Sterilization Techniques

Sterilization Techniques Overview

• Sterilization is a crucial process for creating sterile dosage forms and reprocessing medical devices.
• Several methods exist to achieve proper sterilization
• Each comes with its own advantages and disadvantages.
• All methods require the user to understand the technology, follow appropriate guidelines, and validate the process to ensure quality.
• Inadequate documentation and control can lead to sterilization failure, potentially with fatal consequences.

Terminal Sterilization

• Achieved through heat
• Examples include steam and dry heat.
• Physical sterilization involves radiation.
• Examples include gamma radiation and accelerated electrons (particle radiation).
• Chemical sterilization uses various methods
• Gaseous: Low-temperature steam formaldehyde and gas plasma.
• Liquid: Glutaraldehyde, o-phthalaldehyde, formaldehyde, peracetic acid, and hydrogen peroxide.

Non-Terminal Sterilization

• Filtration is used as a non-terminal sterilization method
• Performed under strict aseptic condition.
• Involves an aseptic procedure.

Physical Sterilisation: Heat

• Thermal damage to biological systems is a function of heat energy absorption
• Dry sterilization inactivates microorganisms through oxidation and follows first-order kinetics.
• Dry sterilization is suitable for materials unharmed by high temperatures, such as glassware, metal instruments, oils, and powders.
• Several sterilizing conditions exist, for dry sterilisation, which depend on time and temperature
• Examples are like, 121°C for at least 16 hours to 180°C for 30 minutes.
• Water is required for moist sterilization
• Hydrating microorganisms increases their sensitivity.
• Protein coagulation disrupts H-bonds in protein 3D structures.
• The four main types of moist sterilization are boiling, autoclaving, tyndallization, and pasteurization:
• Pasteurization is explicitly noted as not a sterilization method.
• Boiling at 100°C at atmospheric pressure for 10 minutes kills bacteria, fungi, and viruses, but not endospores.
• Unpressurized steam is practically equivalent to boiling water at 100°C.
• Autoclaving is the most reliable heat sterilization method
• Saturated steam at the phase boundary offers more latent heat for transfer.
• Sterilisation is achieved under positive pressure with moist steam
• High temperatures of 120 ± 2°C are required to kill spores.
• Common sterilisation temperature is 121°C @ 15 lbs/inch² for 15 minutes
• Autoclaving is suitable for materials not susceptible to heat or moisture.
• Larger items or volumes may require longer processing times.
• In autoclaving an automatic ejector valve is thermostatically controlled and closes on contact with pure steam when air is exhausted.
• Glassware, pipettes, media, lab wares, dressings, surgical instruments, polycarbonates, and polyethylene can be autoclaved.
• Heat-sensitive solutions, sugars, salts, antibiotics, drugs, some plastics, and biologics cannot.
• Tyndallization is used to kill both vegetative and spore-forming bacteria.
• Achieved at a temperature of 100°C or below for 30 minutes over consecutive days.
• Pasteurization is mostly used in the food industry, a mild heat treatment that is not sterilization.
• Cell hydration is a significant factor in determining resistance to heat.
• Resistance varies between species and strains, depending on genetic resistance.
• Enzymatic activity and temperature resistance differ among microorganisms:
  • Psychochrils (0-30°C), mesophils (10-50°C), and thermophils (25-90°C).
• Spores are more resistant than vegetative forms; cell age and chemical composition also affect resistance.
• Prions are the most heat-resistant, surviving 138°C for 1 hour, while bacterial spores show little activation below 80°C.
• Fungal spores are typically eradicated above 80°C, and yeast can survive 20 minutes at 60°C.
• Viruses are unlikely to survive more than 20 minutes at 55-60°C, and algae may survive a few hours at 40-45°C.

Physical Sterilisation: Radiation

• Electromagnetic Radiation includes ɣ-rays, X rays, and UV.
• Waves have deep penetration.
• Particulate Radiation includes α and β particles, positrons, and neutrons.
• α particles are heavy, travel slowly, and have low penetration power.
• β particles are negatively charged with the same mass as an electron, have good penetration but can be stopped by thin aluminum and are generated through radioactive decay.
• Gamma radiation occurs after emission of α and β particles, dissipates energy as short-wave radiation, negligible mass or charge, and travel at the speed of light.
• Pharmaceuticals and disposable medical supplies can be sterilized with radiation.
• Alpha particles travel slowly and have limited penetration.
• Beta particles have 100x more penetrating power than alpha particles.
• Gamma emissions possess short wavelengths, no mass or charge, and can penetrate lead.
• X-rays are generated when a heavy metal target is bombarded with electrons and share similar properties to gamma rays.
• Absorption of radiation depends on the energy of the incident photons.
  • Photoelectric effect: Low-energy radiation absorbed by the atom results in electron ejection or excitation.
  • Compton effect: Photons collide with atoms, absorbing a proton of energy and emitting an electron.
  • Pair production: High energy converts to electrons and positrons upon impact.
• Free radical production also occurs.
• Direct effects target cellular DNA, limiting its ability to repair damage, also creating free radicals
• Indirect effects involve ionizing radiation through water, forming free radicals and peroxides.
• These free radicals and peroxides are highly reactive and destructive, killing microorganisms.
• Resistance to radiation is genetically determined.
• The presence of oxygen increases sensitivity, and moisture influences it.
• Dehydration increases resistance, while freezing enhances radiation resistance by reducing free radical mobility.
• Organic materials and sulfhydryl groups offer protection.
• UV radiation has bactericidal activity at 220-280 nm and excites electrons without ionizing.
• UV is used for sterilizing surfaces and air by forming crosslinks between adjacent pyrimidine bases in DNA and has limited penetration.
• Cell aggregates, wet conditions, and proteins limit UV penetration.

Chemical Sterilisation: Gaseous

• Not many gases are suitable for sterilisation; disinfection is more prevelant.
• Alkylating gases like ethylene oxide (EO), low-temperature steam formaldehyde (LTSF), and gas plasmas can be used.
  • LTSF: uses 37% w/v formalin, at 70-75°C, with steam.
• Alkylating gases are suitable for re-usable surgical instruments, medical, diagnostic, and electrical equipments, and surface sterilisation of powder.
• Alkylation of suphydryl, amino, hydroxyl, and carboxyl groups on proteins and imino groups of nucleic acids, is the fundamental method for chemical sterilisation using gases.
• Limitations include that they are potentially mutagenic, carcinogenic, and acutely toxic, irritating the skin, conjunctiva, and nasal mucosa
• Requires strict atmospheric controls to protect personnel.
• Ethylene Oxide is highly explosive at >3.6% v/v in air, and dried organisms are more resistant because they are protected from inclusion in crystalline or dried organic deposits.

Non-Terminal Sterilisation: Filtration

• Membrane filters with pore diameters of 0.2-0.22 µm are commonly used
• The filter medium must be sterilisable, optimally by steam treatment.
• According to the FDA, "a sterilizing filter is one that, when challenged with microorganism Pseudomonas diminuta at a minimum concentration of 107 organisms per cm² of filter surface, will produce a sterile effluent."
• Under strict aseptic condition, products that cannot be terminally sterilised because they are sensitive to heat and radiation can use non-terminal sterilization with filtration.
• Suitable for aqueous and oily liquids, air, and other gases.
• Depth, surface and membrane filters exis
• Depth filters use fibrous, granular, or sintered material in a maze to trap particles.

Validation of Sterilisation

• The British Pharmacopoeia : product sterility is assured by a validated production process:
  • Sterilization's effect on the product and its packaging is investigated to ensure effectiveness and integrity, that isvalidated before application.
• Documentation for validation is available from international organizations such as ISO and FDA.
• Performance qualification data ensure the equipment meets required sterility levels, and commissioning data describe installation and equipment characteristics.
• Physical, chemical, and biological indicators measure process effectiveness.
• Autoclave tape indicates when appropriate conditions (temperature) have been met, by changing the white stripe to black.
• Temptubes show specific melting points have been reached
• Spores added to a carrier and spore suspensions used to inoculate products, are important components in showing sterilisation.
• Self-contained Indicators are also useful for measuring sterilisation and include :
  • Cap filter
  • Plastic cap
  • Macroporous material
  • Plastic sleeve
  • Glass ampoule
  • Spore strip
  • Label

Selection of Sterilisation Process

• What's the type of product/preparation, its volume and composition?
• Potential damages to both the product and the container must be taken into consideration:
  • Damage to the product can be from Heat on Heat-sensitive preparations, Radiation on water, and general Corrosiveness from oxidizing agents.
  • Damage to the container can be from Water ballasting, general Moisture, the Glass breaking on cooling, a Change in composition post irradiation, and Corrosiveness.
• Other considerations:
  • Toxicity and safety
  • The level of bioburden
  • The sterilisation regimen being used
  • The cost of the actual sterilisation process
  • Validation of instruments both pre and post the sterilisation process.