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Appendix XVIII Methods of Sterilisation

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Methods of Sterilisation

(Methods of Preparation of Sterile Products, Ph. Eur. general texts 5.1.1)

GENERAL INTRODUCTION

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 × 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

Principle

Steam sterilisation is achieved by heat transfer during condensation of water from a saturated vapour phase on the surfaces 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.

Equipment

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 a 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.

Sterilisation cycle

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.

Cycle effectiveness

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 the biological indicators used. Fbio is calculated by the following equation:

F bio = 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.

Routine control

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

Principle

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.

Equipment

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.

Sterilisation cycle

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.

Cycle effectiveness

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.

Routine control

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

Principle

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 preparation of sterile products.
Cycle effectiveness

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.

Routine control

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)

Principle

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.

Sterilising agents

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.

Sterilisation cycle

Specialised equipment may be required for the monitoring of temperature, humidity and gas concentration during both validation and routine operation.

Cycle effectiveness

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.

Routine control
The relevant cycle process parameters (including the results of the biological indicator test) are recorded.

MEMBRANE FILTRATION

Principle

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.

Equipment

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.

Filtration effectiveness

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.

Filtration process

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.

Routine control

Filtration processes are monitored by physical and microbiological determination of parameters established during validation studies. These parameters
include the following: pre-sterilisation microbial contamination, pre-filtration integrity test results, duration of filtration, volume filtered, differential pressure
and post-filtration integrity test results.

ASEPTIC ASSEMBLY
Principle

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:

— environment;

— personnel;

— 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

(Biological Indicators of Sterilisation, Ph. Eur. general texts 5.1.2)

1 INTRODUCTION

The use of biological indicators in this general chapter is intended to cover the sterilisation of finished products and relevant related sterilisation processes
i.e. sterilisation processes for items coming into direct contact with the final sterilised product. Other uses of biological indicators to validate the sterilisation
of other non-terminal units is outside the scope of this general chapter.

Biological indicators are test systems containing viable micro-organisms (usually spores of bacteria) that provide a defined challenge to verify the required
effectiveness of a specified sterilisation process.

Biological indicators are intended for the development and validation of the sterilisation processes and not for routine monitoring unless otherwise stated in
this general chapter.

The validity of the sterilisation process and the validity of the biological indicators can be assured by the use of reduced sterilisation process conditions,
whereby a small proportion of the micro-organisms within the biological indicator will be shown to survive. However, when the validated sterilisation
process is used, there will be no surviving viable micro-organisms (see section 3-1-2).

Bacterial spores are resistant forms of life, they can be produced and standardised, and may be stored for long periods of time under appropriate
conditions.

Commercially available biological indicators typically contain a standardised population of spores of a suitable bacterium. In cases where no suitable
commercial biological indicators are available to characterise the sterilising effect in the product or at a position difficult to penetrate by the sterilant,
custom-made biological indicators may be used. Such biological indicators can be prepared by inoculating a standardised spore suspension onto or into
the item or product to be sterilised, such action may change the characteristics of the biological indicator.

A suspension of vegetative bacterial cells is used to validate the bacterial retention capability of sterilising grade filters when applied as a sterilisation step
in an aseptic production process.

2 BIOLOGICAL INDICATORS FOR STERILISATION PROCESSES

In addition to the physical sterilisation parameters, the effectiveness of a sterilisation process as described in general chapter 5.1.1 is dependent on a large
number of variables, which may include, but is not necessarily restricted to, the number and resistance of contaminating micro-organisms, penetration of
the sterilant, time, temperature, concentration, pH, moisture content, and the chemical composition of the product or item being sterilised.
To validate a sterilisation process, physical conditions are chosen that are expected to sterilise the items in the load to achieve a sterility assurance level

(SAL) equal to or less than 10-6 as described in general chapter 5.1.1. In a physical validation process it is demonstrated that these conditions are
delivered homogeneously to all parts and positions of the load. It is the aim of biological validation to demonstrate the correlation between the predicted
effect of the physical conditions applied during the process and the observed biological effect on biological indicators. Using process parameters that have
been demonstrated to deliver the required biological effect will ensure sterility of the resulting product in routine processing.

The selection of the type of biological indicator used will depend on:

— the nature of the sterilising agent (e.g. heat, gas or radiation);

— the expected effectiveness of the treatment (e.g. the Fphys calculated from the process parameters);

— the process conditions (e.g. temperature, time, relative humidity, gas concentration, radiation dose);

— the characteristics of the pharmaceutical product or item (e.g. product in final container, packaging material, utensils such as tubes or pumps) to be
sterilised.

In the development of a sterilisation process, the load and the product should be assessed to determine the most difficult position to sterilise (e.g. cold
spots, vial-stopper interface, difficult to penetrate areas). When choosing the optimum biological challenge to a sterilisation process, the conditions in the
most difficult position to sterilise in the load and the product should be simulated as closely as possible.

Spores inoculated into a product or onto surfaces are known to react differently to sterilising conditions as compared to biological indicator units. In these
cases, commercially available biological indicator units may not be suitable to test sterilisation effectiveness and an inoculated test product/item prepared
from a well-characterised spore suspension may be a better model to evaluate the effectiveness of the sterilisation cycle.

2-1 DESCRIPTION OF BIOLOGICAL INDICATORS FOR STERILISATION PROCESSES

Depending on the process to be characterised, a suitable biological challenge may consist of biological indicators presented as test micro-organism
suspensions, inoculated carriers, or self-contained biological indicators. The user must establish a high level of confidence in the manufacturer's
compliance to quality standards for the biological indicator (e.g. by means of auditing) in order to rely on the characteristics stated by the manufacturer (see
section 2-2). Alternatively, the labelled characteristics of biological indicators shall be verified by the user or by an independent, contract laboratory that is
formally approved by the user. For custom-made biological indicators (see section 2-1-4), the characteristics shall be verified by the user or by a contract
laboratory.

2-1-1 Inoculated carriers

Inoculated carriers consist of a defined population of bacterial spores inoculated into or onto a suitable carrier, and in most cases, in a protective envelope.
The type of carrier (and the envelope if used) may influence the resistance of the bacterial spores and must be compatible with the chosen sterilisation
process (e.g. strips of filter paper in glassine envelopes are frequently used for steam and ethylene oxide, while metal discs packaged in non-woven fibre
envelopes are used for hydrogen peroxide vapour). After exposure to the sterilisation process, the carrier is aseptically handled according to the
manufacturer's instructions, transferred to a suitable culture medium and incubated for a sufficient period of time at the appropriate temperature.

2-1-2 Self-contained biological indicators

A self-contained biological indicator may be, for example:

— a system consisting of an inoculated carrier and a container (e.g. ampoule) with a nutrient medium suitable for the test micro-organism used; the
system is designed in such a way that the sterilising agent comes into contact with the inoculated carrier (e.g. through a tortuous path or a filter) while
the growth-promoting properties of the nutrient medium are not adversely affected by the sterilisation process. After sterilisation, the carrier is brought
into contact with the nutrient medium by simple manipulation. This type of biological indicator system may be used to characterise moist heat
sterilisation processes including assurance of the penetration of steam into the system;

— a container (e.g. ampoule) of a population of the test micro-organism in an appropriate nutrient medium. After sterilisation, the container is incubated
without further manipulation. This type of biological indicator is sensitive only to an exposure time and temperature and may be used primarily to
monitor sterilisation of aqueous fluids. In order to facilitate detection of growth, the medium may contain an indicator (e.g. a pH indicator).

Self-contained biological indicators might not be suitable for the validation of certain sterilisation processes.

2-1-3 Characterised spore suspensions

Characterised spore suspensions consist of a defined population of bacterial spores, prepared from a clearly characterised and suitably maintained strain
of a spore-forming bacterial species (e.g. of the genera Bacillus or Clostridium) in a stable suspension.

2-1-4 Custom-made biological indicators

Custom-made biological indicators are test items (e.g. rubber stoppers), or products, inoculated with a suitable test micro-organism, usually from a
characterised spore suspension but also from spore suspensions prepared from isolates from environmental monitoring or other microbiological testing
using a well-defined procedure designed to give satisfactory sporulation. The D-value (time for 90 per cent reduction of micro-organisms under the stated
conditions) and, when appropriate, the z-value (see section 3-1-1) of the spore suspension must be determined. Also the D-value and z-value (if
appropriate) of the spores of the inoculated test items/ products must be determined as this may be different from the spores in suspension.

After exposure to the sterilisation cycle, the custom-made biological indicator is enumerated or tested for the presence/absence of surviving test micro-
organisms using a validated, appropriate microbiological technique.

2-2 QUALITY REQUIREMENTS FOR BIOLOGICAL INDICATORS

The following are required to be known by the user per delivery of each batch:

— genus and species of the micro-organism (including the type culture collection number where applicable);

— unique reference (e.g. batch number);

— logarithm of the viable spore count expressed to 1 decimal place in scientific notation;

— recovery method used;

— type of carrier;

— type of packaging (e.g. envelope);

— composition of the recovery medium, if needed (e.g. in case of self-contained biological indicators);

— type of indicator (e.g. pH indicator) for growth, if relevant;

— type of sterilisation process(es) and the conditions for which the biological indicator has been characterised;

— resistance (D-value) per batch of finished biological indicator against the specified sterilisation processes throughout the labelled shelf-life; the D-
value should be stated in applicable units (e.g. time or dose) and expressed to 1 decimal place, together with a 95 per cent confidence interval if
feasible;

— method (inactivation kinetics or fraction negative method) used to determine the resistance (D-value); parameters to verify e.g. exposure conditions,
number of replicates tested, medium and incubation conditions used for recovery after exposure, etc.;

— the z-value (where relevant) for the biological indicator stated in temperature units with a precision of 1 decimal place in scientific notation, including
the range of temperatures used to determine the z-value;

— the storage conditions and the expiry date.

2-2-1 User requirement specification (URS)

The particular sterilisation process (moist heat, dry heat, gas, or ionising radiation) is considered as the basis for the choice of the biological indicator. This
choice includes the selection of the test micro-organism, the type of biological indicator (inoculated carrier, self-contained, or custom-made), the D-value,
and the initial spore count. Moreover, the resistance of the test strain is suitable for the particular sterilisation method and is great compared to the
resistance of micro-organisms potentially contaminating the product.

2-2-2 Quality control

Quality control for biological indicators consists of testing for purity, identity and estimation of the number of viable cells. The biological indicator should be
compliant with the URS. Users employing biological indicators outside of the manufacturer's labelled recommendations should thoroughly characterise the
biological indicators for the particular sterilisation process.

Purity
Examination of the micro-organisms on a suitable culture medium incubated under appropriate conditions shall not show any evidence of contamination.

Identification

Colony morphology and homogeneity of the population are verified, as appropriate.

Viable count

The viable count is performed according to the manufacturer's instructions or by any other validated method.

2-2-3 Suitability for purpose

The user shall ensure that the biological indicator is inactivated to the expected survival rate by the particular range of sterilisation conditions used.

3 BIOLOGICAL INDICATORS FOR HEAT STERILISATION

3-1 PARAMETERS OF BIOLOGICAL INDICATORS FOR HEAT STERILISATION

3-1-1 z-Value

Sterilisation processes can be operated at temperatures lower than the standard 121 °C (for longer exposure times) or at higher temperatures (for shorter
exposure times). The z-value (the temperature difference that leads to a 10-fold change in the D-value of the biological indicator) is used to compare the
efficacy of 2 cycles operated at different temperatures. For a z-value determination, the D-value must be determined at 3 or more temperatures. The
intended process temperature should be within the range of the 3 temperatures. The log10 of the D-value is plotted against the temperature in degrees

Celsius. The z-value is equal to the negative reciprocal of the slope of the best-fit linear curve as determined by log10-linear regression analysis.

3-1-2 Establishment of validation cycle

The characteristics of the sterilisation process (e.g. time- temperature combination, level of sterility assurance or F0 required) are the basis for the choice of

the biological indicator (type of biological indicator, test micro-organism, and initial viable count).

Inactivation of micro-organisms under sterilising conditions can be described by lethality kinetics and statistical probabilities. For a number of biological
indicator units with an initial population of N0 micro-organisms per unit and a given D-value, the exposure time in minutes where all units are expected to

carry survivors (average of 100 surviving spores per unit) is calculated by equation (1).

ts = D× (log10 N0 − 2) (1)

ts = survival time

The exposure time in minutes where all units are expected to be inactivated (average of 10-4 surviving spores per unit) is calculated by equation (2).

tk = D× (log10 N0 + 4) (2)

tk = kill time

The objective of a validation study is to demonstrate that the sterilisation effectiveness anticipated from the physical process parameters is equivalent to
the biological sterilisation effectiveness. As part of that objective, the exposure time during validation tvl, shall not exceed tk. If a too high tvl is chosen, even

a relatively large increase in the D-value would still result in biological indicator units with no surviving micro-organisms. In this case, the suboptimal
sterilising conditions would not be detected. It is considered reasonable to choose a tvl not higher than required to expect 1 in 1000 biological indicator units

having surviving micro-organisms. However, too short a tvl shall not be chosen. If a tvl is chosen such that 50 per cent of the biological indicator units have

surviving micro-organisms, changes in the sterilising conditions (e.g. time, temperature) could still result in 100 per cent of the biological indicator units

having surviving micro-organisms, and the test would be meaningless. For these reasons, a tvl is chosen such that a theoretical survival rate between 10-1

and 10-3 is expected, thus:

 D× (log10 N0 + 1) ≤tvl ≤D× (log10 N0 + 3) (3)

In general, biological indicators are subjected to the intended sterilisation process. However, for highly effective sterilisation processes, the calculated
effectiveness of the cycle may be such that the tk is exceeded by a wide margin. In such instances, biological validation is carried out with reduced

sterilisation cycles. Such reduced cycles may be shorter in time (e.g. half cycle) or be performed at a lower temperature. In the latter case the z-value for
the test micro-organism under the actual sterilising conditions shall be known. A reduced cycle is chosen such that the temperature is not more than 1 z-
value below the reference sterilisation process temperature. Biological indicators of an appropriate resistance for that cycle show an expected micro-
organism survival rate within a window between the lower tvl and the tk (see equation (3)). A decision not to perform this test must be justified.

Depending on the D-value of the test micro-organism and the tvl chosen, biological indicators having surviving micro-organisms can be expected with a low

frequency (not more than 1 in 10). If it can be demonstrated that the frequency of biological indicators having surviving micro-organisms is within the
expected range and is not due to inappropriate sterilising conditions, the process can be accepted.

Following a full sterilisation cycle, all biological indicators in a validation study must be inactivated, thereby proving at least a 106 reduction in micro-
organisms. It can then be concluded, from the resistance of the spore preparation used, that the process has delivered sufficient lethality to achieve the
required sterility assurance level.

3-2 BIOLOGICAL INDICATORS FOR MOIST HEAT STERILISATION

3-2-1 Test micro-organisms

Geobacillus stearothermophilus Is the most widely accepted biological indicator micro-organism for moist heat sterilisation processes. Reported
D121 °C-values for its spores are in the range of 1.5 min to about 4.5 min, depending on sporulation conditions, the carrier material on which the spores are

inoculated, the primary package surrounding the inoculated carrier, and the environment during sterilisation. Strains ATCC 7953, NCTC 10007, CIP 52.81,
NCIMB 8157 and ATCC 12980 (equivalent to NRRL B-4419) have been found to be suitable. Other strains may be used, provided equivalent performance

has been demonstrated. It is recognised that a 105 or 106 population of Geobacillus stearothermophilus may not be suitable for sterilisation processes

delivering an F0 between 8 and 15, therefore a lower spore number (i.e. 103 or 104) or a different test micro-organism may be used. Where a test micro-

organism other than Geobacillus stearothermophilus (e.g. Bacillus subtilis ATCC 35021) is used, the resistance of the test micro-organism is evaluated to
ensure its suitability for the process.

3-3 BIOLOGICAL INDICATORS FOR DRY HEAT STERILISATION

The reference conditions are stated in general chapter 5.1.1. Heat transfer is less effective with dry heat than with steam, and temperature distribution in
dry heat sterilisers is less homogeneous compared to steam sterilisers.

For example, biological indicators available for dry heat sterilisation have D160 °C-values within a range of 1 to 5 min. When exposed to the reference cycle

of 2 h at 160 °C, a biological indicator with a D160 °C-value of 2.5 min would be inactivated by 48 log10 scales. For dry-heat sterilisation processes, z-values

of about 20 °C are typically assumed in calculations of equivalence of cycle effectiveness (FH-calculations). FH is the equivalent time in minutes at a

temperature of 160 °C delivered by the sterilisation process to the product in its final container. For a biological indicator with a D160 °C-value of 5 min, the

D150 °C-value would be about 16 min, and inactivation in the reference cycle would be 7.5 log10 scales. The use of a sterilisation process at a temperature

reduced from the target temperature by 10 °C would give an expected 1 in 30 biological indicator units having surviving micro-organisms.

3-3-1 Test micro-organisms

Spores of Bacillus atrophaeus (e.g. ATCC 9372, NCIMB 8058, NRRL B-4418, or CIP 77.18) have been found to be suitable for use as biological indicators
for dry heat sterilisation processes performed at temperatures between 160 °C and 180 °C. Where a test micro-organism other than Bacillus atrophaeus is
used, to ensure its suitability, the resistance of the test micro-organism for the sterilisation process is evaluated as described in section 3-1-2

4 BIOLOGICAL INDICATORS FOR GAS STERILISATION

The use of biological indicators is necessary for the development, validation and monitoring of all gaseous sterilisation processes. Gas sterilisation is a
multi-factorial process: gas concentration, humidity, temperature, time, surface characteristics interact in a complex manner. A number of gas sterilisation
processes are currently used, including ethylene oxide, hydrogen peroxide and peracetic acid or combinations of the latter.
Gas surface disinfection is widely used for medical devices, isolators, chambers, etc. Use for such purposes is outside the scope of the European
Pharmacopoeia but the use of biological indicators as described in this general chapter may assist in the validation of such disinfection processes.

4-1 TEST MICRO-ORGANISMS

4-1-1 Ethylene oxide sterilisation

The use of spores of Bacillus atrophaeus (e.g. ATCC 9372, NCIMB 8058, NRRL B-4418, or CIP 77.18), or other strains of micro-organism having

demonstrated equivalent performance, is recommended for ethylene oxide sterilisation. The number of viable spores is greater than or equal to 106 per
carrier. Test micro-organisms shall have D-values relevant to the process to be validated. These biological indicators are used routinely during each
sterilisation cycle thus allowing the effectiveness of the process to be checked.

4-1-2 Other processes

It is the responsibility of the user to define the sterilisation cycle and the suitability of any biological indicator used. Geobacillus stearothermophilus has
been found suitable for vaporised hydrogen peroxide processes.

5 BIOLOGICAL INDICATORS FOR IONISING RADIATION STERILISATION

Unless otherwise indicated, biological indicators are not generally considered necessary for validation of the sterilising dose for radiation sterilisation. The
use of biological indicators may however be required for the development and validation of ionising radiation sterilisation e.g. of tissues, cell preparations or
other specific cases (e.g. products with a potential for spore protection).

5-1 TEST MICRO-ORGANISMS

Spores of Bacillus pumilus (e.g. ATCC 27142, NCTC 10327, NCIMB 10692 or CIP 77.25) or other strains of micro-organisms having demonstrated
equivalent or better performance are recommended.

6 MICROBIAL PREPARATIONS FOR STERILISATION GRADE FILTRATION

As stated in general chapter 5.1.1, certain products that cannot be sterilised in their final container may be sterilised by a filtration process. In contrast to
the biological indicators discussed in the previous sections, which assess kill-based sterilisation, the biological challenge assesses the retention of micro-
organisms by the filters.

To validate the sterilisation process, it must be demonstrated that the filtration process (usually in a scaled-down model) is capable of completely retaining

a microbial challenge of at least 107 CFU per square centimetre of effective filter surface using a suitable test micro-organism. This test should mimic the
actual filtration process as closely as possible. Where feasible, the test is carried out in the product using the specified filtration conditions. If this is not
possible, e.g. due to the antimicrobial properties of the product, a medium as similar as possible to the product must be used in the test.

6-1 TEST MICRO-ORGANISMS

For processes using a filtration system with a nominal pore size not greater than 0.22 µm, a suspension of Brevundimonas diminuta (ATCC 19146,
NCIMB 11091 or CIP 103020) is recommended. The Brevundimonas diminuta suspension must be prepared in order to achieve predominantly single cells
of the smallest possible size. Other micro-organisms, for example natural flora isolated from the product or process in question, may be used if presenting
a stronger challenge to the sterile filtration system than Brevundimonas diminuta. For filtration systems with a nominal pore size of 0.1 µm or less, a
suspension of Acholeplasma laidlawii (ATCC 23206) may be used.

Methods of Sterilisation

(Application of the F0 Concept of Steam Sterilisation of Aqueous Preparations, Ph. Eur. general texts 5.1.5)

The following chapter is published for information.

The F0 value of a saturated steam sterilisation process is the lethality expressed in terms of the equivalent time in minutes at a temperature of 121 °C

delivered by the process to the product in its final container with reference to micro-organisms possessing a theoretical Z-value of 10.
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 sterilisation 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 give an SAL of 10-6 or better.

In connection with sterilisation by steam, the Z-value 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 sterilisation (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 (log10 N0 − log10 N ) = D121 log10 I F

D121 = D-value of the reference spores (5.1.2) at 121 °C;

N0 = initial number of viable micro-organisms;

N = final number of viable micro-organisms;

IF = inactivation factor.

T2 −T1
Z =
log D 1 −log D2
10 10

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

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

N0 t/D
IF = = 10
N

t = exposure time;

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

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