Sterile og aseptisk fremstilte legemidler skal oppbevares og utleveres i beholdere eller emballasje som beskytter mot mikrobiell kontaminasjon; salver skal, når det er mulig, oppbevares og utleveres i tuber.
Methods of Preparation of Sterile Products (Ph. Eur. 5.1.1.)
Sterility is the absence of viable micro-organisms, as defined by a sterility assurance level equal to or less than 10-6. Sterility is a critical quality attribute for a wide variety of human and veterinary preparations, including but not restricted to:
- preparations required to be sterile due to their route of administration, such as parenteral, ophthalmic and intramammary preparations, and some inhalation, irrigation and intrauterine preparations;
- preparations applied to severely injured skin, such as semi-solid preparations for cutaneous application
The achievement of sterility for any one item in a population of items submitted to a sterilisation process can neither be guaranteed nor demonstrated. It is essential to study the effect of the chosen sterilisation procedure on the product (including its final container) to ensure its effectiveness and the integrity of the product, and to validate the procedure before it is applied in practice. Failure to follow meticulously a validated process introduces the risk of a non-sterile and/or deteriorated product.
Sterile products are prepared under appropriate conditions and are packed in suitable containers. It is recommended that the choice of container permits application of the optimum sterilisation process for the product. The container and closure system are required to maintain the sterility of the product throughout its shelf life.
Sterilisation process conditions are chosen to achieve the highest level of sterility assurance compatible with the drug product and, wherever possible, a process in which the product is sterilised in its final container (terminal sterilisation) is chosen. When a fully validated terminal sterilisation method by steam (moist heat), dry heat or ionising sterilisation is used, parametric release (i.e. the release of a batch of sterilised items based on process data rather than submission of a sample of the items to sterility testing) may be carried out, subject to the approval of the competent authority. If terminal sterilisation is not possible, aseptic assembly or filtration through a bacterial retentive filter is used. Wherever possible, an appropriate additional treatment (e.g. heating) of the product in its final container is applied to further ensure the sterility assurance level.
Requirements for the use of biological indicators for validation of sterilisation processes are given in general chapter 5.1.2.
The present general chapter provides guidance on conditions, validation and control of sterilisation processes. The methods described here apply mainly to the inactivation or removal of bacteria, yeasts and moulds. For biological products of animal or human origin, or in cases where such material has been used in the production process, it is necessary to demonstrate during validation that the process is capable of the removal or inactivation of any relevant viral contamination. Further guidance is provided in general chapter 5.1.7. Viral safety.
The efficacy of a sterilisation process is dependent on its nature, the processing conditions (e.g. time, temperature, moisture), the pre-sterilisation microbial contamination and the formulation of the product. The inactivation of micro-organisms by physical or chemical means follows an exponential law and hence there is always a non-zero probability that a micro-organism may survive the sterilisation process.
Sterility assurance level (SAL)
In the methods described, reference is made to a sterility assurance level (SAL) where appropriate. The SAL for a given sterilisation process is expressed as the probability of micro-organisms surviving in a product item after exposure to the process. An SAL of 10-6, for example, denotes a probability of not more than 1 non-sterile item in 1 x 106 sterilised items of the final product. The SAL of a process for a given product is established by appropriate validation studies. Microbial contamination may be described by the number, type and resistance of any micro-organisms present. Microbiological monitoring and setting of suitable limits is therefore essential for all components of sterile preparations. Steps designed to reduce microbial contamination, such as filtration prior to sterilisation, will contribute significantly to sterility assurance. The composition of a product can affect the behaviour of micro-organisms present in the product, which in turn can affect the efficacy of the sterilisation process. The water activity (Aw), the pH and the presence of compounds with antimicrobial activity are examples of factors that can influence the resistance of any micro-organisms present. The water activity or the product formulation (including the presence of nutrients) can affect the number of micro-organisms, which in turn can affect the efficacy of the membrane-filtration process.
Methods and Conditions of Sterilisation
Sterilisation may be carried out by one of the methods described hereafter. Modifications to, or combinations of, these methods may be used, provided that the chosen procedure is validated with respect both to its effectiveness and to the integrity of the product including its container. For all sterilisation methods, the critical parameters of the procedure are monitored in order to confirm that any previously determined requirements or conditions are respected throughout the batch during the entire sterilisation process. This applies in all cases, including those where the reference conditions are used. Guidance concerning validation of a steam sterilisation process using the F0 concept is described in general chapter 5.1.5. Biological indicators of sterilisation are used to develop and validate sterilisation processes and also to monitor gas sterilisation processes. Guidance on the use of biological indicators is provided in general chapter 5.1.2.
Precautions shall be taken to prevent contamination of the sterilised articles after the sterilisation phase.
Steam sterilisation is achieved by heat transfer during condensation of water from a saturated vapour phase on the surface of the sterilised items. Where items (open or wrapped) are sterilised in direct contact with steam, the hydrating effect of the condensate adds to the sterilising effect. For direct steam exposure, it is essential that the items are fully penetrated by saturated steam, i.e. free of air and other non-condensable gases. Where items are sterilised in closed-containers, the chamber of the steriliser serves as a steam jacket. Condensation on the surface of the containers still serves as a highly effective mechanism for energy transfer, but has no additional sterilising effect on its own. In closed-container sterilisation, the sterilising effect is determined by the conditions reached within the closed containers, where sterilisation must be achieved in the product itself and in the head-space.
Steam sterilisation is performed in autoclaves, i.e. pressure vessels designed to admit or generate steam continuously and to remove condensate from the chamber to maintain the pressure and temperature at controlled levels.
For equipment used to perform direct steam exposure cycles, the supply of saturated steam, free of non-condensable gases, is assured. In autoclaves intended for the sterilisation of closed containers, steam-air mixtures or superheated water spray can be used to achieve heat transfer. Suitable autoclaves are qualified to achieve homogeneous conditions within the chamber and the load. The principles of operation are appropriate for the items to be sterilised and the loading configuration. The suitability of the equipment for the items to be sterilised and its performance in the chosen cycle is demonstrated in autoclave performance qualification studies. Temperature profiles in the slowest-to-heat items are recorded.
Suitable autoclaves are equipped with temperature and pressure sensors of appropriate sensitivity that are placed in relevant positions to ensure effective process control. Chamber temperature and pressure profiles are recorded for each cycle. There is at least 1 independent thermal probe that controls the load temperature at the slowest-to-heat position or in the slowest-to-heat closed container of the load.
Cooling water sprayed into the chamber at the end of a sterilisation process for closed containers is of sufficient quality not to impact negatively the sterility of the sterilised items.
Suitable sterilisation cycles are chosen to be compatible with the items to be sterilised and the loading configuration. Where air is displaced from the chamber by gravity, the items to be autoclaved are designed to allow the removal of air and are arranged within the autoclave to prevent the formation of inaccessible air pockets. Where air is removed by vacuum cycles followed by steam pulses, it is assured that the items are not affected by the evacuation process. For pressure-sensitive products in closed containers, saturated steam sterilisation may not be possible. Steam-air mixtures may be applied to the chamber in order to balance pressure conditions inside the closed containers. Steam penetration is assured by choosing suitable cycles to remove air from porous loads or hollow bodies. Steam penetration is verified during cycle development by, for example, the use of physical/chemical indicators, while the biological effectiveness of the cycle is verified by the use of biological indicators (5.1.2). Appropriate loading patterns are specified.
The reference cycle for steam sterilisation is 15 min at 121 °C in saturated steam determined in the coldest position of the chamber. Product- and load-specific cycles, e.g. applying another combination of time and temperature, may be adopted based on cycle development and validation. The minimum temperature acceptable for a steam sterilisation process is 110 °C. The minimum F0 calculated in the slowest-to-heat position of the load is not less than 8 min. The calculation of sterilisation effectiveness by the F0 concept is performed according to general chapter 5.1.5.
Calculated effectiveness from physical parameters (Fphys) is correlated with biological effectiveness (Fbio). Fbio expresses the lethality, in minutes, provided by the process in terms of destruction of biological indicators used. Fbio is calculated by the following equation:
Fbio = D121 (log10 N0 – log10 N)
D121 is the D-value of the biological indicator at an exposure temperature of 121 °C, N0 is the number of viable micro-organisms in the biological indicator before exposure, and N is the number of viable micro-organisms in the biological indicator after exposure.
In cycle validation, the relevant positions in the load that are the most difficult to sterilise are determined and adequate biological effectiveness is verified by exposure of biological indicators (5.1.2) in these positions or products, whichever is relevant. Protection of spores from the sterilising effect (e.g. by physical occlusion of steam or by the protective properties of the product) are suitably addressed. The Fbio, determined for the most-difficult-to-sterilise position is used to define the parameters necessary to achieve reliably the required SAL equal to or less than 10-6 for the chosen cycle.
Autoclave cycles are monitored by physical determination of chamber pressure and temperature profiles, at a minimum, in the coldest position of the chamber. For each cycle, pressure, time and temperature are recorded and, if possible, F0 is calculated and recorded.
Dry heat sterilisation
Dry heat sterilisation is a terminal sterilisation method based on the transfer of heat to the articles to be sterilised. Heat may be transferred by means of convection, radiation or direct transfer.
Dry heat sterilisation is carried out in an oven with forced air circulation or using other equipment specifically designed for this purpose, e.g. a tunnel.
The steriliser is loaded in such a way that the specified or required temperature is achieved throughout the load. Knowledge of the temperature within the steriliser during the sterilisation cycle is obtained by means of temperature-sensing elements suitably placed in or on representative items situated in the coolest part (as previously established) of the loaded steriliser. The time and temperature throughout each cycle is suitably recorded.
The reference conditions for this method of sterilisation are a minimum of 160 °C for at least 2 h. Other combinations of time and temperature may be used if it has been satisfactorily demonstrated that the process chosen delivers an adequate and reproducible level of lethality when operated within the established tolerances. The procedures and precautions employed are such as to achieve an SAL equal to or less than 10-6. Dry heat sterilisation processes are validated using a combination of temperature mapping and biological indicator studies (5.1.2).
Dry heat at temperatures greater than 220 °C, for a validated time, is frequently used for depyrogenation of glassware. In this case, demonstration of a 3 log10 reduction in heat-resistant endotoxin can be used as validation criteria and biological indicators will not be needed.
Dry heat sterilisation cycles are monitored by determination of temperature profiles, at a minimum, in the coldest position of the chamber. Time and temperature are recorded for each cycle.
Ionising radiation sterilisation
Sterilisation by irradiation is achieved by exposure of the product to ionising radiation in the form of either gamma rays from a suitable isotopic source (such as cobalt 60), a beam of electrons energised by a suitable electron accelerator, or X-rays resulting from bombarding a suitable target with energised electrons. Ionising radiation may be used for the terminal sterilisation of finished dosage forms, the microbial inactivation of tissues and cells, or the sterilisation of materials or containers to be employed in aseptic processing. Low-energy electrons may be used for the surface sterilisation of materials upon entry to isolators used in the preparations of sterile products.
For this method of sterilisation, the reference absorbed dose is 25 kGy. Other doses may be used if, during validation of the sterilising dose, it has been satisfactorily 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 achieve an SAL equal to or less than 10-6. Biological indicators may be required for the development and validation of the sterilisation of tissues and cell products. They may also be required for products with a potential to prevent spore inactivation.
During the sterilisation process, the sterilisation dose delivered is monitored using a dosimetry system, measurements from which are traceable to national standards.
Gas sterilisation (Vapor phase sterilisation)
Gas sterilisation of surfaces may be used for the sterilisation of primary packaging materials, equipment and some pharmaceuticals.
It is essential that penetration by gas and moisture into the material to be sterilised is ensured, and that it is followed by a process whereby the gas is eliminated under conditions that have been previously established as sufficient to ensure that any residues of gas or related transformation by-products are below concentrations that could give rise to toxic effects during product use.
There are 2 main categories of gaseous sterilising agents as distinguished by their antimicrobial action: alkylating agents and oxidising agents.
Alkylating agents. Alkylating agents are highly reactive compounds and interact with many components, such as amino, sulfhydryl and hydroxyl groups in proteins and purine bases in nucleic acids.
Ethylene oxide is an alkylating agent that is associated with cytotoxic, carcinogenic and mutagenic effects.
Oxidising agents. Oxidising agents are highly reactive, toxic compounds. Such compounds currently used as sterilising agents include hydrogen peroxide and peracetic acid.
Development and validation of sterilisation processes
Gas sterilisation is performed by exposure of the product to the sterilising agent in a leak-proof chamber under specified conditions.
A typical gas sterilisation process consists of 3 phases:
(pre)conditioning, sterilisation and aeration. The parameters necessary for these phases to produce the required SAL are established during process development. A combination of physical and biological methods is used to determine the optimum sterilisation conditions. The cycle shall not compromise the functionality of either product or the container.
Specialised equipment may be required for the monitoring of temperature, humidity and gas concentration during both validation and routine operation.
Validation of microbiological performance shall confirm the effectiveness of the defined process for the product/load combination in the steriliser. The lethality of the cycle may be determined by using an appropriate approach: after time-graded exposures, the rate of inactivation (D-value) of the test organisms can be established by construction of a survivor curve or by using a fraction-negative method.
Biological indicators shall be shown to be, at a minimum, as resistant to the sterilising agent as the microbiological contaminants of the product to be sterilised. They shall be placed within the product at locations where sterilising conditions are most difficult to achieve.
The effectiveness of the process is dependent on a number of parameters, including gas concentration, temperature, humidity, exposure time, load configuration and characteristics of the product and its packaging materials. The effect on the process effectiveness of any change in one or more of these parameters shall be investigated.
The relevant cycle process parameters (including the results of the biological indicator test) are recorded.
Membrane filtration is used for reduction of viable and non-viable particles in gases and fluid products that are not amenable to sterilisation by heat or irradiation. In contrast to other sterilisation methods, the principle of membrane filtration is not inactivation but removal of microorganisms from the product. Removal is achieved by a combination of sieving and surface interaction.
Membrane filters are available as flat stock (discs) in appropriate holders or as cartridges. Pore size ratings are based on the correlation between microbial retention and diffusion characteristics or bubble-point measurement. Many factors contribute to the effectiveness of the filtration process, e.g. shape, pore size, structure, surface properties, the structure and arrangement of the filter unit, interaction of the filter matrix with the product, applied pressure, flow and duration of the process. Filter characteristics have to be determined in a product-specific validation. Suitable integrity test procedures (e.g. diffusive flow measurement, bubble-point determination or water-intrusion testing) are employed, as recommended by filter manufacturers. Chemical and physical compatibility of the membranes with the product to be filtered and the conditions of the filtration process are demonstrated in development studies. The filter size is suitable for the volume of the product to be filtered and the bioburden.
For sterilisation of process gases, an appropriate frequency for physical integrity testing is established.
Microbial challenge tests with a suitable model system shall demonstrate the effectiveness of the filtration process. Where testing with the product is not possible (e.g. due to the antimicrobial properties of the product), a fluid that is representative of the product shall be used, or the test conditions are modified.
It is recommended that the filtration process is carried out as close as possible to the filling point.
Sterilisation of membrane filters
Membrane filters may be sterilised off-line or in-line. If sterilisation is off-line, steam penetration is verified and the filter is suitably protected against contamination. The sterilised filter is aseptically assembled in the production line by means of a validated procedure. For in-line sterilisation, steam penetration throughout the filtration equipment is assured and the pressure difference across the membrane is controlled to prevent damage to the membrane itself.
Sterilisation by membrane filtration is performed by passage of the product through a microporous membrane with a nominal pore size not greater than 0.22 μm.
The pre-sterilisation microbial contamination is determined for each batch of product and process parameters are applied as established and validated in the development of the filtration process.
Where multiple bioburden-reduction filters are used to increase the efficacy of the filtration process, the filter closest to the filling point in the final container is characterised as the sterilising filter
The sterility and integrity of the equipment downstream from the point of filtration, the qualified environmental conditions and the validated aseptic procedures applied in the handling of the filtered product all contribute to preventing recontamination of the product. This is addressed in the section on aseptic assembly.
Filtration processes are monitored by physical and microbiological determination of parameters established during validation studies. These parameters include the following: pre-sterilisation microbiological contamination, pre-filtration integrity test results, duration of filtration, volume filtered, differential pressure and post-filtration integrity test results.
The objective of aseptic assembly is to maintain the sterility of a product that is assembled from components, each of which has been sterilised by one of the above methods. This is achieved by using conditions and facilities designed to prevent microbial contamination.
Aseptic processing may include aseptic filling of products into container/closure systems, freeze-drying under aseptic conditions, aseptic blending of formulations followed by aseptic filling, and aseptic packaging.
Development and validation of aseptic assembly
In order to maintain the sterility of the components and the product during assembly, careful attention needs to be given to the following:
- critical surfaces;
- container/closure sterilisation and transfer procedures;
- the maximum holding period of the product before filling into the final container.
Process validation includes appropriate checks on all of the above and also regular checks on the process, which are carried out by means of process simulation tests using microbial growth media that are then incubated and examined for microbial contamination (media fill tests). In addition, a suitable sample of each batch of any product that is aseptically processed is tested for sterility (2.6.1).
Biological Indicators and related Microbial Preparations used in the Manufacture of Sterile Products
Se Ph. Eur. 5.1.2.
Application of the F0 Concept to Steam Sterilisation of aqueous Preparations
Se Ph. Eur. 5.1.5.
Guidelines for Using the Test for Sterility
Se Ph. Eur. 5.1.9.