7 Concept Of Sterility And Sterilisation Methods Flashcards
What are the terminologies commonly used in sterilisation?
Sterilisation: Removal or destruction of all life of any kind from an object or material
Disinfection: Destruction of microorganisms, but not usually bacterial spores, to non-harmful level to health
Pasteurisation: Heat treatment that kills part, but not all microorganisms present in heat-sensitive materials and usually involves application of temp below 100ºC
Sterility: Absence of viable microorganisms
What are the reasons for sterilisation?
1) To prevent the risk of infection by use of contaminated pharmaceutical products and medical devices
2) To prevent transmission of disease via contaminated items or air
3) To prevent spoilage of materials/ preparations by microorganisms
**4) To remove competition for nutrients in growth media and prevent impairment of growth or metabolic activity of desired microorganisms
5) To enable production of pure cultures for various purposes (Eg. Microbiological tests)**
What are the concepts of sterility?
1) Sterility refers to absence of viable microorganisms
2) Critical quality attribute of sterile products
— “Critical” most important attribute as long as not sterile it cannot be used
3) According to BP, sterility of product cannot be guaranteed by testing. It has to be assured by application of a suitably validated production process
Sterility Assurance Level
— Refers to probability of a treated item remaining contaminated by one or more viable microorganism(s) in the population of treated items
— Major pharmacopoeias require SAL of 10^-6, i.e. not more than one viable microorganisms in 1,000,000 sterilised units or better; which is the target to be achieved for a given sterilisation process
— Good knowledge of the effects of sterilisation processes on microbial populations is required to achieve the target
What is microbial inactivation kinetics?
Regardless of the type of lethality induced by a sterilisation process, microorganisms die according to a logarithmic r/s betw the population of living cells and time of exposure
For most microorganisms, the destruction of microbial population follows 1st order kinetics
— For each fixed increment of exposure time, a constant proportion of a surviving population is inactivated; i.e. For every one half life, population of cells drop by 50%; Therefore, after 3 half lives population would have reduced by 87.5% wrt to the original population
Log N = Log Nº - Kt [Log Nº/N = Kt]
Nº = Initial number of cells (bioburden)
N = No. of cells surviving time, t
K = Death rate constant
What are sterilisation parameters?
D-value (Decimal reduction time): Time of exposure required from a sterilisation process to reduce a microbial population by 90%
Z-value (Temperature coefficient): Is a measure in which D-values change with temp
F-value (Lethality): Is the lethality that is delivered by a sterilisation cycle to the item
Use of parameters enables:
1) Calculation of lethal effects of sterilisation cycle for a given product
2) Comparison of efficiency between different sterilisation cycles
What are the methods of sterilisation?
5 Main methods listed in pharmacopoeias
Choice depends on nature of material to be sterilised
Heat Sterilisation — Steam + Dry heat
1) Steam Sterilisation
2) Dry heat Sterilisation
3) Ionising radiation Sterilisation
4) Gas Sterilisation
5) Sterilisation by Filtration
What is D-value?
D-value (Decimal reduction time): Time of exposure required from a sterilisation process to reduce a microbial population by 90%
— Specific for particular microorganisms
— Affected by:
1) Temp, radiation dose or gas conc employed
2) Surface of exposure
3) Formulation components
— Biological indicator (Standardised preparations of selected microorganisms used to assess the effectiveness of a sterilisation procedure) for sterilisation process is usually adopted
-Kt = Log (N/Nº)
-KD = Log (1/10) ; When t = D, there is 90% reduction in population where N = 0.1 Nº, hence N/Nº = 0.1 = 1/10
D = 1/K = Decimal reduction time
— Significance of D value:
For a bacterial population of 10^6,
At t = 6D, there is only 1 survivor left
At t = 7D, there is only 1/10th of the survivor left which means that the survivor has a 1/10 chance to survive
— Notwithstanding the strength or duration of the sterilisation process, there is always a finite statistical probability of a survivor occurring; This implies that total freedom from all viable life forms can never be achieved in practice
What is Z-value?
Z-value (Temperature coefficient): Is a measure in which D-values change with temp
— Typically refers to temp increase required for one log reduction in D-value (10x faster)
Z = (T2 - T1) / (Log D1 - Log D2)
What is F-value?
F-value (Lethality): Is the lethality that is delivered by a sterilisation cycle to the item
— It is expressed in terms of minutes at a reference temp (Tref)
— Calculated using specified Z-value
— Reference temp = Desired temp
— In case of heat sterilisation, temp-time data is needed for calculation
— F-value is obtained by the integration of the lethal rates throughout the sterilisation process at a specified Z-value (Z-value of biological indicator is generally adopted):
F = ∆t (∑L) = ∆t (∑10^(T-Tref)/Z)
∆t = Time increment betw each temp reading
L = Lethal rate calculated for each temp reading
T = Item temp
Tref = Ref temp
Z = Z-value (Pharmacopoeia recommended at 10ºC)
— For heat sterilisation, items in steriliser will take some time to attain the desired temp delivered by steriliser
— F-value considers the equivalent time, not clock time, that a monitored item is exposed to the desired temp
— For steam sterilisation, reference temp is typically 121ºC and Z-value is typically 10ºC and determined F-value is represented as F0
F0 = D121 (Log N0 - Log NF)
NF = Final number of viable microorganisms with Z value of 10 = 10^-6 (SAL)
— Fphy: Denotes that the F-value was determined from physical data of the sterilisation process
What is Steam Sterilisation?
- Most commonly employed in terminal sterilisation; particularly for Aqueous preparations
Mode of action
- Steam kills microbial cells by causing coagulation or hydrolysis of proteins and other essential components (Eg. Enzymes and Nucleic acids)
- State of hydration of microbial cell can influence its resistance to heat (In absence of water cell is very resistant to heat and chemicals; Eg. Spores)
Principle
- Employs heat involving the heating of the material with steam under pressure in an autoclave (Increased pressure increases BP of water making it hotter as steam)
Equipment/ Operation
Operation:
1) Add water to autoclave to generate steam
2) Set temp and pressure to desired values (Eg. 121ºC and 15 psi)
3) Start heating
Vol of water added
- Sufficient: Eqm betw liquid water and steam is established. Saturated Steam is achieved where temp of steam and water is at the desired value (121ºC)
- Insufficient: All liquid water turns to steam; becomes dry and temp is higher than desired value (>121ºC). Described as Superheated Steam
Superheated steam is less efficient than Saturated steam as it is a poorer conductor of heat
Saturated steam possess properties that promote lethality
- It condenses on the surface of the material and liberates a high % of latent heat, exerting a lethal effect
- Condensation causes steam to contract to a smaller vol; This phenomenon helps to facilitate steam penetration into porous items, resulting in faster and greater increase of temp inside the item
Air impurities
Important to remove air from autoclave chamber before sterilisation (Heat first and release the hot air to reduce air within the autoclave before steaming)
- Mixture of steam and air gives a lower temp than pure steam
Steam Sterilisation Cycle
Is a function of temp and time depending on:
1) Heat stability of product
2) Type of container and pack size
3) Heat permeability into packet
4) Bioburden
BP values are (121ºC for 15mins)
BP Biological indicator: Geobacillus stearothermophilus spores
What is Dry Heat Sterilisation?
Mode of action
Ethylene oxide is a powerful alkylating agent and kills microorganisms by alkylation:
- It reacts with sulfydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic acids
- DNA and RNA are the most likely target molecules
- It is active against all types of bacteria, fungi, viruses and spores
Principle
- Involves exposure to a microbicidal gas in a specialised equipment resembling an autoclave.
Gas: Ethylene Oxide and Formaldehyde
- Complex cycle as certain factors have to be controlled
Equipment/ Operation
Operation
[Ethylene oxide] (mg/L) = 400-1000 (Below explosive conc. ~3.6%, v/v)
Exposure time (h) = 0.5-10
Temp (ºC) = 45-65 (Higher temp = Higher activity)
Relative Humidity (%) = 40-80 (>80% produces dilution effect, <40% inhibits ionisation of essential groups preventing alkylation)
Items should be aired after sterilisation to prevent toxic effects of residual Ethylene Oxide
BP Biological Indicator: Bacillus atrophaeus spores
Adv & Disadv
Adv
- Suitable for materials that are sensitive to heat and radiation
Disadv
- Less reliable than heat and ionising radiation due to difficulty to control variation
— Employed only when there is no appropriate alternative
- Toxic (Irritates eyes and nose, Causes nausea and vomiting, Has vesicant [Blistering] action )
- Forms an explosive mixture with air (>3.6%, v/v). Usually used with inert gas, eg. Carbon dioxide or Nitrogen
Application
What is Ionising Radiation Sterilisation?
- Involves exposure of materials to gamma rays (electromagnetic radiation) or high speed electron beams (particulate radiation)
- Termed as “Cold” sterilisation method as there is no significant rise in temp
- Requirements
1) Good penetration power
2) High sterilising efficiency
3) Min damage to irradiated materials
4) Satisfactory production efficiency
Mode of action
- As radiation passes through matter, they excite electrons or remove electrons from atoms and molecules. Both excitation and ionisation (More effective) of the atoms of essential molecules can cause the death of microorganisms
- Lethal effects may be due to direct or indirect action
— Direct action: Ionising radiation > Target (Eg. DNA)
— Indirect action: Ionising radiation > Water > Free radicals > Target (Eg. DNA)
Principle
Gamma radiation (Surface + Inside)
- Penetration
— Good; Comparatively large items may be sterilised
- Sterilisation time
— Comparatively slow; Several hours of exposure
Electron beam (Better for Surface only sterilisation)
- Penetration
— Comparatively poor; Not appropriate for dense materials
- Sterilisation time
— Faster; Minutes or even seconds of exposure
Lethality of Radiation
Solely dependent on absorbed dose which is affected by:
1) Strength of source
2) Time of exposure
3) Apparent density (Bulk density) of product packages being irradiated
4) Source-to-product geometry
- Unit for absorbed radiation dose is ’Gy’ (Gray)
— Previously, dose unit was ‘rad’
— 1 rad = 0.01J of energy per kg of material
— 1 Gy = 100 rad = 1J per kg
BP absorbed dose: 25k Gy
BP Biological indicator: Bacillus pumilus spores
Equipment/ Operation
Equipment
Gamma Radiation Sterilisers
- Gamma rays from appropriate radioisotopic source
Electron Beam Sterilisers
- Accelerate electrons energised by appropriate electron accelerator
What is Gas Sterilisation?
Mode of action
Ethylene oxide is a powerful alkylating agent and kills microorganisms by alkylation:
- It reacts with sulfydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic acids
- DNA and RNA are the most likely target molecules
- It is active against all types of bacteria, fungi, viruses and spores
Principle
- Involves exposure to a microbicidal gas in a specialised equipment resembling an autoclave.
Gas: Ethylene Oxide and Formaldehyde
- Complex cycle as certain factors have to be controlled
Equipment/ Operation
Operation
[Ethylene oxide] (mg/L) = 400-1000 (Below explosive conc. ~3.6%, v/v)
Exposure time (h) = 0.5-10
Temp (ºC) = 45-65 (Higher temp = Higher activity)
Relative Humidity (%) = 40-80 (>80% produces dilution effect, <40% inhibits ionisation of essential groups preventing alkylation)
Items should be aired after sterilisation to prevent toxic effects of residual Ethylene Oxide
BP Biological Indicator: Bacillus atrophaeus spores
Adv & Disadv
Adv
- Suitable for materials that are sensitive to heat and radiation
- Can penetrate paper, fabrics, plastics and rubber, but not films of organic matter
Disadv
- Less reliable than heat and ionising radiation due to difficulty to control variation
— Employed only when there is no appropriate alternative
- Toxic (Irritates eyes and nose, Causes nausea and vomiting, Has vesicant [Blistering] action )
- Forms an explosive mixture with air (>3.6%, v/v). Usually used with inert gas, eg. Carbon dioxide or Nitrogen
Application
1) Disposable medical devices
2) Surgical instruments (Those that cannot withstand heat)
3) Blankets
4) Surface sterilisation of powders
5) Paper and plastic bags, rubber capped glass bottles and polythene vials are possible choices of containers
What is Sterilisation by Filtration?
Mode of action
- Involves physical removal of microorganisms by adsorption on filter medium or by sieving mechanism
Sieving Mechanism:
Microorganisms/ particles larger than the pore size are removed from the liquid and retained onthe filter surface
Adsorption Mechanisms:
- Microorganisms/particles adsorbed onto the filter surface or to the side of the pore channels
- Entrapment Mechanisms:*
- Microorganisms/ particles are entrapped within the tortuous (twist and turns) channels
Principle
- Does not kill microorganisms
- Filtration can be employed for both clarification and sterilisation of liquids and gases
- Done in a clean room
- Not a terminal sterilisation method
Equipment/ Operation
Equipment - Requirement of filer medium
- Able to remove/ retain particles above stated size
- Remove nothing except microorganism sand solid particles (Remove unwanted particles but keep desired API)
- Yield nothing to filtrate
- Sufficiently porous for adequate flow of liquid through the filter
- Compatible with liquid being filtered
- Ease of assembly in a leak-proof filtration unit
- Possess adequate mechanical strength (To resist pressure)
Types of Filters
Membrane Filters
- Exhibit sieving mechanism
- Increase in pressure does not cause particles to pass through
— Characteristics
1) Made of cellulose derivatives or other polymeric materials
2) Disposable
3) Pore diameter range: 0.2-0.22µm
4) Two major forms: Disc or Cartridge
Depth Filters
- Exhibit mainly adsorption and entrapment mechanism
- Particles stop at point where resistance = driving force
- Increase in pressure drive particles deeper and sometimes through filter
— Characteristics
1) Made of ceramic or glass
2) Resuable
3) Variable porosities
4) May be fused to filter holder
5) High dirt-handling capacity
6) Bacteria may grow in filter if left in contact for more than 18hrs
BP Biological Indicator: Pseudomonas diminuta
Adv & Disadv
Application
What are the Advs and Disadvs + Application of Steam Sterilisation?
Adv & Disadv
Adv
- Lower temp and shorter duration than dry heat
- Good for aq solutions
- No damage to surgical dressings
- Rubber and most plastics can withstand the conditions
- No toxic residues
- High sterilising efficiency
- Very economical
(Steam Sterilisation is the default method, other methods are used when Steam Sterilisation is ineffective)
Disadv
- Not suitable for anhydrous materials (Eg. Oils/ Powders)
— Penetration of water does not take place (Oil), Powder becomes moist which would lower stability/ flowability
- Not suitable for thermolabile medicaments and materials
- Not good for assembled equipment
- Clouding and alkali extraction in glass
— After repeated sterilisation, Alkali substances are found in Glass materials and it could contaminate the pharmaceutical product
- Metallic objects get rusted
Application
1) Aq formulations in sealed containers
2) Glassware (Class A)
3) Containers and closures
4) Dressings
5) Surgical and dental instruments
6) Decontamination of infected materials or lab waste
What are the Advs and Disadvs + Applications of Dry Heat Sterilisation?
Adv & Disadv
Adv
- Good for moisture-sensitive materials
- Good for assembled equipment
- Less damaging to glass and metal
Disadv
- Drastic condition for most medicaments, rubber, plastic materials etc
- Not suitable for aq solutions
— Water will evaporate and pressure accumulated would cause an explosion due to the high temp and long duration
- Slower heat transfer rate and longer processing time than steam sterilisation
Application
1) Oily preparations (Heat stable)
2) Oils, fats and waxes (Heat stable)
3) Powders (Heat stable)
4) Glassware (Sterilisation and Depyrogenation)
— Dry heat at >220ºC (BP)
— Common temp-time combo: 250ºC for 30mins
— Validation criteria: 3 log reduction in heat-resistant endotoxin
5) Metal instruments
6) Apparatus and equipment (Heat stable)
Pyrogens: Substances that cause rise in body temp on injection; Eg. Bacterial endotoxins
Bacterial Endotoxins:
1) Lipopolysaccharides from outer cell wall of Gram-negative bacteria (Lipid A)
2) High MW
3) Heat stable up to ~250ºC
4) Can pass through filter
5) Not affected by bactericide
What are the Advs and Disadvs + Applications of Ionising Radiation Sterilisation?
Adv & Disadv
Adv
- Negligible temp rise (~4ºC)
— Good for heat and moisture labile materials
- Short exposure time (Electron)
- Large amt can be exposed at one time (Gamma)
- Continuous process (Gamma)
- Good for pack items (Gamma)
- Reliable process; may be accurately controlled
Disadv
- Deleterious effects on product and packaging materials
1) Decomposition immediately or after storage
— Heparin, Fats, Antibiotics, Steroids, Insulin, Vitamins, Hormones
2) Colour change
— Polymethyl methacrylate (PMMA) gets slightly coloured
— Polypropylene turns yellow
3) Physical change
— Butyl and chlorinated rubbers get degraded
— Polypropylene becomes brittle
— Teflon/ polytetrafluoroethylene becomes soft
4) Flavour change
— Food may develop unpleasant taste
- High capital and replacement cost (Expensive)
- Elaborate and expensive safety precautions required
- Radioisotope should be used continuously
Methods to reduce undesirable effects of ionising radiation (Indirect effect)
1) Irradiation in frozen state
- Suppresses free radical movement
2) Addition of protectant (Eg. Ascorbic acid, Cysteine, Thiourea)
- Compete for free radicals
3) Removal of Oxygen
- Decreases oxidative effects
4) Use of pulsed radiation
- Intense but short duration
Application
1) Medical devices
2) Surgical materials
3) Pharmaceutical packaging components
4) Medicaments/ Excipients
