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APPENDIX 12 STERILIZATION AND STERILITY ASSURANCE

      Sterilization is the process of rendering an article, or product, free from viable micro-organisms. It may be effected by killing the micro-organisms by physical or chemical methods or by removing them by filtration. Wherever possible, a process in which the product is sterilized in its final container (terminal sterilization) is chosen. If terminal sterilization is not possible, filtration through a bacteria-retentative filter or aseptic processing is used.

      The method of attaining sterility in an article is determined by the nature of the product, the extent and type of any contamination present and the conditions under which the product has been prepared; it is assumed that the principles of good manufacturing practice will have been observed. Materials to be sterilized should be as free as possible from microbial contamination. The effect of the chosen sterilization process on the product (including its final container or package) should be validated before that procedure is applied in practice. Failure to follow a process meticulously involves the risk of a non-sterile or deteriorated product. Proper validation of the sterilization process or the aseptic process requires, however, a high level of knowledge of the field of sterilization and clean room technology. In order to comply with currently acceptable and achievable limits in sterilization parameters, it is necessary to employ appropriate instrumentation and equipment to control the critical parameters such as temperature and time, humidity, and sterilizing gas concentration, or absorbed radiation. An important aspect of the validation programme in many sterilization procedures involves the employment of biological indicators. The validated and certified process should be revalidated periodically; however, the revalidation programme need not necessarily be as extensive as the original programme.

      Within the strictest definition of sterility, an article would be deemed sterile only when there is complete absence of viable micro-organisms from it. However, this absolute definition cannot currently be applied to an entire lot of finished compendial 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 terms, where the 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, if any, under  appropriate current good manufacturing practice, and not by relying solely on sterility testing.

      Sterility assurance level (SAL) The SAL of a sterilizing process is the degree of assurance with which the process in question renders a population of items sterile. The SAL for a given process is expressed as the probability of a non-sterile item in that population. An SAL of 10^–6, for example, denotes a probability of not more than one viable micro-organism in 1 × 10⁶ sterilized items of the final product. The SAL of a process for a given product is established by appropriate validation studies.

Methods of Sterilization

      Methods of terminal sterilization, including removal of micro-organism by filtration, and aseptic processing are described. Modern technological developments, however, have led to the use of additional procedures. The choice of the appropriate process for a given dosage form or component requires a high level of knowledge of sterilization techniques and information concerning any effects of the process on the material being sterilized.

      Steam sterilization The process of thermal sterilization employing saturated steam under pressure is carried out in a chamber called an autoclave. It is probably the most widely employed sterilization process, especially for aqueous preparation. The basic principle of operation is that the air in the sterilizing chamber is displaced by the saturated steam, achieved by employing vents or traps. In order to displace air more effectively from the chamber and from within articles, the sterilization cycle may include air and steam evacuation stages.

      For this method of terminal sterilization, the reference conditions for aqueous preparations are heating at a minimum of 121º for 15 minutes. Other combinations of time and temperature may be used provided that it has been satisfactorily demonstrated that the process chosen delivers an adequate and reproducible level of lethality when operating routinely within the established tolerances. The procedures and precautions employed are such as to give an SAL of 10^–6 or better. A biological assessment of the process may be obtained by including a suitable biological indicator.

      With large batch sizes of aqueous preparations, it is essential to have knowledge of the physical conditions within the autoclave chamber during the sterilization procedure. To obtain this information, recording temperature-sensitive elements inserted into representative containers may be used together with additional elements at the previously-established coolest part of the loaded chamber. It is desirable that each sterilization cycle be recorded on a temperature-time chart. Other types of temperature indicator may be inserted at appropriate positions in the load but total reliance should not be placed on chemical indicators except when they suggest failure to attain sterilizing conditions. 

      When surgical dressings are sterilized by Steam sterilization, the steam used should not contain more than 5 per cent of entrained moisture. Most dressings are sterilized by maintaining at a temperature of 134º to 138º for 3 minutes, but other suitable combinations of temperature and time may be used, the conditions being chosen with regard to the stability of the dressings.

      Apart from that description of sterilization cycle parameters, using a temperature of 121º, the F₀ concept may be appropriate. The F₀, at a particular temperature other than 121º, is the time (in minutes) required to provide the lethality equivalent to that provided at 121º for a stated time.

      The total F₀ of a process takes account of the heating up and cooling down phases of the cycle and can be calculated by integration of lethal rates with respect to time at discrete temperature intervals.

      When a steam sterilization cycle is chosen on the basis of the F₀ concept, great care must be taken to ensure that an adequate assurance of sterility is consistently achieved. In addition to validating the process, it may also be necessary to perform continuous, rigorous microbiological monitoring during routine production to demonstrate that the microbiological parameters are within the established tolerances so as to given as SAL of 10^–6 or better.

      In connection with sterilization by steam, the Zvalue relates the heat resistance of a micro-organism to changes in temperature. The Z-value is the change in temperature required to alter the D-value by a factor of 10.

      The D-value (or decimal reduction value) is the value of a parameter of sterilization (duration or absorbed dose) required to reduce the number of viable organisms to 10 per cent of the original number. It is only of significance under precisely defined experimental conditions.

      The following mathematical relationships apply:

              F₀ = D₁₂₁(logN₀ – logN) = D₁₂₁log IF

where D₁₂₁ = D-value of the reference spores at 121º,

             N₀ = initial number of viable micro-organisms,

              N = final number of viable micro-organisms,

             IF = inactivation factor.

Z = (T₂ – T₁)/(logD₁ – logD₂)

where D₁ = D-value of the micro-organism at temperature T₁,

          D₂ = D-value of the micro-organism at temperature T₂.

IF = N₀ /N = 10^t/D

where t = exposure time,

D = D-value of micro-organism in the exposure conditions.

      Dry-heat sterilization Dry-heat sterilization may be used for heat stable non-aqueous preparations, powders and certain impregnated dressings. For this method of terminal sterilization the reference conditions are a minimum of 160º for at least 2 hours. Other combinations of time and temperature may be used provided that it has been satisfactorily demonstrated that the process chosen delivers an adequate and reproducible level of lethality when operated routinely within the established tolerances. The procedures and precautions employed are such as to give an SAL of 10^–6 or better. A modern oven is supplied with heated, filtered air, distributed uniformly throughout the chamber by convection or radiation and employing a blower system with devices for sensing, monitoring, and controlling the critical parameters.

      Appropriate biological indicators may be employed to demonstrate the effectiveness of the sterilization process. An example of a biological indicator for validating and monitoring dry-heat sterilization is a preparation of Bacillus subtilis spores. For heat-stable articles or components, the conditions of sterilization are not less than 250º. A microbial survival probability of 10^–12 is considered achievable for heat-stable articles or components. Since dryheat at 250º is frequently employed to render glassware or containers free from pyrogens as well as viable microbes, a pyrogen challenge, where necessary, should be an integral part of the validation program, e.g., by inoculating one or more of the articles to be treated with 1000 or more EU of bacterial endotoxin. The test with Limulus lysate could be used to demonstrate that the endotoxic substance has been inactivated to not more than 1/1000 of the original amount (3 log cycle reduction). For the test to be valid, both the original amount and, after acceptable inactivation, the remaining amount of endotoxin should be measured. For additional information on the endotoxin assay, see under the “Test for Bacterial Endotoxins” (Appendix 8.5).

      Gas sterilization This method of sterilization is only to be used where there is no suitable alternative. It is essential that penetration by gas and moisture into the material to be sterilized is ensured and that it is followed by a process of elimination of the gas under conditions that have been previously established to ensure that any residue of gas or its transformation products in the sterilized product is below the concentration that could give rise to toxic effects during use of the product. The active agent generally employed in gaseous sterilization is ethylene oxide of acceptable sterilizing quality or a mixture of ethylene oxide with a suitable inert gas.

      Wherever possible, the gas concentration, relative humidity, temperature and duration of the process are measured and recorded. Measurements are made where sterilization conditions are least likely to be achieved, as determined at validation.

      The effectiveness of the process applied to each sterilization load is checked using a suitable biological indicator. 

      Ionizing radiation sterilization Sterilization by this method is achieved by exposure of the product to ionizing radiation in the form of gamma radiation from a suitable radioisotopic source, such as cobalt-60 (⁶⁰Co) or of a beam of electrons energized by a suitable electron accelerator.

      For this method of terminal sterilization the reference absorbed dose is 25 kGy. Other doses may be used provided that it has satisfactorily been demonstrated that the dose chosen delivers an adequate and reproducible level of lethality when the process is operated routinely within the established tolerances. The procedures and precautions employed are such as to give an SAL of 10^–6 or better.

      During the sterilization procedure the radiation absorbed by the product is monitored regularly by means of established dosimetry procedures that are independent of dose rate. Dosimeters are calibrated against a standard source at a reference radiation plant on receipt from the supplier and at suitable intervals of not longer than one year thereafter.

      Where a biological assessment is carried out, this is obtained using a suitable biological indicator.

      Filtration Sterilization by Filtration may be used for certain medicaments and preparations which are not sufficiently stable to heat to allow sterilization by steam sterilization. Solutions or liquids may be sterilized by passage through a sterile bacteria-retaining filter of a type that has been demonstrated to be satisfactory by means of a microbial challenge test using a suitable test micro-organism. A suspension of Pseudomonas diminuta (ATCC 19146, NCIMB 11091 or CIP 103020) may be suitable. It is recommended that a challenge of at least 10⁷ CFU per cm² of active filter surface is used and that the suspension is prepared in Soybean-casein digest medium which, after passage through the filter, is collected aseptically and incubated aerobically at 30º to 35º. Such products need special precautions. The production process and environment are regularly subjected to appropriate monitoring procedures. The equipment, containers and closures and, wherever possible, the ingredients are subjected to an appropriate sterilization process. It is recommended that the filtration process be carried out as close as possible to the filling point. The operations following filtration are carried out under aseptic conditions.

      Filtration for sterilization is usually carried out with assemblies having membranes of porosity not greater than 0.22 μm; however, membranes of smaller porosities are also used and may be needed for some products. The types of membrane filter which are now available include cellulose acetate, cellulose nitrate, fluorocarbonate, acrylic polymers, polycarbonate, polyester, polyvinyl chloride, and even metal membranes, and they may be reinforced or supported by an internal fabric. A membrane filter assembly should be tested for integrity of the membrane and its effectiveness confirmed before and after use. A typical test is the bubble-point test, whereby it is determined that a prescribed pressure is necessary to force air bubbles through the intact membrane wetted with either product, water or hydrocarbon liquid.

Biological Indicators

      Biological indicators are standardized preparations of selected micro-organisms used to assess the effectiveness of a sterilization procedure. They usually consist of a population of bacterial spores placed on an inert carrier, for example a strip of filter paper, a glass slide or a plastic tube. The inoculated carrier is covered in such a way that it is protected from any deterioration or contamination, while allowing the sterilizing agent to enter into contact with the micro-organisms. Spore suspensions may be presented in sealed ampoules. Biological indicators are prepared in such a way that they can be stored under defined conditions; an expiry date is set. Micro-organisms of the same bacterial species as the bacteria used to manufacture the biological indicators may be inoculated directly into a liquid product to be sterilized or into a liquid product similar to that to be sterilized. In this case, it must be demonstrated that the liquid product has no inhibiting effect on the spores used, especially as regards their germination.

      A biological indicator is characterized by the name of the species of bacterium used as the reference microorganism, the number of the strain in the original collection, the number of viable spores per carrier and the D-value. The D-value is the value of a parameter of sterilization (duration or absorbed dose) required to reduce the number of viable organisms to 10 per cent of the original number. It is of significance only under precisely defined experimental conditions. Only the stated micro-organisms are present. Biological indicators consisting of more than one species of bacteria on the same carrier may be used. Information on the culture medium and the incubation conditions is supplied. It is recommended that the indicator organisms be placed at the locations presumed, or wherever possible, found by previous physical measurement to be least accessible to the sterilizing agent. After exposure to the sterilizing agent, aseptic technique is used to transfer carriers of spores to the culture media, so that no contamination is present at the time of examination. Biological indicators that include an ampoule of culture medium placed directly in the packaging protecting the inoculated carrier may be used.

      A choice of indicator organisms is made such that:

(a) the resistance of the test strain to the particular sterilization method is great compared to the resistance of all pathogenic micro-organisms and to that of micro-organisms potentially contaminating the product;

(b) the test strain is non-pathogenic;

(c) the test strain is easy to culture.

      After incubation, growth of the reference microorganisms subjected to the sterilization procedure demonstrates that this procedure is unsatisfactory.

      Steam sterilization The use of biological indicators intended for steam sterilization is recommended for the validation of sterilization cycles. Spores of Bacillus stearothermophilus (for example, ATCC 7953, NCTC 10007, NCIMB 8157 or CIP 52.81) are recommended. The number of viable spores exceeds 5 × 10⁵ per carrier. The D-value at 121º exceeds 1.5 minutes. It is verified that exposing the biological indicators to steam at 121º±1º for 6 minutes leaves revivable spores, and that there is no growth of the reference micro-organisms after the biological indicators have been exposed to steam at 121º±1º for 15 minutes.

      Dry-heat sterilization Spores of Bacillus subtilis (for example, var. niger ATCC 9372, NCIMB 8058 or CIP 77.18) are recommended for the preparation of biological indicators. The number of viable spores exceeds 1 × 10⁵ per carrier and the D-value at 160º is approximately 5 to 10 minutes. Dry heat at 250º is frequently used for sterilization and depyrogenation of glassware. In this case, demonstration of a 3 log reduction in heat resistant bacterial endotoxin can be used as a replacement for biological indicators.

      Gas sterilization The use of biological indicators is necessary for all gas sterilization procedures, both for the validation of the cycles and for routine operations. The number of viable spores exceeds 5 × 10⁵ per carrier. For hydrogen peroxide and peracetic acid spores of Bacillus stearothermophilus (for example ATCC 7953, NCTC 10007, NCIMB 8157 or CIP 52.81), for ethylene oxide and formaldehyde spores of Bacillus subtilis (for example, var. niger ATCC 9372, NCIMB 8058 or CIP 77.18) are recommended. The parameters of resistance are known for the procedure used: for example, for ethylene oxide, the D-value exceeds 2.5 minutes for a test cycle involving 600 mg per litre of ethylene oxide, at 54º and at 60 per cent relative humidity. It is verified that there is no growth of the reference micro-organisms after the biological indicators have been exposed to the test cycle described above for 60 minutes and that exposing the indicators to a reduced temperature cycle (600 mg per litre at 30º and 60 per cent relative humidity) for 15 minutes leaves revivable spores. It is essential that the biological indicator be able to reveal insufficient humidification in the sterilizer and the product to ensure dehydrated micro-organisms are inactivated. Exposing the indicators to 600 mg per litre of ethylene oxide at 54º for 60 minutes without humidification must leave revivable spores.

      Ionizing radiation sterilization Biological indicators may be used to monitor routine operations, as an additional possibility to assess the effectiveness of the set dose of radiation energy, especially in the case of accelerated electron sterilization. The spores of Bacillus pumilus (for example, ATCC 27.142, NCTC 10327, NCIMB 10692 or CIP 77.25) are recommended. The number of viable spores exceeds 1 × 10⁷ per carrier. The D-value exceeds 1.9 kGy. It is verified that there is no growth of the reference micro-organisms after the biological indicators have been exposed to 25 kGy (minimum absorbed dose).

Aseptic Processing

      While there is general agreement that sterilization of the final filled container as a dosage form or final packaged device is the preferred process for assuring the minimal risk of microbial contamination in a lot, there is a substantial class of products that are not terminally sterilized but are prepared by a series of aseptic steps. These are designed to prevent the introduction of viable micro-organisms into components, where sterile, or once an intermediate process has rendered the bulk product or its components free from viable microorganisms. A review of the principles involved in producing aseptically processed products with a minimal risk of microbial contamination in the finished lot of final dosage forms is hereby provided.

      A product defined as aseptically processed is likely to consist of components that have been sterilized by one of the processes described above. For example, the bulk product, if filterable, may have been sterilized by filtration. The final empty container components would probably be sterilized by heat, dry heat being employed for glass vials and an autoclave being employed for rubber closures. The areas of critical concern are the immediate microbial environment where these presterilized components are exposed during assembly to produce the finished dosage form and the aseptic filling operation.  

      The requirements for a properly designed, validated and maintained filling or other aseptic processing facility are mainly directed to (1) an air environment free from viable micro-organisms, of a proper design to permit effective maintenance of air supply units, and (2) the provision of trained operating personnel who are adequately equipped and gowned. The desired environment may be achieved through the high level of air filtration technology, which contributes to the delivery of air of the requisite microbiological quality. The facilities include both primary (in the vicinity of the exposed article) and secondary (where the aseptic processing is carried out) barrier systems.

      For a properly designed aseptic processing facility or aseptic filling area, consideration should be given to such features as nonporous and smooth surfaces, including walls and ceilings that can be sanitized frequently; gowning rooms with adequate space for personnel and storage of sterile garments; adequate separation of preparatory rooms for personnel from final aseptic processing rooms, with the availability where necessary of such devices as airlocks and/or air showers; proper pressure differentials between rooms, the most positive pressure being in the aseptic processing rooms or areas; the employment of laminar (unidirectional) air flow in the immediate vicinity of exposed product or components, and filtered air exposure there to, with adequate air change frequency; appropriate humidity and temperature environmental controls; and a documented sanitization programme. Proper training of personnel in hygienic and gowning techniques should be undertaken so that, for example, gowns, gloves, and other body coverings substantially cover exposed skin surfaces.

      Certification and validation of the aseptic process and facility is achieved by establishing the efficiency of the filtration systems, by employing microbiological environmental monitoring procedures, and by processing of sterile culture medium as simulated product.

      Monitoring of the aseptic facility should include periodic environmental filter examination as well as routine particulate and microbiological environmental monitoring, and may include periodic sterile culture medium processing.

Sterility Testing of Lots

      It should be recognized that the referee sterility test might not detect microbial contamination if present in only a small percentage of the finished articles in the lot because the specified number of units to be taken imposes a significant statistical limitation on the utility of the test results. This inherent limitation, however, has to be accepted since current knowledge offers no nondestructive alternatives for ascertaining the microbiological quality of every finished article in the lot, and it is not a feasible option to increase the number of sample s significantly.

      The primary means of supporting the claims that a lot of finished articles purporting to be sterile meets the specifications consist of the documentation of the actual production and sterilization record of the lot and of the additional validation records that the sterilization process possesses the capability of totally inactivating the established product microbial burden or a more resistant challenge. Further, it should be demonstrated that any processing steps involving exposed product following the sterilization procedure are performed in an aseptic manner, to prevent contamination. If data derived from the manufacturing process sterilityassurance validation studies and from inprocess controls are judged to provide greater assurance that the lot meets the required low probability of containing a contaminated unit (compared to sterility testing results from finished units drawn from that lot), any sterility test procedures adopted may be minimal, or dispensed with on a routine basis. However, assuming that all of the above production criteria have been met, it may still be desirable to perform sterility testing on samples of the lot of finished articles. Such sterility testing is usually carried out directly after the lot is manufactured as a final product quality control test. Sterility tests employed in this way in manufacturing control should not be confused with those described under the other section of “Sterility Test” (Appendix 10.1). The procedural details may be the same with regard to media, inocula and handling of samples, but the number of units and/or incubation time(s) selected for testing may differ. The number should be chosen relative to the purpose to be served, i.e., according to whether greater or lesser reliance is placed on sterility testing in the context of all the measures for sterility assurance in manufacture. Also, longer times of incubation would make the test more sensitive to slow-growing microorganisms. In the growth promotion tests for media, such slow growers, particularly if isolated from the product microbial burden, should be included with the other test strains.

      Negative or satisfactory sterility test results serve only as further support of the existing evidence concerning the quality of the lot if all of the pertinent production records of the lot are in order and the sterilizing or aseptic process is known to be effective. Unsatisfactory test results, however, in manufacturing quality control indicate a need for further action.

      Interpretation of quality control tests Quality control sterility tests (either according to the official referee test or modified tests) may be carried out in two separate stages in order to rule out false positive results.

      First stage Regardless of the sampling plan used, if no evidence of microbial growth is found, the results of the test may be taken as indicative of absence of intrinsic contamination of the lot.

      If microbial growth is found, proceed to the Second stage (unless the First stage test can be invalidated). Evidence for invalidating a First stage test in order to repeat it as a First stage test may be obtained from a review of the testing environment and the relevant records there to. Finding of microbial growth in negative controls need not be considered the sole grounds for invalidating a First stage test. When proceeding to the Second stage, particularly where depending on the results of the test for lot release, concurrently initiate and document a complete review of all applicable production and control records. In this review consideration should be paid to the following:

      a. A check on monitoring records of the validated sterilization cycle applicable to the product.

      b. Sterility test history relating to the particular product for both finished and in-process samples, as well as sterilization records of supporting equipment, con-tainers/closures, and sterile components, if any.

      c. Environmental control data, including those obtained from media fills, exposure plates, filtering records, any sanitization records and microbial monitoring records of operators, gowns, gloves, and garbing practices.

      Failing any lead from the above review, the current microbial profile of the product should be checked against the known historical profile for possible change. Records should be checked concurrently for any changes in source of product components and/or inprocessing procedures that might be contributory. Depending on the findings, and in extreme cases, consideration may have to be given to revalidation of the total manufacturing process.

      Second stage For the Second stage it is not possible to specify a particular number of sample to be taken for testing. It is usual to select double the number specified for the First stage under “Sterility Tests” (Appendix 10.1), or other reasonable number. The minimum volumes tested from each sample, the media, and the incubation periods are the same as those indicated for the First stage.

      If no microbial growth is found in the Second stage, and the documented review of appropriate records and the indicated product investigation does not support the possibility of intrinsic contamination, the lot may be considered to meet the requirements of a test for sterility. If growth is found, the lot fails to meet the requirements of the test. As was indicated for the First stage test, the Second stage test may similarly be invalidated with appropriate evidence, and, if so done, repeated as a Second stage test.