Manual – 039 Sterilization Process Validation

5.1 Steam Sterilization

Steam Sterilization is the most common type of sterilization employed in the pharmaceutical manufacturing environment. The principles of steam sterilization are applicable to processes conducted within autoclaves as well as sterilization-in-place (SIP) processes.

For the sterilization of fluids in e.g. vials and ampoules, a fluids load autoclave cycle is used. The steam (or superheated water) is used as a heat transfer medium to heat the contents of the vials/ampoules. The moisture required for sterilizations derived from the contents in the vial/ampoule.

For the sterilization of processing equipment, tubing, garments etc. a porous load (equipment or hard goods) autoclave cycle is used. Air must be removed from the chamber and load prior to the sterilization phase of the cycle. The steam issued to heat the load and to provide the moisture needed for sterilization. SIP processes are unique because of the equipment configurations involved.

The system must be correctly designed to ensure adequate air and condensate removal, sequencing of the process and maintenance of sterility post-cycle. The steam is used to heat the equipment and to provide the moisture needed for sterilization. Steam must be of suitable quality.

Some EU regulatory authorities place great emphasis on the quality of steam used for sterilization.

For porous load cycles (product contact equipment and hard goods cycles) and SIP processes the steam, when condensed, should be of WFI quality with low levels of non-condensable gases (typically <0.35%v/v), a dryness fraction of 0.95 and show less than 25°Csuperheat. Methods to determine these parameters are described in BS EN 285. High levels of non-condensable gases may prevent the attainment of sterilizing conditions.

Dryness fractions of <0.95 may lead to slow heat up times and wet loads. Superheated steam is a dry gas and is not suitable due the lack of water, which is needed for sterilization. Generally, the development and validation of steam sterilization cycles follows one of two methods:

Overkill Cycle Method

The overkill cycle method is applicable to processing equipment and to vials and ampoules containing heat stable fluids. It is the preferred validation method.

Overkill cycles are easier to validate and ensure substantially greater than a 12-log reduction of any native bioburden present in or on the items to be sterilized. Minimal information is therefore necessary about the native bioburden, since any bioburden present will have substantially less resistance (and a lower population) than the Biological Indicators used in this type of cycle.

A typical overkill cycle would deliver a minimum of 15 minutes at 121°C. Cycle times must first be established by considering the time required to inactivate the resistant biological indicators and adjusting the cycle time as necessary to achieve a minimum 12-log reduction. Validation of steam sterilization processes must be designed to incorporate elements of worst-case conditions. For example, during the validation of an overkill cycle it is possible to use reduced cycle times and temperatures e.g.

Reduce the sterilization temperature by 1ºC and shorten the exposure time by several minutes. Terminally sterilized drug product cycles generally include an upper limit for the time/temperature at which the product can be exposed to ensure that unacceptable degradation of the product does not occur.

Product Specific Method

The Product Specific Method (PSM) applies to sterilization cycles where an overkill approach is not possible because the product cannot withstand the temperatures and/or exposure times required for an overkill cycle. The PSM is more applicable to the validation of heat sensitive fluids, for example, where the sterilization cycle is developed to adequately destroy the microbial load and yet not result in product degradation.

The PSM relies on determining the population and heat resistance of the environmental, product, or items-to-be-sterilized bioburden along with ongoing monitoring and control over the bioburden during routine operation. A PS cycle would deliver less lethality than an overkill cycle, but would still deliver a Sterility Assurance Level of at least 10-6 (for the most heat resistant bioburden in the product).

In the PSM, once the population of the overall bioburden and the heat resistance(generally only for spore-forming environmental or product/item isolates) has been determined, these values (plus additional safety margins based on professional judgment, the extent of the bioburden data, and the degree of product bioburden testing that will be conducted on an ongoing basis) are used to determine the lethality necessary to achieve a minimum SAL of 10-6.The safety margin selected inversely correlates to the frequency and magnitude of ongoing tests conducted for bioburden population and resistance.

For example, if the observed worst-case product spore bioburden resistance is 0.3 minutes and, for lethality determination, a D-value of 0.4 minutes is selected, then extensive ongoing bioburden resistance testing would be necessary. However, if a D-value of 1.0 minute were selected for lethality determination, then minimal ongoing bioburden resistance testing would be necessary. Ongoing monitoring of bioburden population must be conducted. Once the bioburden data has been used to determine the lethality required for these derived cycle, the attributes (population, resistance) of the BI challenge system can be determined. The semi-logarithmic survivor curve equation is used for determination of both the required cycle lethality and the attributes of the Challenge.

When the resulting log reduction for the BI challenge system is compared to theolog reduction of the typical bioburden organism (whose D value is substantially less) the log reduction delivered for the actual bioburden is significantly higher than the demonstrated log reduction using the BI challenge system.

The F-value for steam sterilization (F0) is a measurement tool used to demonstrate accumulated lethality and must be used in both validation and routine monitoring of cycles to demonstrate that acceptance criteria for F0 has been met. For terminally sterilized drug products validated using the PSM approach, reductions in cycle parameter set points during the validation are less common.

Terminally sterilized drug product cycles generally include an upper limit for the time/temperature at which the product can be exposed to ensure that unacceptable degradation of the product does not occur. Load definition and load size are parameters that can be varied in the validation design. An acceptable validation approach is to perform studies that include both the minimum and maximum contents of loads with a defined loading pattern.

5.1.1 Tests to be performed during the validation of a porous load (equipment or wrapped goods) cycle

For equipment or wrapped goods cycles, the orientation of the items within the load is critical to ensure that condensate can drain out of the items. Special racks or holders are often used to ensure proper equipment orientation and assist condensate draining. If condensate is not drained away, lower temperatures will result and the proper sterilizing conditions will not be experienced at these locations.

The use of racks or holders or specific orientation should be specified as a requirement in the routine loading operations instructions(SOPs). Similarly, any equipment load valves (e.g. filter housing valves) or covers that must be left open or ajar to permit air removal and steam penetration (and therefore sterilization), must be specified as a requirement in the routine loading operations instructions.

Any wrapping that is used for items to be sterilized must allow for the removal of air from and the penetration of steam into the item. Use of aluminium foil or sealed containers must not be used as a wrap for dry goods or equipment to be steam sterilized. The temperature equilibration phase (‘come up time’) is a parameter that must be studied as part of the validation exercise for porous (equipment or wrapped goods cycles).

The equilibration time is defined as ‘the period that elapses between the attainment of sterilizing conditions in the chamber and the attainment of the sterilization temperature in all parts of the load’. Some EU regulatory authorities consider any equilibration time in excess of 15 seconds for chambers of less than 800 litres (or 30 seconds for chambers of greater than 800 litres volume) to be indicative of an air removal or steam penetration problem.

For any process where temperature measurements are taken in the chamber or drain to control or record data during the validation, the placement of an independent validation probe near teach of these recording probes is essential.

5.1.1.1 Empty Chamber Temperature Distribution

This study must be performed to demonstrate that uniform heating is occurring throughout the chamber. Temperature measuring probes, e.g. thermocouples, must be located in free space throughout the chamber. One probe is located next to the controlling temperature sensor.

Empty Chamber Temperature Distribution studies should be able to meet an acceptance criterion for the temperature to be within + 1°C of the mean chamber temperature after one minute of the exposure stage. The difference between the control probe, recording chart probe and independent sensor (usually a thermocouple) during the exposure stage should not exceed + 1.0°C.

5.1.1.2 Loaded Chamber Temperature Distribution

This study must be included to demonstrate that the equipment loading patterns do not significantly change the chamber temperature distribution within the chamber.

Typically, thermocouples are distributed throughout the chamber (not in contact with load items) as for the Empty Chamber Temperature Distribution study and cycles may be run using both the maximum and minimum loads.

Loaded Chamber Temperature Distribution studies should meet an acceptance criterion for the temperature to be within + 1°C of the mean loaded chamber temperature after one minute of the exposure stage. The difference between the control probe, recording chart probe and independent sensor during the exposure stage should not exceed + 1.0°C.

5.1.1.3 Heat Penetration Studies

Loaded chamber heat penetration studies must be performed to demonstrate that the pre-required time at temperature criteria are met for the loads being validated. The heat penetration locations to be monitored are assessed using both temperature probes and BIs.

The thermocouples and the BIs should be placed at the same locations wherever possible. Special emphasis is placed on those locations identified during cycle development studies as being difficult for steam too penetrate/difficult to heat (cold spot determination).

Such locations typically include the interior of hoses, filter housings, large objects, filling apparatus and items with multiple layers of protective wrapping. The placement of temperature probes and BIs within the load must not enhance the penetration of steam into the load item. Where the load includes multiple items of the same configuration in the load (e.g. bags of stoppers), BIs should be placed in a second item adjacent to that containing the thermocouple.

This is to prevent the presence of the thermocouple from enhancing the penetration of steam to the Bowered items in the load are unique, the BIs must be placed in the load item near the probe and precautions taken to prevent enhanced steam penetration. Heat penetration cycles performed, as part of an initial validation exercise must be repeated several times, e.g. three times, to demonstrate consistency.

These studies should be performed using established load patterns, though minimum and maximum load may be used to represent each particular load pattern for qualification purposes.

The purpose of the heat penetration study is to document that the load items (including the cold spot) receives the minimum required pre-determined time at temperature/F0.  The BIs must be placed throughout the load, adjacent to the temperature probes, and include locations that are expected to be slow to heat (such as inside tubing, within filters, etc.).

5.1.2 Tests to be performed during the validation of a fluids cycle

5.1.2.1 Empty Chamber Temperature Distribution

This study must be performed to demonstrate that uniform heating is occurring throughout the chamber. Temperature measuring probes, e.g. thermocouples, must be located in free space throughout the chamber.

One probe is located next to the controlling temperature sensor. Empty Chamber Temperature Distribution studies should be able to meet an acceptance criterion for the temperature to be within + 1°C of the mean chamber temperature after one minute of the exposure stage. This is particularly important where the sterilizer is being used for terminal sterilization of drug products.

The difference between the control probe, recording chart probe and independent sensor (usually a thermocouple) during the exposure stage should not exceed +1.0°C.

5.1.2.2 Loaded Chamber Temperature Distribution

This study must be performed to demonstrate that the proposed loading patterns do not significantly change the chamber temperature distribution within the chamber. Typically, thermocouples are distributed throughout the chamber (not in contact with load items) as for the Empty Chamber Temperature Distribution study and cycles are run using both the maximum and minimum loads.

Loaded Chamber Temperature Distribution studies should meet an acceptance criterion for the temperature to be within + 1°C of the mean loaded chamber temperature after one minute of the exposure stage. The difference between the control probe, recording chart probe and independent sensor during the exposure stage should not exceed + 1.0°C.

5.1.2.3 Heat Penetration Studies – Terminal Sterilization of Drug Products and Fluid Loads

The heat penetration studies for the terminal sterilization of drug products in sealed containers must include an assessment of any cold spot(s) within an individual container (container mapping) as well as in containers within each load (product load mapping).

These studies are usually part of cycle development. Smaller containers may not have a discernible container cold spot. In larger containers, fill volume can have an appreciable influence on the location of the container cold spot. Care must be taken to ensure that the placement of thermocouples does not enhance heating of the product during both container and product load mapping.

Small volume parenteral drug product loads may not have load cold spot due to the relatively uniform temperature rise across the load. Using defined maximum and minimum load patterns may be useful in elucidating the presence of a product load cold spot.

Once the container and product load cold spots (if any) have been determined, consistency of the cycle must be demonstrated by repeat runs with temperature probes and BIs located at the container and load cold spots.

The purpose of consistency study is to document that any load or container cold spot receives the minimum required pre-determined F0and/or time at temperature.

5.1.3 Demonstrating Biological Lethality of the Cycle

The use of BIs to demonstrate biological lethality must be included in both porous (equipment or hard goods) cycles and fluids load cycles, whether validating using the overkill cycle method or the PSM. BIs used in steam sterilization are bacterial spores, typically Debacles stereo thermophiles, having defined heat resistance that is significantly greater than the native bioburden of the items/product to be sterilized.

For PSM cycles, other BI challenge systems such as Clostridium supergenes, Bacilluscoagulans or Bacillus atropheus may be used. BIs provide a direct measure of biological lethality and allow the targeted placement of highly heat resistant microorganisms in situ.

BIs respond tooth heat and moisture and therefore provide proof that the required sterilizing conditions have been achieved (temperature probes alone cannot do this).  The destruction of large numbers of these resistant organisms supports the efficacy of the sterilization process for the total destruction of any native bioburden organisms present.

Is are available commercially as pre-prepared spore strips or coupons, self- contained units, or aspire suspensions. The heat resistance of BIs is characterized by the specific D-value and Z-value for the preparation determined under defined conditions. Spore strips or coupons should be placed within the load at locations where steam penetration is deemed to be most difficult (sometimes referred to as the ‘cold spot’).

The manufacturer of the strips, coupons or self-contained unit provides information on the number of organisms present on the inoculated carrier(population) and an expected resistance level (D-value) under defined conditions.

Spore strips or coupons should only be stored, used and incubated as directed by the manufacturer. They should never be immersed in liquid or altered or abused in any way with since this may dramatically change their resistance from the labelled value.

The end user should verify the population of commercially obtained spore strips or coupons. Self-contained Bus include both the organism and the culture media in a single unit. The end user positions the BI in the load, runs the cycle, recovers the unit and then incubates according to the manufacturer’s instructions.

Where these units are used in accordance with the manufacturer’s instructions, the population and D-value provided may be used directly, though it is good practice to determine the population as part of the quality control of these units. Spore suspensions can be used to directly inoculate items to be sterilized.

The suspension is allowed today on the surface of the item and then the item is positioned in the sterilizer load. Following the validation cycle, the item is recovered aseptically and tested in a manner similar to that used for the spore strip or coupon. Spore suspensions may also use during the validation of terminal sterilization of drug products or other liquids by direct inoculation into the liquid filled container.

The container is placed in the load, subjected to the validation cycle, recovered, and its contents subject to filtration and incubation similar to a sterility test. Spore suspensions can be used to inoculate fluid pathway surfaces such as the interface between a vial stopper and the stopper-seating surface of the vial. Use of spore suspensions to inoculate surfaces or liquids requires that the population and D-value be determined for the conditions and substrates involved.

The BI manufacturer’s labelled D-value should only be considered a very rough estimate of performance. The BI manufacturer may offer a service to determine the D value of the spores in the product to be sterilized.

5.2 Sterilize-In-Place

Sterilize-In-Place (SIP) processes follow the general principles covered for conventional steam sterilization validation with some unique considerations. Atreus saturated steam environment is not always obtained in a SIP system and air and condensate removal present design and operational challenges.

The overkill cycle design is typically implemented for SIP processes. Cycle development studies must take into consideration the necessary controls to ensure that air and condensate are removed from the system and that the steam quality characteristics, particularly non-condensable gasses are controlled.

Controls and instrumentation to monitor and/or control the pressure differential across filters, steam flow direction across filters, monitoring of temperature at various locations and verification of proper steam trap operation, the cooling process and proper conditions in the system post-sterilization to maintain sterility are also parameters to be addressed. Purge times, steam valve pressure settings, temperature ranges, time for heat-up, exposure time, exposure temperature and cooling/drying times are typical cycle parameters to define and control.

The validation includes both the physical and biological assessments already covered for steam sterilization.

5.3 Filtration Sterilization

The use of microbial retentive filtration to achieve sterilization of product solutions or process gasses must be validated. The current widely accepted definition of a sterilizing grade filter is one that is designated as nominally 0.22µm porosity or smaller.

The EMEA requires that product solutions, before filtration, contain no more than10 colony-forming units (CFU) per 100 milk If the pre- filtration bioburden limitis greater than 10 CFU per 100mL, then a bioburden reduction filtration is required to reduce the bioburden down to not more than 10 CFU per mL.

The product solutions then filtered using a sterilizing grade filter. In addition, it is common to use a second sterilizing grade filter immediately before final filling. The manufacturing process must be designed to minimize the pre filtration bioburden. Products that are subject to microbial proliferation should be held fora’s short a time as possible.

If long holding times are anticipated, consider the options of sterile filtering the solution into a sterile holding tank or holding the product in either a chilled or a heated state to reduce the probability of an increased bioburden. Sterile holding tanks and any contained liquids must be held under positive pressure or appropriately sealed to prevent microbial contamination. Filtration validation should be product-specific.

The use of a “matrix approach “or “family approach” allowing grouping of products or the selection of a worst-case product to represent a number of products is discouraged due to the extensive justification needed.

Even with such justification, the acceptance by regulatory authorities would be on a case-by-case basis and in the absence of any official guidance for industry. All sterilizing grade filters must be integrity tested before use (preferably after sterilization) and then again following use.

The integrity test parameters must be clearly defined and, along with the test limits, be correlated to the microbial validation of the filter.

5.3.1 Validation of Filtration Process

The validation of sterile filtration processes must demonstrate microbial retention as well as filter compatibility and filter extractable relative to the solution being sterilized. The microbial retention studies required are highly specialized and the industry in general, contract this work out to the laboratories of the filter manufacturers.

Filtration validation is process specific and process parameters must be provided to the filter manufacturer in order to assure that the laboratory-based validation conditions reflect both actual and elements of worst-case use conditions in our facilities. Filters must never be subjected to conditions beyond those mandated by the filter manufacturer.

Process parameters such as pressures, maximum use time, hydraulic shock, flow rates etc. as well as product solution characteristics such as pH, temperature, osmolality, etc. Are some of the parameters that must be factored into the validation exercise.

While the actual work to validate microbial retention may be contracted out, the responsibility for the validation lies with the sponsor company. A copy of the validation documentation must be held at the sponsor site.

5.3.2 Microbial Retention Test

Sterilizing grade filters must pass a microbial retention test using Brevundimonas diminuta as the challenge organism. The requirements for culturing the organism as well as the conditions of the test are rigorous and highly specialized. It is particularly important to perform this test by directly inoculating the drug product solution if at all possible. If the drug product is bactericidal then a simulation test must be run.

The simulated drug should match the drug product as closely as possible without affecting the challenge microorganism. Production process parameters such as pH, viscosity, osmolality, temperature, pressure, flow rate and time should match the product as closely as possible. The filter manufacturer performs these tests with oversight as described inspection 5.2.1 above.

5.3.3 Product/Filter Compatibility & Extractable Testing

This test determines that the filter under consideration is not additive, sportive or reactive beyond predetermined specifications with respect to the active product, preservatives or other additives.

Specific tests to consider include oxidisable substances test, weight change, extractables, integrity test, flow rate and a physical inspection of the filter. The filter manufacturer provides limits for extractables. Both compatibility and extractable testing must be performed under worst-case conditions.

One key objective in the extractable testing is to determine the initial flush volume of the filter apparatus prior to capturing product solution filtrate for filling. The filter manufacturer may perform these tests.

5.3.3.1 Filter Integrity Testing

Before and after each run the filter apparatus must be integrity tested. The integrity test acceptance criteria must correlate with the microbial retention. The pre-filtration integrity test should be performed after sterilization of the filter.

Filter integrity test equipment is readily available that simplifies and automates the testing operation. When initially performing this work the manufacturer of the integrity test equipment should be consulted. In general manufacturers offer excellent technical support in setting up these operations.

5.3.3.2 Sterilization of Filter and Housing

Consider how the filter and housing are going to be sterilized. A SIP (sterilize in place) system is strongly preferred because it minimizes the manipulation of the filter assembly. However, sterilizing the filter assembly in an autoclave followed by aseptic assembly may be acceptable if strict controls are in place to minimize the possibility of contamination being introduced into the system as a result of the assembly operation.

Again, filter manufacturers provide very useful advice on performing this operation. Validation of the filter assembly sterilization process, using temperature distribution, heat penetration and biological indicators, is required. Similar to an autoclave chamber study the coldest spot in the filter assembly has to be identified and challenged. Biological testing can use spores trips of Geobacillus stearothermophilus on the downstream side of the filter.

5.4 Dry Heat Sterilization and Depyrogenation

Dry heat sterilization processes are typically used for equipment parts while dry heat-based depyrogenation processes are used for glass containers for drug products. Dry heat sterilization and depyrogenation can be performed in ‘batch ‘dry heat ovens or continuous sterilizing tunnels. Rubber closures are also subject to a validated depyrogenation process using a washing/rinsing process – See section 5.3.4 below). There is limited use for dry heat in sterilizing drug products, though raw materials are sometimes sterilized using dry heat.

The validation of these dry heat based cycles follows many of the general principles outlined for steam sterilization. Concepts such as equilibration time, temperature mapping, heat penetration, biological lethality and worst-case cycle parameters (shortened times, lower temperatures, faster belt speeds, etc.) are relevant, though in different context.

It is common practice to determine FH values for dry heat processes in a similar manner to F0 for steam sterilization. However, most applications of dry heat are to dehydrogenate and not just to sterilize. The depyrogenation process is not as well understood as the sterilization process. Some evidence indicates that inactivation of endotoxin is a second-order reaction whereas the standard uses off, D and Z values assumes a single-order reaction.

Therefore, for depyrogenation cycles there is even more reliance on the biological proof (inactivation ofendotoxin) as opposed to only the accumulation of physical data. Even with these limitations it is valuable to collect the FH data to demonstrate reproducibility.

The temperature range allowed in both empty and loaded chamber studies for dry heat cycles is significantly greater than for steam sterilization. In particular, for empty chamber studies, USP 29 states that ± 15ºC is a typical acceptable temperature range when the oven is operating at not less than 250ºC. For loaded chamber studies, a ± 5ºC temperature range is generally achievable. Depyrogenation processes must be validated to achieve a minimum 3-logreduction of bacterial endotoxin.

The endotoxin must be spiked onto the item in a liquid state and allowed to air dry prior to being subject to the depyrogenation cycle. Recovery studies and appropriate controls are necessary part of these studies.

5.4.1 Dry Heat Ovens

The validation of a dry heat process in an oven, whether it is used for sterilization or depyrogenation, is similar to the validation of a steam sterilization process. Biological indicators, typically Bacillus atrophies spores (for sterilization processes) or bacterial endotoxin (for depyrogenation processes) must be used in conjunction with temperature measurements.

A successful endotoxin challenge in the validation of a depyrogenation process assures that sterilization has also occurred and there is no need for additional studies with bacterial spores. The load size and configuration are very important in dry heat processing.

These must be carefully designed and duplicated between the validation studies and routine operation. Fans or blowers are generally used to help assure uniform distribution of heat throughout the chamber.

Fan speed should be determined during validation and thereafter periodically monitored to assure operation within the acceptable range. Typical test during the validation of a dry heat oven include:

5.4.1.1 Empty Chamber Temperature Distribution

Temperature probes (thermocouples) are distributed throughout the empty chamber (in free space) and a temperature profile is produced. A uniform temperature profile is expected. A probe should be located next to the controlling sensor. Note the come-up time to temperature and cool-down times, as these should be consistent in an empty chamber.

5.4.1.2 Loaded Chamber Temperature Distribution and Heat Penetration

In dry heat applications loaded chamber mapping and heat penetration studies must be performed. Typically, these studies may be performed at the same time. Thermocouples must be distributed throughout the chamber (in free space) for heat distribution information. Thermocouples must also be placed inside of the container, equipment or component being treated for the heat penetration information.

The penetration thermocouples need to make contact with the surface of the item. This is because, due to the mass of the item, the time to reach sterilization/depyrogenation temperature can substantially lag behind the temperature of the surrounding air.

Biological indicators or endotoxin-spiked vials should be located adjacent to the penetration thermocouples. Heat penetration studies, conducted as part of the initial validation, should be repeated several times (e.g. three times) to demonstrate consistency. Product containers should have container mapping performed similar to that described for steam sterilization. Load patterns are important as air is a poor conductor of heat and the distribution of mass can greatly affect heating characteristics.

Assigning a temperature profile requirement is more problematic as the profile is load specific. The temperature profile for a specific load should how good reproducibility

5.4.2 Dry Heat Tunnels

Validation of dry-heat tunnels is demonstrated by both temperature measurements and inactivation of bacterial endotoxins (depyrogenation). Similar studies to dry-heat ovens are performed, i.e., empty tunnel temperature distribution and loaded tunnel endotoxin challenge. The temperature variation within the sterilization/depyrogenation zone may be greater than seen in an oven.

Higher temperatures are usually selected – ranging from 270° – 350°C, due to the shorter exposure time required and the greater temperature variation. Points to consider for physical measurements include:

* Belt Speed – determines the exposure time

* Temperature – determines the time required for inactivation

* Blowers/Fans – for proper air balance and the differential pressure between the tunnel and the room environment

* Room pressure differentials

5.4.2.1 Empty Tunnel Temperature Distribution

Temperature probes (thermocouples) are distributed across the width of the tunnel and a temperature profile is produced across the conveyor and along the tunnel during operation.

A reasonably uniform temperature profile is expected. Aerobe needs to be located next to the controlling sensor. Note the come-up time to temperature and cool-down times, as these should be consistent in an empty tunnel.

5.4.2.2 Loaded Chamber Heat Penetration

In dry heat tunnel applications heat penetration studies must be performed. Thermocouples must be placed in contact with vials distributed across the tunnel. The temperature of the vial is usually lower than the air temperature.

Test should be performed with all vial sizes. Endotoxin spiked vials should be located adjacent to the penetration thermocouples. Heat penetration studies, conducted as part of the initial validation, must be repeated several times (e.g. Three times) to demonstrate consistency. Consider the loading arrangement of the tunnel i.e. at the start, middle and end of a run. The temperature profile for a specific load should show good reproducibility.

5.4.3 Demonstrating Biological Lethality

The use of BIs to demonstrate biological lethality must be included in the validation approach. For dry heat sterilization the indicator of choice is Bacillus atrophaeusspores. However, dry heat is also used to depyrogenate. In this case endotoxin (E. coli) issued to validate the depyrogenation activity.

If endotoxin is used then Bacillusatrophaeusspore strips are not required as endotoxin is more heat resistant than Bacillus atrophies. These biological studies can be performed at the same time as the loaded chamber temperature mapping. If this is done the carriers should be located as close as possible to the thermocouples.

5.4.4 Washing Process for Depyrogenation

In some cases, the depyrogenation of items that cannot be dry heat sterilised (e.g. Rubber stoppers) is carried out in a washing/rinsing process. It must be demonstrated that a 3-log reduction in the endotoxin concentration on spiked stoppers can be achieved. Recovery studies on the spiked stoppers must be performed. The stoppers must be subsequently sterilized using a validated steam sterilization cycle.

5.5 Radiation Sterilization

Radiation sterilization (usually60Co) is not generally used for dosage form sterilization as the free radicals formed during processing can cause chemical changes to occur.

The historical standard for radiation sterilization is 25 key (2.5 Murad) and it is still used for many applications. The use of lower doses is finding wider acceptability for sterilization – following the principles in ISO11137. See ISO11137:1995.

Irradiation Sterilization (and validation of the process) is usually performed by an irradiation contractor. The critical parameters for radiation sterilization are the source activity and time. The dose delivered is determined by the activity of the 60Co source in the irradiator (which is generally not variable from run to run – a monthly adjustment to account for decay may be made).

On a day-to-day basis the critical parameters the speed of the conveyor system, which determines the time the box of product is in front of the radiation source. As the source activity is fixed, the dose received is directly related to the length of time in the irradiator. The most important aspect of validation is dose mapping of boxes of product (using dosimeters) to determine the regions of high and low dose within the box.

Dosimeters must be uniformly distributed throughout the product load as well as in the geometric centre of the load (as this is expected to be the most difficult to sterilize location). Dosimeters are required during routine processing. Product must only be released on the basis of dosimeter results.

Biological lethality is demonstrated using the procedure detailed in ISO 11137.This involves determining the bioburden on items to be sterilized and then subjecting the items to a sub-lethal dose of radiation (based on the level of the bioburden) to determine the inherent radiation resistance of the bioburden.

The minimum dose required to achieve a SAL 10-6can then be determined forms 11137 for the level of bioburden (or a minimum dose of 25 key may be delivered). In summary, the most significant points for the validation of a radiation sterilization process are:

 – The loading pattern for the box of product must be kept constant

 – Dose mapping must be performed for each product using calibrated dosimeters

 – Timer settings should be recorded

 – Bioburden and sub-process dose experiments (to demonstrate that the sterilizing dose is adequate to achieve a SAL 10-6)