Validation and In-Process Monitoring of Sterilization Procedures

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Chapter: Pharmaceutical Microbiology : Sterilization Procedures And Sterility Assurance

There are several definitions of ‘validation’ but, in simple terms, the word means demonstrating that a process will consistently produce the results that it is intended to. Thus, with respect to sterile products, validation would be necessary for each of the individual aspects of the manufacturing process.



There are several definitions of ‘validation’ but, in simple terms, the word means demonstrating that a process will consistently produce the results that it is intended to. Thus, with respect to sterile products, validation would be necessary for each of the individual aspects of the manufacturing process, e.g. environmental monitoring, raw materials quality assessment, the sterilization process itself and the sterility testing procedure. Of these, it is the sterilization process that is likely to be subject to the most detailed and complex validation procedures, and these will be used to exemplify the factors to be considered. A typical validation procedure for a steam sterilization process is likely to incorporate most, or all, of the following features:


                              Calibration and testing of all the physical instruments used to monitor the process, e.g. thermocouples, pressure gauges and timers

                              Production of evidence that the steam is of the desired quality (e.g. that the chamber temperature is that expected for pure steam at the measured pressure)

              Conduct of leak tests and steam penetration tests using both an empty chamber and a chamber filled with the product to be sterilized in the intended load conformation

              Use of biological indicators either alone or in combination with bioburden organisms to demonstrate that the sterilization cycle is capable of producing an acceptable level of sterility assurance under ‘worst case’ conditions

              Production of data to demonstrate repeatability of the above (typically for three runs)

              Testing of software associated with parametric and operational monitoring

              Comprehensive documentation of all of these aspects.

There are different approaches to the demonstration of adequate sterility assurance in steam sterilization depending upon the thermostability and knowledge of the pre-sterilization bioburden. Where the product is known to be stable, an overkill approach may be adopted in which biological indicators  containing 106 test organisms are inactivated in half the proposed exposure time (thus achieving a 12-log reduction and a sterility assurance level of 10−6 in the full exposure period). For a marginally thermostable product the cycle could be validated on the basis of measurements of the worst case bioburden level and the heat resistance of the known bioburden organisms; such an approach would necessitate rigorous control of the bioburden during routine manufacturing. In the UK, biological indicators are used primarily in validation rather than routine monitoring of heat sterilization processes, although their use in routine manufacturing may be required in other countries. Chemical indicators of sterilization are more convenient to use than biological indicators, but as they provide no direct measure of the efficacy of the process in terms of microbial killing they are considered to be less useful. In certain instances these are no longer routinely used. Physical measurements of temperature, pressure, time, relative humidity, etc. are of such fundamental importance to the assurance of sterility that records of these parameters are retained for each batch of sterilized product.


a) Physical Indicators


In heat sterilization processes, a temperature record is made of each sterilization cycle with both dry and moist heat (i.e. autoclave) sterilizers; this chart/digital record forms part of the batch documentation and is compared against a master temperature record (MTR). It is recommended that the temperature be taken at the coolest part of the loaded sterilizer. Further information on heat distribution and penetration within a sterilizer can be gained by the use of thermocouples placed at selected sites in the chamber or inserted directly into test packs or bottles. For gaseous sterilization procedures, elevated temperatures are monitored for each sterilization cycle by temperature probes, and routine leak tests are performed to ensure gas-tight seals. Pressure and humidity measurements are recorded. Gas concentration is measured independently of pressure rise, often by reference to weight of gas used. In radiation sterilization, a plastic (often Perspex) dosimeter which gradually darkens in proportion to the radiation absorbed gives an accurate measure of the radiation dose and is considered to be the best technique currently available for following the radio sterilization process.


Sterilizing filters are subject to a bubble point pressure test, which is a technique employed for determining the pore size of filters, and may also be used to check the integrity of certain types of filter device (membrane and sintered glass) immediately after use. The principle of the test is that the wetted filter, in its assembled unit, is subjected to an increasing air or nitrogen gas pressure differential. The pressure difference recorded when the first bubble of gas breaks away from the filter is related to the maximum pore size. When the gas pressure is further increased slowly, there is a general eruption of bubbles over the entire surface. The pressure difference here is related to the mean pore size. A pressure differential below the expected value would signify a damaged or faulty filter. A modification to this test for membrane filters involves measuring the diffusion of gas through a wetted filter at pressures below the bubble point pressure (diffusion rate test); a faster diffusion rate than expected would again indicate a loss of filter integrity. In addition, a filter is considered ineffective when an unusually rapid rate of filtration occurs.


Efficiency testing of HEPA filters used for the supply of sterile air to aseptic workplaces  is normally achieved by the generation upstream of dioctylphthalate (DOP) or sodium chloride particles of known dimension followed by detection in downstream filtered air. Retention efficiency is recorded as the percentage of particles removed under defined test conditions. Microbiological tests are not normally done.


b)  Chemical Indicators


Chemical monitoring of a sterilization process is based on the ability of heat, steam, sterilant gases and ionizing radiation to alter the chemical and/or physical characteristics of a variety of chemical substances. Ideally, this change should take place only when satisfactory conditions for sterilization prevail, thus confirming that the sterilization cycle has been successfully completed. In practice, however, the ideal indicator response is not always achieved and so a necessary distinction is made between (1) those chemical indicators which integrate several sterilization parameters (i.e. temperature, time and saturated steam) and closely approach the ideal; and those which measure only one parameter and consequently can only be used to distinguish processed from unprocessed articles. Thus, indicators which rely on the melting of a chemical substance show that the temperature has been attained but not necessarily maintained.


Chemical indicators generally undergo melting or colour changes, the relationship of this change to the sterilization process being influenced by the design of the test device (Table 21.6). It must be remembered, however, that the changes recorded do not necessarily correspond to microbiological sterility and consequently the devices should never be employed as sole indicators in a sterilization process. Nevertheless, when included in strategically placed containers or packages, chemical indicators are valuable monitors of the conditions prevailing at the coolest or most inaccessible parts of a sterilizer.


c)  Biological Indicators


Biological indicators (BIs) for use in thermal, chemical or radiation sterilization processes consist of standardized bacterial spore preparations which are usually in the form either of suspensions in water or culture medium or of spores dried on paper, aluminium or plastic carriers. As with chemical indicators, they are usually placed in dummy packs located at strategic sites in the sterilizer. Alternatively, for gaseous sterilization these may also be placed within a tubular helix (Line–Pickerill) device. After the sterilization process, the aqueous suspensions or spores on carriers are aseptically transferred to an appropriate nutrient medium, which is then incubated and periodically examined for signs of growth. Spores of stearothermophilus in sealed ampoules of culture medium are used for steam sterilization monitoring, and these may be incubated directly at 55 °C; this eliminates the need for an aseptic transfer. Aseptic transfers are also avoided by the use of self-contained units where the spore strip and nutrient medium are present in the same device ready for mixing after use.


The bacterial species to be used in a BI must be selected carefully, as it must be non-pathogenic and should possess above-average resistance to the particular sterilization process. Resistance is adjudged from the spore destruction curve obtained upon exposure to the sterilization process; recommended BI spores and their decimal reduction times (D-values) are shown in Table 21.7. Great care must be taken in the preparation and storage of BIs to ensure a standardized response to sterilization processes. Indeed, while certainly offering the most direct method of monitoring sterilization processes, it should be realized that BIs may be less reliable monitors than physical methods and they are not recommended for routine use, except in the case of gaseous sterilization.


One of the long-standing criticisms of BIs is that the incubation period required in order to confirm a satisfactory sterilization process imposes an undesirable delay on the release of the product. This problem has been overcome, with respect to steam sterilization at least, by the use of a detection system in which a spore enzyme, α-glucosidase (reflective of spore viability), converts a non-fluorescent substrate into a fluorescent product in as little as 1 hour.


Filtration sterilization requires a different approach from biological monitoring, the test effectively measuring the ability of a filter to produce a sterile filtrate from a culture of a suitable organism. For this purpose, Serratia marcescens, a small Gram-negative rod-shaped bacterium (minimum dimension 0.5 μm), has been used for filters of 0.45 μm pore size, and a more rigorous test involving Brevundimonas diminuta (formerly Pseudomonas diminuta) having a minimum dimension of 0.3 μm is applied to filters of 0.22 μm pore size. The latter filters are defined as those capable of completely removing Brev. diminuta from suspension. In this test, using this organism, a realistic inoculum level must be adopted, as the probability of bacteria appearing in the filtrate rises as the number of Brev. diminuta cells in the test challenge increases; a standardized inoculum size of 107 cells cm−2 is normally employed. The extent of the passage of this organism through membrane filters is enhanced by increasing the filtration pressure. Thus, successful sterile filtration depends markedly on the challenge conditions. Such tests are used as part of the filter manufacturer’s characterization and quality assurance process, and a user’s initial validation procedure. They are not employed as a test of filter performance in use.


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