Parenteral Sterilisation Methods PDF

MSOP1016 Sterilisation Techniques/methods Dr Vivek Trivedi Sterilisation Techniques Learning objectives: At the end of this lecture, you will be able to: Discuss the principles of the five main techniques of terminal sterilisation Identify their advantages and...

MSOP1016 Sterilisation Techniques/methods Dr Vivek Trivedi Sterilisation Techniques Learning objectives: At the end of this lecture, you will be able to: Discuss the principles of the five main techniques of terminal sterilisation Identify their advantages and disadvantages Identify appropriate sterilisation methods for different products Sterilisation Techniques Contextualisation "Sterility" is the absence of viable microorganisms Vital for some pharmaceutical products Products can be "terminally sterilised" or "non-terminally sterilised" Terminally sterilised products are manufactured in a non- sterile form, then sterilised in their final containers Non-terminally sterilised products are not sterilised in their final container, but are prepared under aseptic conditions from previously sterilised materials Aseptic manipulation  Sterility Fundamental concepts "Sterility" is the absence of viable microorganisms "Sterility" is an "absolute" concept "Sterilisation" is a process intended to achieve sterility There are two methods of sterilisation: "Inactivation methods" "Removal methods" The BP recognises five sterilisation techniques  Sterility Fundamental concepts In practice, it is extremely difficult to achieve or demonstrate sterility Exposure for an infinite time to an inactivating process is required to ensure sterility A mathematical probability function is used to assess the effectiveness of the sterilisation procedure The "Sterility Assurance Level (SAL)"  Sterility Assurance Level (SAL) SAL is a measure of the probability of survival of micro- organisms after a sterilisation process The BP states that a minimum SAL of 10-6 is required for pharmaceutical products to be considered "sterile" An SAL of 10-6 is defined as the sterility assurance level that is equivalent to a probability of not more than one viable micro-organism occurring in 1 x 106 sterilised items of product The probability of a non-sterile unit of product is 1 x 10-6, ie a 1 in a million chance  Sterility Assurance Level (SAL) Factors affecting SAL Initial "bioburden" The more viable micro-organisms are present initially, the longer it will take to inactivate them Types of micro-organisms present Some micro-organisms are easier to kill than others Resistance of the micro-organisms present Linked to the type of micro-organism and the process used Sterilisation process used  Sterilisation Techniques Inactivation methods The micro-organisms are being killed (inactivated) Bodies or fragments may Removal methods remain in or on the product The micro-organisms Steam (moist heat) are being removed sterilisation (autoclaving) - from the product, but most common may remain intact Dry heat sterilisation Filtration Ionising radiation sterilisation Gas sterilisation  Gas Sterilisation Uses a gas which is toxic to micro-organisms (and potentially humans) Applies the gas to the product in a sealed container Ethylene oxide - most common Formaldehyde Peracetic acid Hydrogen peroxide  Gas Sterilisation Ethylene oxide Volatile gas, with a boiling point of 10.8oC Easily liquefied at low pressures Readily miscible with water Forms an explosive mixture with air Usually used in mixtures of approx. 10% ethylene oxide with an inert gas such as CO2 or halogenated hydrocarbons (e.g., CCl2F2) Penetrates well into porous materials, especially at higher humidities  Gas Sterilisation Ethylene oxide Alkylating agent - affects proteins, nucleic acids etc All types of organisms are susceptible Needs to be dissolved in water to penetrate cell membranes Will hydrolyse in the presence of too much water to give ethylene glycol (less effective)  Gas Sterilisation Ethylene oxide Process Wrap the product in a steriliser bag and place in the sterilising chamber Apply vacuum to the chamber, add water vapour and ethylene oxide Remove ethylene oxide from the chamber at the end of the cycle by drawing a vacuum and replace with sterile air Typical conditions: 400 mg / L ethylene oxide; 40°C, 30 to 60% RH, 1.5 bar, 2 hours  Gas Sterilisation - Ethylene oxide Advantages: Disadvantages: Effective against ALL Ethylene oxide is TOXIC to humans organisms: bacteria, Safe maximum concentration in moulds, viruses the atmosphere is 10 ppm Odour is detectable at 100 ppm Effective at low Safe disposal of gas is vital humidities and EtO and any decomposition temperatures products must be allowed to disperse from the product Risk of product degradation low Some products need to be aseptically wrapped after sterilisation Good penetration into porous loads and High cost in comparison to other plastics methods  Gas Sterilisation - EtO Uses: Limited application Single use medical devices e.g., catheters, pacemaker wires, surgical dressings, etc Heat sensitive equipment e.g., plastic disposable syringes NOT for products which can absorb and retain ethylene oxide  Ionising Radiation Sterilisation Uses gamma rays, e.g., from 60Cobalt or 137Caesium Uses high energy electron beams from a linear particle accelerator Generates free radicals and ionises cell contents Causes structural damage in enzymes and DNA, eventually killing the micro-organism Requires an absorbed dose into the product of 25 kGy (kilogray) 1 Gy = 1 J of energy absorbed per kilogram of material irradiated  Ionising Radiation Sterilisation Disadvantages: Advantages: Need specialist equipment for Effective against a large generation of the radiation number of different Cost types of bacteria, Availability yeasts, moulds and Large installation some viruses Disposal of waste Highly efficient Ineffective against some viruses Can be used for and prions thermolabile samples Protection of operators from radiations. Ionising Radiation Sterilisation Uses: Limited application Used for products which can be made and sterilised well before use, e.g., syringes, gloves, clean room clothing, bottles Heat sensitive equipment e.g., plastic disposable syringes  Dry Heat Sterilisation Uses dry heat Essentially a hot air oven with forced air circulation high efficiency particulate air (HEPA) filtered air required Causes cell death by dehydration and oxidative processes Proteins and DNA are most affected HEPA filter  Dry Heat Sterilisation Process notes: Must ensure that the temperature within the oven is uniform Validate heat distribution via multiple thermocouples Must ensure adequate air circulation and heat transfer to items Walls are the hottest place in the oven, therefore avoid contact Remember radiation (walls) and convection (fan) effects Packing of items into the oven must be even Items shouldn't touch each other All material to be sterilised in one load should be of the same dimensions and type Containers should be sealed or covered but screw caps of containers should be unscrewed half a turn to Prevent distortion of closure or bursting of containers.  Dry Heat Sterilisation Process conditions BP specifies a range of conditions: 160°C for 2 hours 170°C for 1 hour 180°C for 30 minutes BP default specification is 160°C for 2 hours Theoretically, other temperature / time conditions can be used if validated  Dry Heat Sterilisation Advantages Good for moisture-sensitive Uses: products Good for pre-assembled Thermostable dry apparatus, e.g., syringes powders Less damaging than moist heat to glass and metal Oils, fats, waxes, glycerol, equipment paraffins and their products Disadvantages Devices Slow and less efficient cf moist heat sterilisation Packaging components, Can't be used for thermo- e.g., glass ampoules labile materials  Moist Heat Sterilisation Uses moist heat, i.e., steam "Clean steam" required, from Purified Water BP Steam kills by denaturation of essential proteins and nucleic acids via hydrolysis Must be over 100°C to kill spores Highly energetic Requires high pressure to generate the high temperatures needed Performed in an autoclave  Moist Heat Sterilisation Steam 1 atmosphere pressure = 101 kPa = 101 kN/m2 = 1.01 bar = 14.7 psi = 760 mmHg Boiling point of water At normal atmospheric pressure, the boiling point 0 psi – 100°C of water ~100°C 10 psi – 115 °C At higher pressures, the boiling point is elevated, 15 psi – 121 °C as it is more difficult for water molecules to break away from the bulk water 30 psi – 136 °C  Moist Heat Sterilisation Steam – total energy content, effect of pressure and temperature Enthalpy due to temperature + Enthalpy of evaporation (latent heat) Examples: At 100°C and 0 psi above atmospheric Enthalpy due to temperature = 420 kJ / kg Enthalpy of evaporation (latent heat) = 2,260 kJ / kg Total = 2,680 kJ / kg At 121°C and 15 psi above atmospheric Enthalpy due to temperature = 505 kJ / kg Enthalpy of evaporation (latent heat) = 2,200 kJ / kg Total = 2,705 kJ / kg Increase in P & T will increase the total energy in the system.  Moist Heat Sterilisation Process notes - general Material to be sterilised is loaded into the autoclave chamber The chamber may be heated up (jacketed) A vacuum may be applied (modern equipment) Dry saturated steam is generated separately from Purified Water BP and applied into the chamber, displacing air which must be removed After sterilisation, the chamber must be cooled and air returned Use HEPA filtered air Use Purified Water BP if water spray cooling is required Moist Heat Sterilisation Process notes - general Must ensure that the temperature within the oven is uniform Validate heat distribution via multiple thermocouples Must ensure adequate heat transfer to items Steam distribution must be uniform Packing of items into the oven must be even Items shouldn't touch each other All material to be sterilised in one load should be of the same dimensions and type  Moist Heat Sterilisation Process notes - bottles Steam condenses on the outside surface of the bottles, releasing the enthalpy of evaporation (condensation) Heat is transferred through the glass to the contents, which are then heated to the correct temperature BP specifies 121°C for 15 minutes Care with glass shattering Pre-heating and vacuum are not required Correct autoclave loading pattern vital for heat distribution  Moist Heat Sterilisation Process notes - flexible bags (plastics) Flexible bags would burst in normal autoclaves because of the pressure Add in HEPA-filtered air to give "ballast" (support) Need to ensure adequate mixing of air and steam Most common conditions are: 121°C for 15 minutes or 115°C for 30 minutes Correct autoclave loading pattern vital for heat distribution  Moist Heat Sterilisation Process notes - porous loads Steam penetrates into the material E.g., theatre gowns, dressings, linen, some medical devices Items are sealed inside a special type of paper bag A vacuum is applied initially, then pulses of steam followed by evacuation to ensure steam penetration into the material Most common conditions are: 134°C for 3 minutes Correct autoclave loading pattern vital for heat distribution  Moist Heat Sterilisation Hugo and Russell’s Pharmaceutical Microbiology, Blackwell 7th Edition  Moist Heat Sterilisation Advantages Energetic process, therefore quick Kills bacteria, spores and viruses Disadvantages Not suitable for thermo-labile or water-sensitive materials Not suitable for sealed containers of oil-based materials Uses Most products  Filtration Sterilisation Removes micro-organisms from solutions Used when other methods are not suitable Size of micro-organisms (m) Candida 2 to 4 Acetobacter 1 to 1.5 Escherichia 1.1 to 1.5 Staphylococcus 0.5 to 1.5 Klebsiella 0.3 to 1.5 Pseudomonas 0.22 to 1.0 BP specifies filtration through a 0.22 m filter Viruses may require a nano-filter for removal  Filtration Sterilisation Depth filters Surface filters Act to retain material within them Act as sieves The filters are a mesh network of fibres Defined by physical pore size Channels between fibres are tortuous Changing the pore size will affect the retention of No defined pore size bacteria Retention is due to the tortuosity of the Commonly used for channel and any interaction between product sterilisation the micro-organism and the filter components E.g., Membrane filters Defined by an effective cut-off diameter  Filtration Sterilisation Advantages Disadvantages Good for thermo- Can't be used for suspensions labile materials Viruses and mycoplasms are not Removes bacterial removed from the products. bodies from the product Mycoplasms: form of bacteria that lack rigidity due to lack of cell walls, No endotoxins hence can squeeze through pores remain in the in the filter. product Necessitates aseptic manipulation Can be used for of the product subsequently small and large volumes Life-time of filter may be limited  How do you choose a sterilisation method? What is the product? Is the product heat-sensitive? Is the product moisture-sensitive? Apply logic - does the process involve heat or water?  Sterilisation decision trees (as defined in EMA guidance – Guideline on the sterilisation of the medicinal product, active substance, excipient or primary container, effective 01 October 2019, https://www.ema.europa.eu/en/documents/scie ntific- guideline/guideline-sterilisation-medicinal- product-active- substance-excipient-primary- container_en.pdf)  What sterilisation method would you choose for? An aqueous solution of a heat-stable drug An aqueous solution of a heat-labile drug An oily solution of a heat-stable drug A powder which will later be reconstituted into an intravenous injection Surgical dressings Plastic disposable syringes  Summary Terminal sterilisation is the method of choice if possible Five methods of terminal sterilisation Steam (moist heat) sterilisation (autoclaving) Dry heat sterilisation Ionising radiation sterilisation Gas sterilisation Filtration sterilisation Each method has its own advantages and disadvantages Choose the method which best suits the product  Additional References Aulton’s Pharmaceutics: the design and manufacture of medicinces – 6th edition (K.Taylor) Pharmaceutical preformulation and formulation, 2nd edition (CRC press, edited by Mark Gibson) Parenteral Drug Association (www.pda.org) EMA sterilisation guidance (https://www.ema.europa.eu/en/sterilisation-medicinal-product- active- substance-excipient-primary-container-scientific-guideline)