consider other options and:
• Are they all terminal?
• Are these suitable for the drug/excipients and primary packaging?
• Are these accessible and cost-effective? Etc.
What does the D value represent?
Decimal reduction number:
value of a parameter of sterilisation (duration/absorbed dose) required to reduce num of viable organisms by 90%. i.e. To 10% of the original number, i.e. 1 log reduction.
If organisms still there but not able to reproduce, less worried about that
Log scale: 1 log reduction (90% reduction)
HOW TO CALC inactivation faactor?
and do it for…
D value of microorganisms exposed to a temperature of 121°C in an autoclave for 20 min is 5 min.
reduction in the N of viable organisms produced by sterilising process
= 10^t/D
= 10^20/5
= 10^4 or 10,000
whats a biological indicator and how is it chosen?
confirms whether decontamination equipment is operating as it should be.
It is made up of bacterial spores presented on/in some form of carrier which can vary depending on the specific application.
The spores themselves have been selected for a particular process due to their inherent resistance. Usually Bacillus stearothermophilic as it’s a very heat resistant non-pathogen
Used to measure D value etc so know how many microorganisms are there and prefer those harder to kill. If confident method kills hard to kill microorganisms, can be sure easier to kill ones die too.
hows a biological indicator chosen?
- nature of the sterilising agent (e.g. heat, gas or radiation);
- expected effectiveness of treatment (e.g. the Fphys calculated from the process parameters);
- process conditions (e.g. temperature, time, relative humidity, gas concentration, radiation dose);
- characteristics of pharm product/ item (e.g. product in final container, packaging material, utensils such as tubes or pumps) to be sterilised.
Most biological indicators are used at a bioburden of 106 spores.
How many log reductions are required to bring the bioburden to 1?
1 = 10^0
So to go to 10^6 = 6
How many log reductions are required to bring the bioburden to 1 if a preparation had a bioburden of 540 cfu?
log(540)
=2.73
Initial, final and the log the difference/division
A steam sterilisation process has a D-value of 2.5 min.
How long should a preparation be autoclaved for if 10^6 spores have been included as biological indicators and a SAL of 10^-6 is targeted.
- D = 2.5 minutes = 1 lg reduction
- Total 12 = log reduction (from spores->SAL 6->-6)
- time to autoclave = log reds x D value (1 log red)
12 x 2.5 = 30 minutes
whats SAL and what values wanted?
one microorganism out of a million= shows risk of sterilisation process, not effective. Want low values as low risk of having one product not sterile.
Why is it important to minimise environmental microbial and particulate contamination during sterilisation production, prior to sterilisation?
efficiency of sterilisation depends on keeping microbial level of sterile product to a minimum (i.e. not to introduce more microbes into the product).
Also lower sterilisation time, bioburden at start already low so less effort to decrease as already controlling it. Less time in autoclave.
Limits in particles in sterilisation. Depending on route, levels will change.
