UNIT OPERATION / PRODUCTION PROCESS


Autoclaving and Sterilization

Steam sterilization or the exposure of microorganisms to saturated steam under pressure in an autoclave is done to achieve destruction by the irreversible denaturation of enzymes and structural proteins. The denaturation temperature varies inversely with the amount of water present, and so sterilization in saturated steam requires precise control of temperature, time, and pressure. The air should be evacuated from the autoclave before the admission of steam since the displacement of air by steam is unlikely to be readily achieved. This method is necessary for aqueous preparations, surgical dressings and medical devices.

The parameters for sterilization in an autoclave are 15-30 minutes, at 121-124 °C, at 100 kPa. The temperature should be used to control and monitor the process; the pressure is mainly used to obtain the required steam temperature. Alternative conditions with different time and temperature combinations are specified below.

Temperature (°C)

Approximate corresponding pressure (kPa)

Minimum sterilization time (min)

126-129

250  (-2.5 atm)

10

134-138

300 (-3.0 atm)

5

Measurement of the minimum sterilization time starts from the moment when all the materials to be sterilized have reached the required temperature. It is essential to monitor the physical conditions within the autoclave during sterilization, and the usage of temperature-monitoring probes inserted into representative containers;  with additional probes placed in the load at the potentially coolest part of the loaded chamber (as established in the course of the validation program) is used to provide the required information. Each cycle should be recorded on a time-temperature chart or by other suitable means wherein the conditions should be within ±2 °C and ±10 kPa (±0.1 atm) of the required values.

Aqueous solutions in glass containers usually reach thermal equilibrium within 10 minutes (for solutions up to 100 mL) and 20 minutes (for solutions up to 1000 mL). Surgical dressings and related porous products, should be processed in an apparatus that ensures steam penetration. Maintaining most dressings at a temperature of 134 - 138 °C for 5 minutes is enough for them to be adequately sterilized. In certain medical devices, glass, porcelain, or metal articles are sterilized at 121 - 124 °C for 20 minutes. Fats and oils may be sterilized at 121 °C for 2 hours but, whenever possible, should be sterilized by dry heat.

In certain cases, like thermolabile substances, sterilization may be carried out at temperatures below 121 °C, given that the chosen combination of time and temperature has been validated. Lower temperatures may be satisfactory if this is evaluated in combination with the known microbe of the material before sterilization. Specific conditions of temperature and time for certain preparations are stated in individual monographs. Individual monographs state different and specific conditions of temperature and time for certain preparations. The bioindicator strain indicated for the validation of this sterilization process, for which the D-value (i.e. 90% reduction of the microbial population) is 1.5-2 minutes at 121 °C, using about 106 spores per indicator are the spores of Bacillus stearothermophilus.

Dry-heat sterilization

The primary lethal process is considered to be the oxidation of cell constituents in a dry-heat process. Dry-heat sterilization requires a much higher temperature and a longer exposure time than moist heat sterilization. This method is determined to be more convenient for non-aqueous and heat-stable materials that cannot be sterilized with the use of steam. Examples of these materials include glassware, powders, oils, and some oil-based injectables.

Preparations to be sterilized by dry heat are sealed or temporarily closed for the process. Other conditions may be necessary for different preparations to ensure the effective removal of all unwanted microorganisms. The entire content in each container is maintained in the oven for the temperature and time stated below. Specific conditions of temperature and time for certain preparations are stated in individual monographs.

Temperature (°C)

Minimum sterilization time (min)

160

180

170

60

180

30

The oven where the process is being conducted should be validated for its defined specifications and should be normally equipped with a forced air system to ensure even distribution of heat throughout the whole process. Containers that have been temporarily closed during the sterilization procedure are sealed after sterilization using aseptic techniques to prevent microbial recontamination.

Filtration

Filtration sterilization uses bacteria-retaining filters such as membrane filters (cellulose derivatives, etc.), plastic, porous ceramic, or suitable sintered glass filters, or combinations of these and are employed mainly for heat labile pharmaceuticals. It should be noted that asbestos-containing filters should not be used.

Suitable filters should be used to avoid loss of solute by adsorption onto the filter and to prevent the release of contaminants from the filter, and to prevent the passage of microorganisms. The filtration must be then 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. If larger pore sizes are to be used, the effectiveness of the filtration must be first validated.

A bubble point or similar test should be used to confirm the integrity of the filters, both before and after filtration to follow the filter manufacturer’s instructions. This procedure measures the pressure needed to be applied to the upstream side of a filter causing bulk or open pore flow through the largest pores of a wetted filter.

The filters, tubes, and equipment to be used in the downstream process must be sterile. Filters that can stand 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, and must be then validated. The same filter must not be used for procedures lasting longer than one working day for filtration of a liquid in which microbial growth is possible.

Exposure to ionizing radiation

There are certain drug products, active ingredients, and medical devices that differs in ways of sterilizing them, and can be treated with procedures which can kill microorganisms and viruses by exposure to ionizing radiation, in the form of gamma radiation from a suitable radioisotopic source (cobalt 60) or electrons energized by a suitable electron accelerator. This can kill or cause mutations in human cells, as well as damages DNA and produces peroxides, which can act as powerful oxidizing agents in cells.

Mostly, gamma radiation and electron beams are used to effect ionization procedures in the molecules of organisms. Thus, mutations are formed in the DNA and these reactions alter replication that comes afterward. Only well-trained and experienced staff should decide upon the desirability of their use and should ensure monitoring of the processes for these processes are very dangerous. Specified equipment and specially designed, purpose-built installations should be used.

Specific dosimeters should be used upon monitoring the radiation doses during the entire process. They should be calibrated and validated against a standard source on receipt from a supplier at appropriate intervals, depending on its specifications. The radiation system should be reviewed and validated whenever the source material is changed and, in any case, at least once a year.

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

The bioindicator strains proposed for validation of this sterilization process are the spores of Bacillus pumilus 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 C1 A) are used.

Gas sterilization

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.

In gas sterilization, ethylene oxide or any other highly volatile substance is used as the active ingredient, which is highly flammable and has a potentially explosive nature. These properties can be a disadvantage unless they are mixed with suitable inert gases to reduce their toxic properties. The whole process is difficult to control which means only well-trained personnel should perform the process, and should only be considered if no other sterilization procedure can be used.

Factors influencing gas sterilization include time of exposure, gas concentration, penetration of the gas, and temperature and humidity in the sterilizing chamber. Automatically controlled ethylene oxide sterilizers are usually heated to a temperature of 54°C (130°F). A humidity level of 35 to 70 per cent is recommended.

The efficiency of ethylene oxide sterilization depends on the concentration of factors such as gas, humidity, time of exposure, temperature, and the nature of the material. It is also necessary to ensure that the nature of the packaging can withstand the gas exchange. Maintaining sufficient humidity and temperature is also important during sterilization. Appropriate sterilization conditions should be determined experimentally for each type of material.

When the sterilization process is done, time should be allowed for the elimination of residual sterilizing agents and other volatile residues which can be confirmed by specific tests. Efficiency must be monitored in each time using the proposed bioindicator: the spores of Bacillus subtilis or of Bacillus stearothermophilus since there is difficulty in controlling the process. The same quantity should also be used in sterilizing in an autoclave (steam sterilization) or an oven (dry-heat sterilization).