Sterility or Sterilization Assurance BY Piyush Tripathi

Sterility or Sterilization Assurance Sterility assurance, a specimen deemed sterile only when there is complete absence of viable microorganisms from it. This absolute definition cannot be applied to an entire lot of finished compendia articles because of limitations in testing. Absolute sterility cannot be practically demonstrated without complete destruction of every finished article. The sterility of a lot purported to be sterile is therefore defined in probabilistic reasoning terms, where likelihood of a contaminated unit or article is acceptably remote. Such a state of sterility assurance can be established only through the use of adequate sterilization cycles and subsequent aseptic processing. The basic principle of sterilizing process with reference to USP is as follows. 1. Establish that the process equipment has capability of operating within the required parameters. 2. Demonstrate that the critical control equipment and instrumentation are capable of operating within the prescribed parameters for the process equipment. 3. Perform replicate cycles representing the required operational range of the equipment and employing actual or simulated product. Demonstrate that the processes have been carried out within the prescribed protocol limits and finally that the probability of microbial survival in the replicate processes completed is not greater than the prescribed limits. 4. Monitor the validated process during routine operation. Periodically as needed, re-qualify and recertify the equipment. 5. Complete the protocols, and document steps (1) through (4) above. Aseptic process or sterilization process requires a complete knowledge of the field of sterilization and clean room technology. It is important to employ appropriate instrumentation and equipment to control critical parameters such as temperature, time, humidity and sterilization monitoring controls in order to comply with currently acceptable and achievable limits in sterilization parameters. All processes are required to be timely revalidated extensively as the original program. An important aspect of validation involves biological indicators. Bacterial spores are the most resistant of all living organisms because of their capacity to withstand external destructive agents. Although the physical or chemical process by which all pathogenic and non-pathogenic microorganisms, including spores, are destroyed is not absolute, supplies, product and equipments are considered sterile when necessary conditions have been met during a sterilization process. Reliable sterilization depends on contact of the sterilizing agent with all surfaces of the product / item to be sterilized. Selection of the agent to achieve sterility depends primarily upon the nature of the product/item to be sterilized. Time required to kill spores in the equipment available for the process then becomes critical. Sterilization Methods: Steam Sterilization Heat destroys microorganisms, but this process is hastened by the addition of moisture. Steam in itself is inadequate for sterilization. Pressure, greater than atmospheric, is necessary to increase the temperature of steam for thermal destruction of microbial life. Death by moist heat in the form of steam under pressure is caused by the denaturation and coagulation of protein or the enzyme-protein system within the cells. These reactions are catalyzed by the presence of water. Steam is water vapour; it is saturated when it contains a maximum amount of water vapour. Direct saturated steam contact is the basis of the steam process. Steam, for a specified time at required temperature, must penetrate every fibber and reach every surface of items to be sterilized. When steam enters the sterilizer chamber under pressure, it condenses upon contact with cold items. This condensation liberates heat, simultaneously heating and wetting all items in the load, thereby providing the two requisites: moisture and heat. No living thing can survive direct exposure to saturated steam at 250 F (120 C) longer than 15 minutes. As temperature is increased, time may be decreased. A minimum temperature-time relationship must be maintained throughout all portions of load to accomplish effective sterilization. Exposure time depends upon size and contents of load, and temperature within the sterilizer. At the end of the cycle, re-evaporation of water condensate must effectively dry contents of the load to maintain sterility. Ethylene Oxide Sterilization Ethylene oxide is used to sterilize products/items that are heat or moisture sensitive. Ethylene oxide (EO) is a chemical agent that kills microorganisms, including spores, by interfering with the normal metabolism of protein and reproductive, processes, (alkylation) resulting in death of cells. Used in the gaseous state, EO gas must have direct contact with microorganisms on or in products/items to be sterilized. Because EO is highly flammable and explosive in air, it must be used in an explosion-proof sterilizing chamber in a controlled environment. When handled properly, EO is a reliable and safe agent for sterilization, but toxic emissions and residues of EO present hazards to personnel and patients. Also, it takes longer than steam sterilization, typically, 16-18 hrs. to complete the sterilization cycle. EO gas sterilization is dependent upon four parameters: EO gas concentration, temperature, humidity, and exposure time. Each parameter may be varied. Consequently, EO sterilization is a complex multi-parameter process. Each parameter affects the other dependent parameters. Others Sterilization Processes: Dry Heat Sterilization Dry heat in the form of hot air is used primarily to sterilize anhydrous oils, petroleum products, and bulk powders that steam and ethylene oxide gas cannot penetrate. Death of microbial life by dry heat is a physical oxidation or slow burning process of coagulating the protein in cells. In the absence of moisture, higher temperatures are required than when moisture is present because microorganisms are destroyed through a very slow process of heat absorption by conduction. Microwaves The nonionizing radiation of microwaves produces hyperthermic conditions that disrupt life processes. This heating action affects water molecules and interferes with cell membranes. Microwave sterilization uses low-pressure steam with the nonionizing radiation to produce localized heat that kills microorganisms. The temperature is lower than conventional steam, and the cycle faster, as short as 30 seconds. Metal instruments can be sterilized if placed under a partial vacuum in a glass container. Small tabletop units may be useful for flash sterilizing a single or small number of instruments, when technology is developed for widespread use. Formaldehyde Gas Formaldehyde kills microorganisms by coagulation of protein in cells. Used as a fumigant in gaseous form, formaldehyde sterilization is complex and less efficacious than other methods of sterilization. It should only be used if steam under pressure will damage the item to be sterilized and ethylene oxide and glutaraldehyde are not available. Its use for sterilization has been almost abandoned in the United States, Canada, and Australia. The method dates back to 1820, and it is still used in Europe and Asia with is also slowly abandoning the process. Hydrogen Peroxide Plasma Hydrogen peroxide is activated to create a reactive plasma or vapor. Plasma is a state of matter distinguishable from solid, liquid, or gas. It can be produced through the action of either a strong electric or magnetic field, somewhat like a neon light. The cloud of plasma created consists of ions, electrons, and neutral atomic particles that produce a visible glow. Free radicals of the hydrogen peroxide in the cloud interact with the cell membranes, enzymes, or nucleic acids to disrupt life functions of microorganisms. The plasma and vapor phases of hydrogen peroxide are highly sporicidal even at low concentrations and temperature. Ozone Gas Ozone, a form of oxygen, sterilizes by oxidation, a process that destroys organic and inorganic matter. It penetrates membrane of cells causing them to explode. Ozone is an unstable gas, but can be easily generated from oxygen. A generator converts oxygen, from a source within the hospital, to ozone. A 6 to 12 percent concentration of ozone continuously flows through the chamber. Penetration of ozone may be controlled by vacuum in the chamber, or enhanced by adding humidity. At completion of exposure time, oxygen is allowed to flow through chamber to purge the ozone. Cycle time may be up to 60 minutes depending on the size of the chamber or load. Chemical Solutions Liquid chemical agents registered by the EPA as sterilants provide an alternative method for sterilizing heat sensitive items if a gas or plasma sterilizer is not available, or the aeration period makes ethylene oxide sterilization impractical. To sterilize items, they must be immersed in a solution for the required time specified by the manufacturer to be sporicidal. All chemical solutions have advantages and disadvantages; each sterilant has specific assets and limitations. These chemicals are: peracetic acid, glutaraldehyde, and formaldehyde. Ionizing Radiation Some products commercially available are sterilized by irradiation. It is the most effective sterilization method but is limited for commercial use only. Ionizing radiation produces ions by knocking electrons out of atoms. These electrons are knocked out so violently that they strike an adjacent atom and either attach themselves to it, or dislodge an electron from the second atom. The ionic energy that results becomes converted to thermal and chemical energy. This energy causes the death of microorganisms by disruption of the DNA molecule, thus preventing cellular division and propagation of biologic life. The principal sources of ionizing radiation are beta particles and gamma rays. Beta particles, free electrons, are transmitted through a high-voltage electron beam from a linear accelerator. These high-energy free electrons will penetrate into matter before being stopped by collisions with other atoms. Thus, their usefulness in sterilizing an object is limited by density and thickness of the object and by the energy of the electrons. They produce their effect by ionizing the atoms they hit, producing secondary electrons that, in turn, produce lethal effects on microorganisms. Cobalt 60 is a radioactive isotope capable of disintegrating to produce gamma rays. Gamma rays are electromagnetic waves. They have the capability of penetrating to a much greater distance than beta rays before losing their energy from collision. Because they travel with the speed of light, they must pass through a thickness measuring several feet before making sufficient collisions to lose all of their energy. Cobalt 60 is the most commonly used source for irradiation sterilization. The product is exposed to radiation for 10 to 20 hours, depending on the strength of the source. Establishing Sterility Assurance A typical Validation program, as outlined below is one designed for steam autoclave, but the principles are applicable to other sterilization procedures. The installation qualification stage to establish the controls and other instrumentation check properly designed and calibrated or not. Documentation should be on file demonstrating the quality of the required inputs or feeds Utilities Viz. Steam, Water and Air. The operational qualification confirms that the empty chamber functions within the parameters of temperature at all of the key locations prescribed. It is appropriate to develop heat profile records, i.e. simultaneous temperatures in the chamber employing multiple temperature sensing devices. A typical acceptable range of temperature in the empty chamber if + 1o when the chamber temperature is not less than 121o. The confirmatory stage of the validation is actual sterilization of product or material. This determination requires the employment of temperature sensing devices inserted into samples of the articles, as well as either samples of the articles to which appropriate concentrations of suitable test microorganisms have been added. The effectiveness of heat delivery or penetration into the actual articles and the time of the exposure are the two main factors that determine the level or lethality of the sterilization process. The final stage of validation program requires the documentation of the supporting data developed in executing the program. It is generally accepted that terminally sterilized injectable articles or critical devices purporting to be sterile, when processed in the autoclave, attain a 10–6 microbial survivor probability, i.e., assurance of less than 1 chance in 1 million that viable microorganisms are present in the sterilized article or dosage form. With heat-stable articles, the approach often is to considerably exceed the critical time necessary to achieve the 10–6 microbial survivor probability (overkill). However, with an article where extensive heat exposure may have a damaging effect, it may not be feasible to employ this overkill approach. In this latter instance, the development of the sterilization cycle depends heavily on knowledge of the microbial burden of the product, based on examination, over a suitable time period, of a substantial number of lots of the presterilized product.