Sterilization is necessary for the complete destruction or removal of all microorganisms (including spore-forming and non-sporeforming bacteria, viruses, fungi, and protozoa) that could contaminate pharmaceuticals or other materials and thereby constitute a health hazard. Since the achievement of the absolute state of sterility cannot be demonstrated, the sterility of a pharmaceutical preparation can be defined only in terms of probability. The efficacy of any sterilization process will depend on the nature of the product, the extent and type of any contamination, and the conditions under which the final product has been prepared. The requirements for Good Manufacturing Practice should be observed throughout all stages of manufacture and sterilization.

Classical sterilization techniques using saturated steam under pressure or hot air are the most reliable and should be used whenever possible. Other sterilization methods include filtration, ionizing radiation (gamma and electron-beam radiation), and gas (ethylene oxide, formaldehyde).

For products that cannot be sterilized in the final containers, aseptic processing is necessary. Materials and products that have been sterilized by one of the above processes are transferred to presterilized containers and sealed, both operations being carried out under controlled aseptic conditions.

Whatever method of sterilization is chosen, the procedure must be validated for each type of product or material, both with respect to the assurance of sterility and to ensure that no adverse change has taken place within the product. Failure to follow precisely a defined, validated process could result in a non-sterile or deteriorated product. A typical validation programme for steam or dry-heat sterilization requires the correlation of temperature measurements, made with sensory devices to demonstrate heat penetration and heat distribution, with the destruction of biological indicators, i.e. preparations of specific microorganisms known to have high resistance to the particular sterilization process. Biological indicators are also used to validate other sterilization methods (see specific methods), and sometimes for routine control of individual cycles. Periodic revalidation is recommended.

A widely used method for heat sterilization is the autoclave, sometimes called a converter or steam sterilizer. Autoclaves use steam heated to 121–134 °C (250–273 °F) under pressure. To achieve sterility, the article is placed in a chamber and heated by injected steam until the article reaches a temperature and time setpoint. Almost all the air is removed from the chamber, because air is undesired in the moist heat sterilization process (this is one trait that differs from a typical pressure cooker used for food cooking). The article is held at the temperature setpoint for a period of time which varies depending on what bioburden is present on the article being sterilized and its resistance (D-value) to steam sterilization. A general cycle would be anywhere between 3 and 15 minutes, (depending on the generated heat) at 121 °C (250 °F) at 100 kPa (15 psi), which is sufficient to provide a sterility assurance level of 10⁻⁴ for a product with a bioburden of 10⁶ and a D-value of 2.0 minutes. Following sterilization, liquids in a pressurized autoclave must be cooled slowly to avoid boiling over when the pressure is released. This may be achieved by gradually depressurizing the sterilization chamber and allowing liquids to evaporate under a negative pressure, while cooling the contents.

Proper autoclave treatment will inactivate all resistant bacterial spores in addition to fungi, bacteria, and viruses, but is not expected to eliminate all prions, which vary in their resistance. For prion elimination, various recommendations state 121–132 °C (250–270 °F) for 60 minutes or 134 °C (273 °F) for at least 18 minutes. The 263K scrapie prion is inactivated relatively quickly by such sterilization procedures; however, other strains of scrapie, and strains of Creutzfeldt-Jakob disease (CKD) and bovine spongiform encephalopathy (BSE) are more resistant. Using mice as test animals, one experiment showed that heating BSE positive brain tissue at 134–138 °C (273–280 °F) for 18 minutes resulted in only a 2.5 log decrease in prion infectivity.

Most autoclaves have meters and charts that record or display information, particularly temperature and pressure as a function of time. The information is checked to ensure that the conditions required for sterilization have been met. Indicator tape is often placed on the packages of products prior to autoclaving, and some packaging incorporates indicators. The indicator changes color when exposed to steam, providing a visual confirmation.

Bioindicators can also be used to independently confirm autoclave performance. Simple bioindicator devices are commercially available, based on microbial spores. Most contain spores of the heat-resistant microbe Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), which is extremely resistant to steam sterilization. Biological indicators may take the form of glass vials of spores and liquid media, or as spores on strips of paper inside glassine envelopes. These indicators are placed in locations where it is difficult for steam to reach to verify that steam is penetrating there.

For autoclaving, cleaning is critical. Extraneous biological matter or grime may shield organisms from steam penetration. Proper cleaning can be achieved through physical scrubbing, sonication, ultrasound, or pulsed air.

Pressure cooking and canning is analogous to autoclaving, and when performed correctly renders food sterile.

Moist heat causes the destruction of microorganisms by denaturation of macromolecules, primarily proteins. This method is a faster process than dry heat sterilization.^[19]

To sterilize waste materials that are chiefly composed of liquid, a purpose-built effluent decontamination system can be utilized. These devices can function using a variety of sterilants, although using heat via steam is most common

Dry heat was the first method of sterilization and is a longer process than moist heat sterilization. The destruction of microorganisms through the use of dry heat is a gradual phenomenon. With longer exposure to lethal temperatures, the number of killed microorganisms increases. Forced ventilation of hot air can be used to increase the rate at which heat is transferred to an organism and reduce the temperature and amount of time needed to achieve sterility. At higher temperatures, shorter exposure times are required to kill organisms. This can reduce heat-induced damage to food products.

The standard setting for a hot air oven is at least two hours at 160 °C (320 °F). A rapid method heats air to 190 °C (374 °F) for 6 minutes for unwrapped objects and 12 minutes for wrapped objects.Dry heat has the advantage that it can be used on powders and other heat-stable items that are adversely affected by steam

Sterilization by filtration is employed mainly for thermolabile solutions. These may be sterilized by passage through sterile bacteria-retaining filters, e.g. membrane filters (cellulose derivatives, etc.), plastic, porous ceramic, or suitable sintered glass filters, or combinations of these. Asbestos-containing filters should not be used.

Appropriate measures should be taken to avoid loss of solute by adsorption onto the filter and to prevent the release of contaminants from the filter. Suitable filters will prevent the passage of microorganisms, but the filtration must be followed by an aseptic transfer of the sterilized solution to the final containers which are then immediately sealed with great care to exclude any recontamination.

Usually, membranes of not greater than 0.22 μm nominal pore size should be used. The effectiveness of the filtration method must be validated if larger pore sizes are employed.

To confirm the integrity of filters, both before and after filtration, a bubble point or similar test should be used, in accordance with the filter manufacturer’s instructions. This test employs a prescribed pressure to force air bubbles through the intact membrane previously wetted with the product, with water, or with a hydrocarbon liquid.

All filters, tubes, and equipment used “downstream” must be sterile. Filters capable of withstanding heat may be sterilized in the assembly before use by autoclaving at 121 °C for 15 – 45 minutes depending on the size of the filter assembly. The effectiveness of this sterilization should be validated. For filtration of a liquid in which microbial growth is possible, the same filter should not be used for procedures lasting longer than one working day.

Several FDA-cleared liquid chemical sterilants include indications for sterilization of medical devices (Tables 4 and 5)⁶⁹. The indicated contact times range from 3 hours to 12 hours. However, except for a few of the products, the contact time is based only on the conditions to pass the AOAC Sporicidal Test as a sterilant and not on simulated use testing with devices. These solutions are commonly used as high-level disinfectants when a shorter processing time is required. Generally, chemical liquid sterilants cannot be monitored using a biological indicator to verify sterility.

The survival kinetics for thermal sterilization methods, such as steam and dry heat, have been studied and characterized extensively, whereas the kinetics for sterilization with liquid sterilants are less well understood. The information that is available in the literature suggests that sterilization processes based on liquid chemical sterilants, in general, may not convey the same sterility assurance level as sterilization achieved using thermal or physical methods. The data indicate that the survival curves for liquid chemical sterilants may not exhibit log-linear kinetics and the shape of the survivor curve may vary depending of the formulation, chemical nature and stability of the liquid chemical sterilant. In addition, the design of the AOAC Sporicidal Test does not provide quantification of the microbial challenge. Therefore, sterilization with a liquid chemical sterilant may not convey the same sterility assurance as other sterilization methods.

One of the differences between thermal and liquid chemical processes for sterilization of devices is the accessibility of microorganisms to the sterilant. Heat can penetrate barriers, such as biofilm, tissue, and blood, to attain organism kill, whereas liquids cannot adequately penetrate these barriers. In addition, the viscosity of some liquid chemical sterilants impedes their access to organisms in the narrow lumens and mated surfaces of devices⁹²². Another limitation to sterilization of devices with liquid chemical germicides is the post-processing environment of the device. Devices cannot be wrapped or adequately contained during processing in a liquid chemical sterilant to maintain sterility following processing and during storage. Furthermore, devices may require rinsing following exposure to the liquid chemical sterilant with water that typically is not sterile. Therefore, due to the inherent limitations of using liquid chemical sterilants, their use should be restricted to reprocessing critical devices that are heat-sensitive and incompatible with other sterilization methods.

Several published studies compare the sporicidal effect of liquid chemical germicides against spores of Bacillus and Clostridium.

Several FDA-cleared liquid chemical sterilants include indications for sterilization of medical devices (Tables 4 and 5)⁶⁹. The indicated contact times range from 3 hours to 12 hours. However, except for a few of the products, the contact time is based only on the conditions to pass the AOAC Sporicidal Test as a sterilant and not on simulated use testing with devices. These solutions are commonly used as high-level disinfectants when a shorter processing time is required. Generally, chemical liquid sterilants cannot be monitored using a biological indicator to verify sterility.

The survival kinetics for thermal sterilization methods, such as steam and dry heat, have been studied and characterized extensively, whereas the kinetics for sterilization with liquid sterilants are less well understood. The information that is available in the literature suggests that sterilization processes based on liquid chemical sterilants, in general, may not convey the same sterility assurance level as sterilization achieved using thermal or physical methods. The data indicate that the survival curves for liquid chemical sterilants may not exhibit log-linear kinetics and the shape of the survivor curve may vary depending of the formulation, chemical nature and stability of the liquid chemical sterilant. In addition, the design of the AOAC Sporicidal Test does not provide quantification of the microbial challenge. Therefore, sterilization with a liquid chemical sterilant may not convey the same sterility assurance as other sterilization methods.

One of the differences between thermal and liquid chemical processes for sterilization of devices is the accessibility of microorganisms to the sterilant. Heat can penetrate barriers, such as biofilm, tissue, and blood, to attain organism kill, whereas liquids cannot adequately penetrate these barriers. In addition, the viscosity of some liquid chemical sterilants impedes their access to organisms in the narrow lumens and mated surfaces of devices⁹²². Another limitation to sterilization of devices with liquid chemical germicides is the post-processing environment of the device. Devices cannot be wrapped or adequately contained during processing in a liquid chemical sterilant to maintain sterility following processing and during storage. Furthermore, devices may require rinsing following exposure to the liquid chemical sterilant with water that typically is not sterile. Therefore, due to the inherent limitations of using liquid chemical sterilants, their use should be restricted to reprocessing critical devices that are heat-sensitive and incompatible with other sterilization methods.

Several published studies compare the sporicidal effect of liquid chemical germicides against spores of Bacillus and Clostridium.

Sterilization of certain active ingredients, drug products, and medical devices in their final container or package may be achieved by exposure to ionizing radiation in the form of gamma radiation from a suitable radioisotopic source such as 60Co (cobalt 60) or of electrons energized by a suitable electron accelerator. Laws and regulations for protection against radiation must be respected.

Gamma radiation and electron beams are used to effect ionization of the molecules in organisms. Mutations are thus formed in the DNA and these reactions alter replication. These processes are very dangerous and only well-trained and experienced staff should decide upon the desirability of their use and should ensure monitoring of the processes. Specially designed and purposebuilt installations and equipment must be used.

It is usual to select an absorbed radiation level of 25 kGy1 (2.5 Mrad)2 , although other levels may be employed provided that they have been validated.

Radiation doses should be monitored with specific dosimeters during the entire process. Dosimeters should be calibrated against a standard source on receipt from the supplier and at appropriate intervals thereafter. The radiation system should be reviewed and validated whenever the source material is changed and, in any case, at least once a year.

The bioindicator strains proposed for validation of this sterilization process are: spores of Bacillus pumilus (e.g. ATCC 27142 or CIP 77.25) with 25 kGy (2.5 Mrad) for which the D-value is about 3 kGy (0.3 Mrad) using 107-108 spores per indicator; for higher doses, spores of Bacillus cereus (e.g. SSI C 1/1) or Bacillus sphaericus (e.g. SSl C1A) are used.

The active agent of the gas sterilization process can be ethylene oxide or another highly volatile substance. The highly flammable and potentially explosive nature of such agents is a disadvantage unless they are mixed with suitable inert gases to reduce their highly toxic properties and the possibility of toxic residues remaining in treated materials. The whole process is difficult to control and should only be considered if no other sterilization procedure can be used. It must only be carried out under the supervision of highly skilled staff.

The sterilizing efficiency of ethylene oxide depends on the concentration of the gas, the humidity, the time of exposure, the temperature, and the nature of the load. In particular, it is necessary to ensure that the nature of the packaging is such that the gas exchange can take place. It is also important to maintain sufficient humidity during sterilization. Records of gas concentration and of temperature and humidity should be made for each cycle. Appropriate sterilization conditions must be determined experimentally for each type of load.

After sterilization, time should be allowed for the elimination of residual sterilizing agents and other volatile residues, which should be confirmed by specific tests.

Because of the difficulty of controlling the process, efficiency must be monitored each time using the proposed bioindicator strains: spores of Bacillus subtilis (e.g. var. niger ATCC 9372 or CIP 77.18) or of Bacillus stearothermophilus, (e.g. ATCC 7953 or CIP 52.81). The same quantity of spores should be used as for “Heating in an autoclave” and “Dry-heat sterilization”.

Microwaves are used in medicine for disinfection of soft contact lenses, dental instruments, dentures, milk, and urinary catheters for intermittent self-catheterization. However, microwaves must only be used with products that are compatible (e.g., do not melt). Microwaves are radio-frequency waves, which are usually used at a frequency of 2450 MHz. The microwaves produce friction of water molecules in an alternating electrical field. The intermolecular friction derived from the vibrations generates heat and some authors believe that the effect of microwaves depends on the heat produced while others postulate a nonthermal lethal effect. The initial reports showed microwaves to be an effective microbicide. The microwaves produced by a “home-type” microwave oven (2.45 GHz) completely inactivate bacterial cultures, mycobacteria, viruses, and G. stearothermophilus spores within 60 seconds to 5 minutes depending on the challenge organism.

Another study confirmed these resuIts but also found that higher power microwaves in the presence of water may be needed for sterilization. Complete destruction of Mycobacterium bovis was obtained with 4 minutes of microwave exposure (600W, 2450 MHz). The effectiveness of microwave ovens for different sterilization and disinfection purposes should be tested and demonstrated as test conditions affect the results (e.g., presence of water, microwave power). Sterilization of metal instruments can be accomplished but requires certain precautions.. Of concern is that home-type microwave ovens may not have even distribution of microwave energy over the entire dry device (there may be hot and cold spots on solid medical devices); hence there may be areas that are not sterilized or disinfected. The use of microwave ovens to disinfect intermittent-use catheters also has been suggested. Researchers found that test bacteria (e.g., E. coli, Klebsiella pneumoniae, Candida albicans) were eliminated from red rubber catheters within 5 minutes . Microwaves used for sterilization of medical devices have not been FDA cleared.

Hydrogen peroxide solutions have been used as chemical sterilants for many years. However, the VHPâ was not developed for the sterilization of medical equipment until the mid-1980s. One method for delivering VHP to the reaction site uses a deep vacuum to pull liquid hydrogen peroxide (30-35% concentration) from a disposable cartridge through a heated vaporizer and then, following vaporization, into the sterilization chamber. A second approach to VHP delivery is the flow-through approach in which the VHP is carried into the sterilization chamber by a carrier gas such as air using either a slight negative pressure (vacuum) or slight positive pressure. Applications of this technology include vacuum systems for industrial sterilization of medical devices and atmospheric systems for decontaminating for large and small areas. VHP offers several appealing features that include rapid cycle time (e.g., 30-45 minutes); low temperature; environmentally safe by-products (H₂O, oxygen [O₂]); good material compatibility; and ease of operation, installation and monitoring. VHP has limitations including that cellulose cannot be processed; nylon becomes brittle; and VHP penetration capabilities are less than those of ETO. VHP has not been cleared by FDA for sterilization of medical devices in healthcare facilities.

The feasibility of utilizing vapor-phase hydrogen peroxide as a surface decontaminant and sterilizer was evaluated in a centrifuge decontamination application. In this study, vapor-phase hydrogen peroxide was shown to possess significant sporicidal activity . In preliminary studies, hydrogen peroxide vapor decontamination has been found to be a highly effective method of eradicating MRSA, Serratia marcescens, Clostridium botulinum sporesand Clostridium difficile from rooms, furniture, surfaces and/or equipment; however, further investigation of this method to demonstrate both safety and effectiveness in reducing infection rates are required

Ozone has been used for years as a drinking water disinfectant. Ozone is produced when O₂ is energized and split into two monatomic (O₁) molecules. The monatomic oxygen molecules then collide with O₂ molecules to form ozone, which is O₃. Thus, ozone consists of O₂ with a loosely bonded third oxygen atom that is readily available to attach to, and oxidize, other molecules. This additional oxygen atom makes ozone a powerful oxidant that destroys microorganisms but is highly unstable (i.e., half-life of 22 minutes at room temperature).

A new sterilization process, which uses ozone as the sterilant, was cleared by FDA in August 2003 for processing reusable medical devices. The sterilizer creates its own sterilant internally from USP grade oxygen, steam-quality water and electricity; the sterilant is converted back to oxygen and water vapor at the end of the cycle by a passing through a catalyst before being exhausted into the room. The duration of the sterilization cycle is about 4 h and 15 m, and it occurs at 30-35°C. Microbial efficacy has been demonstrated by achieving a SAL of 10^-6 with a variety of microorganisms to include the most resistant microorganism, Geobacillus stearothermophilus.

The ozone process is compatible with a wide range of commonly used materials including stainless steel, titanium, anodized aluminum, ceramic, glass, silica, PVC, Teflon, silicone, polypropylene, polyethylene and acrylic. In addition, rigid lumen devices of the following diameter and length can be processed: internal diameter (ID): > 2 mm, length ≤ 25 cm; ID > 3 mm, length ≤ 47 cm; and ID > 4 mm, length ≤ 60 cm.

The process should be safe for use by the operator because there is no handling of the sterilant, no toxic emissions, no residue to aerate, and low operating temperature means there is no danger of an accidental burn. The cycle is monitored using a self-contained biological indicator and a chemical indicator. The sterilization chamber is small, about 4 ft³ (Written communication, S Dufresne, July 2004).

A gaseous ozone generator was investigated for decontamination of rooms used to house patients colonized with MRSA. The results demonstrated that the device tested would be inadequate for the decontamination of a hospital room.

Low-temperature steam with formaldehyde is used as a low-temperature sterilization method in many countries, particularly in Scandinavia, Germany, and the United Kingdom. The process involves the use of formalin, which is vaporized into a formaldehyde gas that is admitted into the sterilization chamber. A formaldehyde concentration of 8-16 mg/l is generated at an operating temperature of 70-75°C. The sterilization cycle consists of a series of stages that include an initial vacuum to remove air from the chamber and load, followed by steam admission to the chamber with the vacuum pump running to purge the chamber of air and to heat the load, followed by a series of pulses of formaldehyde gas, followed by steam. Formaldehyde is removed from the sterilizer and load by repeated alternate evacuations and flushing with steam and air. This system has some advantages, e.g., the cycle time for formaldehyde gas is faster than that for ETO and the cost per cycle is relatively low. However, ETO is more penetrating and operates at lower temperatures than do steam/formaldehyde sterilizers. Low-temperature steam formaldehyde sterilization has been found effective against vegetative bacteria, mycobacteria, B. atrophaeus and G.

Formaldehyde vapor cabinets also may be used in healthcare facilities to sterilize heat-sensitive medical equipment⁹⁵⁰. Commonly, there is no circulation of formaldehyde and no temperature and humidity controls. The release of gas from paraformaldehyde tablets (placed on the lower tray) is slow and produces a low partial pressure of gas. The microbicidal quality of this procedure is unknown.

Reliable sterilization using formaldehyde is achieved when performed with a high concentration of gas, at a temperature between 60^o and 80°C and with a relative humidity of 75 to 100%.

Studies indicate that formaldehyde is a mutagen and a potential human carcinogen, and OSHA regulates formaldehyde. The permissible exposure limit for formaldehyde in work areas is 0.75 ppm measured as a 8-hour TWA. The OSHA standard includes a 2 ppm STEL (i.e., maximum exposure allowed during a 15-minute period). As with the ETO standard, the formaldehyde standard requires that the employer conduct initial monitoring to identify employees who are exposed to formaldehyde at or above the action level or STEL. If this exposure level is maintained, employers may discontinue exposure monitoring until there is a change that could affect exposure levels or an employee reports formaldehyde-related signs and symptoms. The formaldehyde steam sterilization system has not been FDA cleared for use in healthcare facilities.

A gaseous chlorine dioxide system for sterilization of healthcare products was developed in the late 1980s. Chlorine dioxide is not mutagenic or carcinogenic in humans. As the chlorine dioxide concentration increases, the time required to achieve sterilization becomes progressively shorter. For example, only 30 minutes were required at 40 mg/l to sterilize the 10⁶ B. atrophaeus spores at 30^o to 32°C⁹⁵⁴. Currently, no gaseous chlorine dioxide system is FDA cleared.

The sporicidal activity of peracetic acid vapor at 20, 40, 60, and 80% relative humidity and 25°C was determined on Bacillus atrophaeus spores on paper and glass surfaces. Appreciable activity occurred within 10 minutes of exposure to 1 mg of peracetic acid per liter at 40% or higher relative humidity. No vaporized peracetic acid system is FDA cleared

An infrared radiation prototype sterilizer was investigated and found to destroy B. atrophaeus spores. Some of the possible advantages of infrared technology include short cycle time, low energy consumption, no cycle residuals, and no toxicologic or environmental effects. This may provide an alternative technology for sterilization of selected heat-resistant instruments but there are no FDA-cleared systems for use in healthcare facilities .

The other sterilization technologies mentioned above may be used for sterilization of critical medical items if cleared by the FDA and ideally, the microbicidal effectiveness of the technology has been published in the scientific literature. The selection and use of disinfectants, chemical sterilants and sterilization processes in the healthcare field is dynamic, and products may become available that are not in existence when this guideline was written. As newer disinfectants and sterilization processes become available, persons or committees responsible for selecting disinfectants and sterilization processes should be guided by products cleared by FDA and EPA as well as information in the scientific literature.