Sterilization Techniques - 1 PDF

# Sterilisation techniques ## Dr Laura Urbano * F154 Hillside House * Email: [email protected] * Tel: 01707 281372 ## Learning objectives 1. Commonly employed sterilisation techniques and their applications 2. Critically evaluate the different types of sterilisation techniques 3. Describe...

# Sterilisation techniques ## Dr Laura Urbano * F154 Hillside House * Email: [email protected] * Tel: 01707 281372 ## Learning objectives 1. Commonly employed sterilisation techniques and their applications 2. Critically evaluate the different types of sterilisation techniques 3. Describe the role of sterilisation assurance and application of biological indicators ## Sterilization technologies for pharmaceutical preparations and medical devices | Type | Principle | Examples | | --------------- | ---------------------------- | -------------------------------------------------------------------------------- | | Terminal sterilization | Heat | Steam, dry heat | | Physical | Radiation | ɣ-radiation, accelerated electrons (particle radiation), ethylene oxide | | Chemical | Gaseous | Low-temperature steam formaldehyde, gas plasma | | | Liquid | Glutaraldehyde, o-phthalaldehyde, formaldehyde, peracetic acid, hydrogen peroxide | | Nonterminal sterilization | Filtration | | | | Aseptic procedure | | ## Physical sterilisation ### Heat sterilisation #### Moist heat The image shows a geyser with steam erupting from it. #### Dry heat The image shows a desert landscape. ### Dry sterilisation The image shows a desert landscape. * Thermal damage to biological system as a function of absorption of heat energy. * Inactivation of microorganism through *oxidation*. * Usually follows first order kinetics. The image shows bacteria under a microscope. * Use for materials not damaged by high temperatures: Sterilisation of glassware, metal instruments, oils and powders. ### Dry Sterilisation The image shows a desert landscape. The image shows a graph with Temperature on the y-axis and Time on the x-axis showing the heating, holding and cooling stages of sterilisation. * Sterilising conditions: * 121°C for at least 16 hours * 160°C for at least two hours * 171°C for at least one hour * 180°C for 30 minutes The image shows bacteria under a microscope. ### Moist sterilisation The image shows a geyser with steam erupting from it. * Presence of water: * hydrating microorganisms and making them more sensitive * "coagulation of proteins" disrupts H-bonds critically for protein 3D structure * Four main types: * Boiling * Autoclaving * Tyndallization * Pasteurization * *not sterilisation ### Boiling The image shows a geyser with steam erupting from it. The image shows a boiling pot of water. * Boiling at 100°C at atmospheric pressure for 10 minutes will kill: * bacteria * fungi * viruses BUT will not kill: * endospores * "unpressurized" i.e., free-flowing steam is practically equivalent to the prevalent temperature of boiling water (i.e., 100°C). *not sterilisation ### Autoclaving The image shows a geyser with steam erupting from it. The image shows a phase diagram for water where steam pressure is plotted against temperature, showing the phase change from liquid to gas. * Most reliable heat sterilization method. * Saturated steam at the phase boundary has more latent heat available for transfer. The image shows the same geyser image as before, and an autoclave. * To kill spores temp has to be increased >100°C * High temperatures [120 ± 2°C] are achieved by moist steam under positive pressure * Steam under pressure at 121°C @ 15 lbs/inch² for 15 minutes * Used for materials to be sterilized are not susceptible to heat or moisture * Large items and large volumes may take longer The image shows a diagram of an autoclave, highlighting components such as the exhaust valve, steam jacket, steam chamber, safety valve, pressure gauge, operating valve, and door. * Automatic ejector valve is thermostatically controlled and closes on contact with pure steam when air is exhausted. The image shows a graph with temperature on the y-axis and time on the x-axis plotting the different stages of the autoclaving process. ### Autoclaving The image shows a geyser with steam erupting from it. The image shows an autoclave. * What materials can be autoclaved? * Glassware * Pipettes * Media * Lab wares * Dressings * Surgical instruments * Polycarbonates * Polyethylene * What materials cannot be autoclaved? * Heat sensitive solutions * Sugars * Salts * Antibiotics * Drugs * Plastics (some) * Biologics? ### Autoclaving vs Dry Heat Sterilisation | Process | Min temperature (°C) | Minimum holding time (min) | | ------------------- | --------------------- | --------------------------- | | Steam sterilisation | 115 | 30 | | (Autoclaving) | 121 | 15 | | | 126 | 10 | | | 134 | 3 | | Dry heat sterilisation | 140 | 180 | | | 150 | 150 | | | 160 | 120 | | | 170 | 60 | | | 180 | 30 | ### Tyndallisation The image shows a geyser with steam erupting from it. The image shows a diagram of a Tyndallisation setup (used for sterilisation). * For killing both vegetative and spore forming bacteria. * Temperature of 100C or below for 30 min on the consecutive days. ### Pasteurisation The image shows a geyser with steam erupting from it. The image shows a pasteurisation setup. * Mostly used in food industry * Mild heat * NOT sterilisation!! ## Physical sterilisation ### Resistance to Heat Sterilisation * Cell hydration an important factor in determining its resistance to heat. * Differences between species and strains: * source - genetic resistance present? * Enzymatic activity and temperature resistance: * psychochrils (0-30°C) * mesophils (10-50°C) * thermophils (25-90°C) The image shows a movie poster for the 1959 film "Some Like It Hot", featuring Marilyn Monroe, Tony Curtis, and Jack Lemmon. * Spore vs vegetative forms of the same organism * Age of cell (lag, stationary, exponential phase) * Chemical Composition: * Culture medium * pH (most prefer neutral although some acidophiles) * Large quantities of essential nutrients = rapid growth * C, O, N, S, P, (Fe, Co, K, Na) * anaerobe v aerobe * lithotrophs, organotrophs The image shows a movie poster for the 1959 film "Some Like It Hot", featuring Marilyn Monroe, Tony Curtis, and Jack Lemmon. 1. Prions - most heat resistant, can survive 138 ° C for 1 h 2. Bacterial spores - little or no activation below 80 ° C 3. Fungal spores - usually irradicated above 80° C 4. Yeast - can usually survive for 20 mins at 60 ° C 5. Viruses - unlikely to survive more than 20 mins at 55-60 ° C 6. Algae - may survive a few hours at 40-45 ° C ## Physical sterilisation ### Radiation Sterilisation #### Electromagnetic * ɣ-rays, X rays, UV * Waves - deep penetration #### Particulate * α and β particles * Positrons * Neutrons #### Particulate Radiation * Nuclear disintegration of radioactive elements * α particles- heavy, travel slowly and have low penetration power. * β particles-negatively charged and same mass as electron, good penetration power but can be stopped by thin sheet of aluminium, generated through radioactive decay. * No real application in pharmaceutical sciences. The image shows a diagram highlighting the relative penetration powers of alpha, beta and gamma radiation. * Gamma radiation occurs after emission of a and β particles. * Energy is dissipated in form of very short wave length radiation with no mass or charge, travel at speed of light. * ɣ-radiation is highly penetrative, negligible heating of sterilized product at normal doses and induces no radioactivity in the final product. * Sterilize pharmaceuticals and disposable medical supplies. ### Radiation Sterilisation | PARTICLE RADIATION | ELECTROMAGNETIC RADIATION | | ------------------------- | ----------------------------------------------------------- | | • 3H - alpha particles | • 99Tc - gamma emission | | • Travel slowly | • Short wavelength | | • Limited penetration | • No mass | | | • No charge | | | • Can penetrate lead | | | | | • 14C - beta particles | • X rays | | • 100 x more penetrating than alpha particles | • Generated when a heavy metal target is bombarded with electrons | | | • Similar properties to gamma rays | ## Physical sterilisation ### Radiation Sterilisation #### Impact of radiation on materials * Absorption of radiation depends on the energy of the incident photons. * Photoelectric effect - low energy radiation absorbed by the atom of the material resulting the ejection or excitation of an electron. * Compton effect - photons colloid with the atoms and a proton of the energy is absorbed with the ejection of an electron. * Pair production - very high energy, converted on impact into electrons and positrons. * Free radical production. #### Impact of radiation on microorganisms * Direct effects: * Target cellular DNA: organism's ability to repair the DNA is the limiting factor. * Further damage due to free radicals produced within the cells. * Indirect effects: * Passage of ionizing radiation through water causes formation of free radicals and peroxides. * Free radicals and peroxides are highly reactive, destructive and responsible for killing of microorganism. ## Physical sterilisation ### Resistance to Radiation Sterilisation * Resistance to radiation is genetically determined * Presence of oxygen increases sensitivity (hydroperoxide radical formation) * Presence of moisture influences sensitivity: * Dehydration increases resistance by a direct impact on the formation of free radicals * Freezing increases radiation resistance: * Reduces mobility of free radicals * Protection via organic materials: * Sulfhyldryl groups in proteins and amino acids ## Physical sterilisation ### UV Sterilisation * Bactericidal activity 220-280 nm * Excites electrons non ionising radiation. * Used for destruction of microorganisms on surfaces and in the air. * Formation of crosslinks between adjacent pyrimidine bases in DNA. The image shows a UV sterilisation lamp. * UV wavelengths have limited penetration power. * Cell aggregates - protection to core cells. * Wet vs dry state - water limits penetration. * Proteins - limit penetration (e.g. cells suspended in broth). The image shows several microscopic pictures, showing cells in different states and a beaker containing a yellow solution. ## Chemical sterilisation ### Gaseous Sterilisation * Disinfection- not many gases used for sterilisation. * Alkylating gases: * e.g. Ethylene oxide (EO) * low-temperature steam formaldehyde (LTSF): 37% w/v formalin, at 70-75°C, with steam * Gas plasmas * Applications: * Re-usable surgical instruments, medical, diagnostic and electrical equipments, surface sterilisation of powder. * Mechanism: * alkylation of suphydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic acids ### Gaseous Sterilisation * Limitations: * Potentially mutagenic and carcinogenic. * Acute toxicity including irritation of skin, conjunctiva and nasal mucosa. * Strict control of atmosphere is necessary to protect personnel. * Ethylene Oxide: * Highly explosive in mixture of >3.6% v/v in air. * Organisms are more resistant in dried state as they are protected from the gas by inclusion in crystalline or dried organic deposits. ## Non-terminal sterilisation ### Filtration * Membrane filters of 0.2-0.22 µm nominal pore diameters are chiefly used. * Filter medium employed must be sterilisable, ideally by steam treatment. * According to FDA "a sterilizing filter is one which, when challenge with microorganism Pseudomonas diminuta, at a minimum concentration of 107 organisms per cm² of filter surface, will produce a sterile effluent". The image shows various filtration equipment. ### Filtration * Key features: * Non-terminal sterilization used under strict aseptic condition. * Used products that cannot be terminally sterilised or sensitive to heat and radiation (e.g. vitamins, heparin). * Used to sterilize aqueous and oily liquids, air and other gases. * Filters: * Depth, surface and membrane filters exist. * Depth filters are made of fibrous, granular or sintered material bonded into maze of channel that trap the particles. The image shows various filtration equipment. ## Validation of a sterilization process The British Pharmacopoeia (British Pharmacopoeia Commission, 2017a) states: "The sterility of a product cannot be guaranteed by testing; it has to be assured by the application of a suitably validated production process. It is essential that the effect of the chosen sterilization procedure on the product (including its final container or package) is investigated to ensure effectiveness and the integrity of the product and that the procedure is validated before being applied in practice." * Documentation for validation provided by international organisations as ISO, FDA. * Commissioning data (installation and characteristics of the equipment). * Performance qualification data (ensure that the equipment will produce the required sterility assurance level). ### Process indicators #### Physical, chemical and biological indicates * Autoclave tape * The white stripes on the tape change to black when the appropriate conditions (temperature) have been met. * Temptubes - specific melting point. The image shows an autoclave tape before and after sterilisation. The image shows Temptubes (containing a melting point indicator) that are used for sterilisation. * Spores that are added to a carrier (a disk or strip of filter paper, glass, plastic, or other materials) and packaged to maintain the integrity and viability of the inoculated carrier * Spore suspension that is inoculated on or into representative units of the product to be sterilized. * Self-contained indicator The image shows a schematic of a 3M™ Attest™ 1292 Rapid Readout Biological Indicator. The components of a self-contained 3M™ Attest™ 1292 Rapid Readout Biological Indicator: * Cap filter * Plastic cap * Macroporous material * Plastic sleeve * Glass ampoule * Spore strip * Label ## Sterilization technologies for pharmaceutical preparations and medical devices | Type | Principle | Examples | | --------------- | ---------------------------- | -------------------------------------------------------------------------------- | | Terminal sterilization | Heat | Steam, dry heat | | Physical | Radiation | ɣ-radiation, accelerated electrons (particle radiation), ethylene oxide | | Chemical | Gaseous | Low-temperature steam formaldehyde, gas plasma | | | Liquid | Glutaraldehyde, o-phthalaldehyde, formaldehyde, peracetic acid, hydrogen peroxide | | Nonterminal sterilization | Filtration | | | | Aseptic procedure | | ## Selection of sterilisation process * What's the type of product/preparation? * Volume * Composition * Possible damage to the preparation/product? * Heat (heat-sensitive preparations) * Radiation (water) * Corrosiveness (oxidizing agents) * Possible damage to the product/container? * Water ballasting * Moisture * Glass breaking (on cooling) * Change in composition (irradiation) * Corrosiveness * Other considerations? * Toxicity/safety * Level of bioburden * Sterilisation regimen * Cost of sterilization process * Validation ## Summary * Sterilization is an essential process for the manufacture of sterile dosage forms, and reprocessing medical devices and products. * Several processes can be used to achieve appropriate sterilization. * Each of these processes has advantages and disadvantages, although steam sterilization remains the reference standard. * Common to all these processes is the need for the user to understand the technology, its activity and limitations, to follow the appropriate guidelines but also, importantly, to ensure the validation of the process. * Failure to provide the appropriate documentation and to control a sterilization process adequately might result in failure of the process, with potentially fatal consequences. ## Learning objectives 1. Commonly employed sterilisation techniques and their applications 2. Critically evaluate the different types of sterilisation techniques 3. Describe the role of sterilisation assurance and application of biological indicators ## References and additional resources 1. Pharmaceutics: the design and manufacture of medicines / edited by Michael E. Aulton., 3rd edi, Churchill Livingstone, 2007. 2. Hugo and Russell's pharmaceutical microbiology edited by Stephen P. Denyer, Norman A. Hodges, Sean P. Gorman, 7th Ed., Blackwell Science, 2004 3. [https://www.ema.europa.eu/documents/scientific-guideline/decision-trees-selection-sterilisation-methods-cpmp/qwp/054/98-annex-note-guidance-development-pharmaceutics-cpmp/qwp/155/96_en.pdf](https://www.ema.europa.eu/documents/scientific-guideline/decision-trees-selection-sterilisation-methods-cpmp/qwp/054/98-annex-note-guidance-development-pharmaceutics-cpmp/qwp/155/96_en.pdf)