Commission Implementing Regulation (EU) 2025/2091 ANNEX I

STERILE PRODUCTS AND ASEPTIC MANUFACTURING

ANNEX ISupplementary provisions

ANNEX I STERILE PRODUCTS AND ASEPTIC MANUFACTURING SECTION I SCOPE The additional requirements set out in this Annex shall apply to the manufacture of sterile products and products where aseptic manufacturing is required. SECTION II GENERAL PRINCIPLES II.1. The manufacture of sterile products is subject to special requirements in order to minimise risks of microbial, particulate and endotoxin/pyrogen contamination. The following aspects shall be specifically considered: (a) premises, equipment and processes shall be appropriately designed, qualified and/or validated and, where applicable, subjected to ongoing verification. The use of appropriate technologies (e.g. restricted access barriers systems, isolators, robotic systems, rapid/alternative methods and continuous monitoring systems) shall be considered to increase the protection of the product from potential extraneous sources of endotoxin/pyrogen, particulate and microbial contamination, and assist in the rapid detection of potential contaminants in the environment and in the product; (b) personnel shall have adequate qualifications and experience and training with a specific focus on the principles involved in the protection of sterile products; (c) processes and monitoring systems for the manufacture of sterile products shall be designed, commissioned, qualified, monitored and regularly reviewed by personnel with appropriate knowledge (including on aspects related to the process and relevant engineering and microbiological knowledge); (d) raw materials and packaging materials shall be adequately controlled and tested to ensure that the level of bioburden and endotoxin/pyrogen are suitable for use; (e) processes associated with the finishing and storage of sterile products shall not compromise the sterility of the product. Aspects to be considered in this regard include container integrity and maintenance of adequate storage conditions; (f) all non-conformities, such as sterility test failures, environmental monitoring excursions or deviations from established procedures shall be adequately investigated before certification/release of the batch. The investigation shall determine the potential impact on the process and product quality and whether any other processes or batches are potentially impacted. The reason for including or excluding a product or batch from the scope of the investigation shall be clearly justified and recorded. II.2. Processes, equipment, premises and manufacturing activities shall be managed in accordance with quality risk management principles so as to proactively identify, evaluate and control potential risks to quality. Monitoring or testing alone are not considered sufficient – on their own – to ensure sterility. II.3. A contamination control strategy shall be developed by the manufacturer and be implemented in the site. The contamination control strategy shall aim at avoiding contamination by identifying all the critical control points and assessing the effectiveness of all the controls (design, procedural, technical and organisational) and monitoring measures implemented to manage the risks. The effectiveness of the contamination control strategy shall be periodically reviewed and, where appropriate, updated and shall also drive continual improvement of the manufacturing and control methods. II.4. While the contamination control strategy includes a series of interrelated measures that are typically assessed, controlled and monitored individually, the effectiveness of the implemented measures shall be assessed altogether. II.5. The development of the contamination control strategy requires detailed technical and process knowledge. Potential sources of contamination are attributable to microbial and cellular debris (e.g. pyrogen, endotoxin) as well as particulate (e.g. glass and other visible and sub-visible particles). Elements to be considered within a contamination control strategy include but are not limited to: — plant and processes design, including the associated documentation; — premises and equipment; — personnel; — utilities; — raw material controls, including in-process controls; — product containers and closures; — approval of key component suppliers and critical service providers; — management of outsourced activities and availability/transfer of critical information between parties; — process validation, including validation of sterilisation processes; — preventive maintenance: maintaining equipment, utilities and premises (planned and unplanned maintenance) so as to minimise the risk of contamination; — cleaning and disinfection; — monitoring systems, including an assessment of the feasibility of the introduction of scientifically sound, alternative methods that optimise the detection of environmental contamination; — prevention mechanisms: trend analysis, detailed investigation, root cause determination, corrective and preventive actions and the need for comprehensive investigational tools; — continuous improvement based on information derived from the above. II.6. Changes to the systems in place shall be assessed for any impact on the contamination control strategy before and after implementation. II.7. The manufacturer shall take all the steps and precautions necessary to ensure the sterility of the products manufactured within its facilities. Sole reliance shall not be placed on any terminal process or finished product test. SECTION III PREMISES III.1.    General requirements III.1.1. The manufacture of sterile products shall be carried out in appropriate cleanrooms, entry to which shall be through change rooms that act as airlocks for personnel and airlocks for equipment and materials. III.1.2. Cleanrooms and change rooms shall be maintained to an appropriate cleanliness standard and supplied with air that has passed through filters of an appropriate efficiency. Controls and monitoring shall be scientifically justified and shall effectively evaluate the state of environmental conditions of cleanrooms, airlocks and pass-through hatches. III.1.3. The various operations of component preparation, product preparation and filling shall be carried out with appropriate technical and operational separation measures within the cleanroom or the premises to prevent mix up and contamination. III.1.4. Restricted Access Barrier Systems (RABS)  ( 1 ) or isolators can minimise microbial contamination associated with direct human interventions in the critical zone  ( 2 ) . Their use shall therefore be considered as part of the contamination control strategy; the use of alternative approaches shall be justified. III.1.5. The following grades of cleanroom/zone shall be used: (a) Grade A: for high-risk operations, such as aseptic processing line, filling zone, stopper bowl, open primary packaging or for making aseptic connections under the protection of first air  ( 3 ) . Grade A conditions are usually provided by a localised airflow protection, such as unidirectional airflow  ( 4 ) workstations within RABS or isolators. The maintenance of unidirectional airflow shall be demonstrated and qualified across the whole of the grade A area. Direct intervention (e.g. without the protection of barrier and glove port technology) into the grade A area by operators shall be minimised. (b) Grade B: this is the background cleanroom for grade A for aseptic preparation and filling (except for isolators). Air pressure differences shall be continuously monitored. Cleanrooms of lower grade than grade B may be considered where isolator technology is used (see Section III.3.3 of this Annex). (c) Grade C and D: for less critical stages in the manufacture of aseptically filled sterile products or as a background for isolators. They may also be used for the preparation/filling of terminally sterilised products. III.1.6. In cleanrooms and critical zones, all exposed surfaces shall be smooth, impervious and unbroken in order to minimise the shedding or accumulation of particles or micro-organisms. III.1.7. To reduce the accumulation of dust and to facilitate cleaning there shall be no recesses that are difficult to clean effectively. Therefore, projecting ledges, shelves, cupboards and equipment shall be kept to a minimum. Doors shall be designed to avoid recesses that cannot be cleaned. Sliding doors are generally undesirable for this reason. III.1.8. Materials used in cleanrooms, both in the construction of the room and for items used within the room, shall be selected to minimise generation of particles and to permit the repeated application of cleaning, disinfectant and sporicidal agents as appropriate. III.1.9. Ceilings shall be designed and sealed to prevent contamination from the space above them. III.1.10. Sinks and drains are not allowed in the grade A and grade B areas. In other grades, air breaks shall be fitted between the machine or sink and the drains. Floor drains in lower grade cleanrooms shall be fitted with traps or water seals designed to prevent back flow and shall be regularly cleaned, disinfected and maintained. III.1.11. Cleanrooms shall be supplied with a filtered air supply that maintains a positive pressure and/or an airflow relative to the background environment of a lower grade under all operational conditions and shall flush the area effectively. Adjacent rooms of different grades shall have an air pressure difference of a minimum of 10 Pascals (guidance value). Particular attention shall be paid to the protection of the critical zone. III.1.12. The above-referred requirements regarding air supplies and pressures may be modified where necessary to contain certain materials (e.g. pathogenic, highly toxic or radioactive products or live viral or bacterial materials). The modification may include positively or negatively pressurised airlocks that prevent the hazardous material from contaminating the surrounding areas. Where for reasons of containment, it is necessary for the air to flow into a critical zone, the source of the air shall be from an area of the same or higher grade. III.1.13. Decontamination of facilities (e.g. the cleanrooms and the heating, ventilation, and air-conditioning (HVAC) systems) and the treatment of air leaving a clean area, may be necessary for some operations based on a risk assessment (e.g. in the context of production involving pathogenic, highly toxic or radioactive materials or live viral or bacterial materials, when there is a risk of spreading to the environment, or when contamination has been detected). III.1.14. Airflow patterns within cleanrooms and zones shall be visualised and it shall be demonstrated that there is no ingress from lower grade to higher grade areas and that air does not travel from less clean areas (such as the floor), operators or equipment so that contamination may be transferred to the higher-grade areas. In particular, the following applies: (a) Where unidirectional airflow is required, visualisation studies shall be performed to determine compliance. (b) Where filled, closed products are transferred to an adjacent cleanroom of a lower grade via a small egress point, airflow visualisation studies shall demonstrate that air does not ingress from the lower grade cleanrooms to the grade B area. (c) Where air movement is shown to be a contamination risk to the clean area or critical zone, corrective actions, such as design improvement, shall be implemented. (d) Airflow pattern studies shall be performed both at rest and in operation (e.g. simulating operator interventions). Video recordings of the airflow patterns shall be retained. The outcome of the air visualisation studies shall be documented and be duly considered when establishing the facility’s environmental monitoring programme. III.1.15. Indicators of air pressure differences shall be installed between cleanrooms and/or between isolators and their background. Set points and the criticality of air pressure differences shall be addressed as part of the contamination control strategy. Air pressure differences identified as critical shall be continuously monitored and recorded. A warning system shall be in place to instantly indicate and warn operators of any failure in the air supply or reduction of air pressure differences (below set limits for those identified as critical). The warning signal shall not be overridden without an assessment and a procedure shall be available to outline the steps to be taken when a warning signal is given. Where alarm delays are set, these shall be assessed and justified. Other air pressure differences shall be monitored and recorded at regular intervals. III.1.16. Facilities shall be designed to permit observation of production activities from outside the grade A and B areas (e.g. through the provision of windows or remote cameras with a full view of the area and processes to allow observation and supervision without entry). This requirement shall be implemented when designing new facilities or during refurbishment of existing facilities. III.2.    Transfer of equipment and materials and movement of personnel III.2.1. The transfer of equipment and materials into and out of the cleanrooms and critical zones is one of the greatest potential sources of contamination and appropriate controls shall be therefore implemented. In particular, the transfer of materials, equipment, and components into the grade A or B areas shall be carried out via a unidirectional process. Where possible, items shall be sterilised and passed into these areas through double-ended sterilisers (e.g. through a double-door autoclave or a depyrogenation oven/tunnel) sealed into the wall. Where sterilisation upon transfer of the items is not possible, a validated procedure which achieves the same objective of not introducing contamination shall be implemented (e.g. using an effective transfer disinfection process, rapid transfer systems for isolators or, for gaseous or liquid materials, a bacteria-retentive filter). The removal of items from the grade A and B areas (e.g. materials, waste, environmental samples) shall be carried out via a separate unidirectional process. If this is not possible, a time-based separation of movement (incoming/exiting material) shall be considered and adequate controls shall be applied to avoid potential contamination. III.2.2. Only materials and equipment that have been included on an approved list, which is developed on the basis of an assessment during the validation of the transfer process, shall be transferred into the grade A or grade B areas via an airlock or pass-through hatches. Any unapproved items that require transfer shall be pre-approved as an exception. III.2.3. The movement of material or equipment from a lower grade or unclassified area to a higher-grade clean area shall be subject to cleaning and disinfection commensurate with the risks. Equipment and materials (intended for use in the grade A area) shall be protected when transiting through the grade B area. Appropriate risk assessment and mitigation measures shall be applied and recorded, including a specific disinfection and monitoring programme approved by the department responsible for quality assurance. III.2.4. Airlocks shall be designed and used to provide physical separation and to minimise microbial and particle contamination of the different areas and shall be used for material and personnel moving between different grades. Wherever possible, airlocks used for personnel movement shall be separated from those used for material movement. Where this is not possible, time-based separation of movement (personnel/material) shall be considered. Airlocks shall be flushed effectively with filtered air to ensure that the grade of the cleanroom is maintained. The final stage of the airlock shall, in the ‘at rest’ state, be of the same cleanliness grade (viable and total particle) as the cleanroom into which it leads. The use of separate change rooms for entering and leaving the grade B area is desirable. Where this is not possible, time-based separation of activities (ingress/egress) shall be considered. Where the risk of contamination is high, separate change rooms for entering and leaving production areas shall be used. III.2.5. The following aspects shall be considered in the design of airlocks: — Personnel airlocks  ( 5 ) : In general, hand-washing facilities shall be provided only in the first stage of the changing room and not be present in changing rooms directly accessing the grade B area. — Material airlocks  ( 6 ) : Airlock and pass-through hatches shall be designed to protect the higher-grade environment, for example by effective flushing with an active filtered air supply. For pass-through hatches and airlocks (for material and personnel), the entry and exit doors shall not be opened simultaneously. For airlocks leading to the grade A and grade B areas, an interlocking system shall be used. For airlocks leading to grade C and D areas, at least a visual and/or audible warning system shall be implemented. Where required to maintain the segregation of the area, a time delay between the closing and opening of interlocked doors shall be implemented. III.3.    Barrier technologies III.3.1. Isolators and RABS and associated processes shall be designed to provide protection through the separation of the grade A environment from the environment of the surrounding room. The hazards introduced from the entry or removal of items during processing shall be minimised by the implementation of appropriate technologies or validated systems. III.3.2. The design of the technology and processes used shall ensure that appropriate conditions are maintained in the critical zone to protect the exposed product during the operations. (a) Requirements for isolators: — The design of open isolators shall ensure grade A conditions with first air protection in the critical zone and unidirectional airflow that sweeps over and away from the exposed products during processing. — The design of closed isolators shall ensure grade A conditions with adequate protection for the exposed products during processing. Airflow may not be fully unidirectional in closed isolators where simple operations are conducted. However, any turbulent airflow  ( 7 ) shall not increase the risk of contamination of the exposed product. Where processing lines are included in closed isolators, grade A conditions shall be ensured with first air protection in the critical zone and unidirectional airflow that sweeps over and away from the exposed products during processing. — Negative pressure isolators shall only be used when containment of the product is considered essential (e.g. radiopharmaceutical products) and specialised risk control measures shall be applied to ensure that the critical zone is not compromised. (b) Requirements for RABS: The design of RABS shall ensure grade A conditions with unidirectional airflow and first air protection in the critical zone. A positive airflow from the critical zone to the supporting background environment shall be maintained. III.3.3. The background environment for isolators or RABS shall ensure that the risk of transfer of contamination is minimised. (a) Requirements for isolators: — The background classification applied shall be based on a risk assessment and justified as part of the contamination control strategy. The background environment for open isolators shall generally correspond to a minimum of grade C, while the background for closed isolators shall correspond to a minimum of grade D. — Key considerations when performing the risk assessment for the contamination control strategy of an isolator include the bio-decontamination programme, the extent of automation, the impact of glove manipulations that may potentially compromise ‘first air’ protection of the critical process points, the impact of potential loss of barrier/glove integrity, transfer mechanisms used and activities such as set-up or maintenance that may require the doors to be opened prior to the final bio-decontamination of the isolator. Where additional process risks are identified, a higher grade of background shall be implemented unless appropriately justified in the contamination control strategy. — Airflow pattern studies shall be performed at the interfaces of open isolators to demonstrate the absence of air ingress. (b) Requirements for RABS: The background environment for RABS used for aseptic processing shall correspond to a minimum of grade B and airflow pattern studies shall be performed to demonstrate the absence of air ingress during interventions, including door openings if applicable. III.3.4. The materials used for glove systems (for both isolators and RABS) shall have appropriate mechanical and chemical resistance. The frequency of glove replacement shall be defined as part of the contamination control strategy. (a) Requirements for Isolators: — Leak testing of the glove system shall be performed using a suitable methodology having regard to the intended use and the risks involved. The testing shall be performed at defined intervals. In general, glove integrity testing shall be performed at least at the beginning and at the end of each batch or campaign. Additional glove integrity testing may be necessary depending on the campaign length. Glove integrity monitoring shall include a visual inspection associated with each use and following any manipulation that may affect the integrity of the system. For manual aseptic processing activities (i.e. the operator manually compounds, fills, places and/or seals an open container with sterile product) where a single unit or a small-size batch is produced, the frequency of integrity verification may be based on other criteria, such as the beginning and end of each manufacturing session. — Integrity/leak testing of the isolator system shall be performed at defined intervals. (b) Requirements for RABS: Gloves used in the grade A area shall be sterilised before installation and sterilised or effectively bio-decontaminated by a validated method prior to each manufacturing campaign. If during operation there is exposure to the background environment, there shall be disinfection using an approved methodology after each exposure. Gloves shall be visually examined with each use, and integrity testing shall be performed at periodic intervals. III.3.5. Decontamination methods (cleaning and bio-decontamination, and – where applicable – inactivation for biological materials) shall be duly documented. The cleaning process prior to the bio-decontamination step is essential as any residues that remain may inhibit the effectiveness of the decontamination process. It shall be demonstrated that the cleaning and bio-decontamination agents used do not have an adverse impact on the product produced within the RABS or isolator. (a) Requirements for isolators: The bio-decontamination process of the interior shall be automated, validated and controlled within defined cycle parameters and shall include a sporicidal agent in a suitable form (e.g. gaseous or vaporised form). Gloves shall be appropriately extended with fingers separated to ensure contact with the agent. Methods used (cleaning and sporicidal bio-decontamination) shall render the interior surfaces and critical zone of the isolator free from viable microorganisms. (b) Requirements for RABS: The sporicidal disinfection shall include the routine application of a sporicidal agent using a method that has been validated and demonstrated to cover all areas of the interior surfaces and ensure a suitable environment for aseptic processing. III.4.    Cleanroom and clean air equipment qualification III.4.1. Cleanrooms and clean air equipment such as unidirectional airflow units  ( 8 ) , RABS and isolators, used for the manufacture of sterile products/aseptic manufacturing, shall be qualified according to the required characteristics of the environment. Each manufacturing operation requires an appropriate environmental cleanliness level in the operational state in order to minimise the risk of contamination of the product or materials being handled. Appropriate cleanliness levels in the ‘at rest’ and ‘operational’ states shall also be maintained. III.4.2. Cleanrooms and clean air equipment shall be qualified in accordance with Annex V. Through the qualification of cleanrooms and clean air equipment, the level of compliance of a classified cleanroom or clean air equipment with the relevant requirements having regard to the intended use is assessed  ( 9 ) . The following is part of the qualification requirements (where relevant to the design/operation of the installation): — installed filter system leakage and integrity testing; — airflow tests – volume and velocity; — air pressure difference test; — airflow direction test and visualisation; — microbial airborne and surface contamination; — temperature measurement test; — relative humidity test; — recovery test; — containment leak test. III.4.3. Cleanroom classification is part of the cleanroom qualification. Through the cleanroom classification the level of air cleanliness is assessed by measuring the total particle concentration. Classification activities shall be scheduled and performed so as to avoid any impact on process or product quality. For example, initial classification shall be performed during simulated operations and reclassification performed during simulated operations or during aseptic process simulation. III.4.4. For cleanroom classification, the total amount of particles equal to or greater than 0,5 and 5 μm shall be measured. This measurement shall be performed both at rest and in simulated operations in accordance with the limits specified in Table 1: — ‘at rest’ state is the condition whereby the installation of all the utilities is complete including any functioning HVAC, with the main manufacturing equipment installed as specified but not operating and without personnel present in the room. — The total particle limits given in Table 1 for the ‘at rest’ state shall be achieved after a ‘clean up’ period on completion of operations and line clearance/cleaning activities. The ‘clean up’ period (guidance value of less than 20 minutes) shall be determined during the qualification of the rooms, documented and adhered to in procedures to reinstate a qualified state of cleanliness if disrupted during operation. — ‘in operation’ state is the condition where the installation of the cleanroom is complete, the HVAC system fully operational, equipment installed and functioning in the manufacturer’s defined operating mode with the maximum number of personnel present performing or simulating routine operational work. Table 1 Maximum permitted total particle concentration for classification   Maximum limits for total particle ≥ 0,5 μm/m 3 Maximum limits for total particle ≥ 5 μm/m 3 Grade at rest in operation at rest in operation A 3 520 3 520 Not specified  ( 10 ) Not specified  ( 10 ) B 3 520 352 000 Not specified  ( 10 ) 2 930 C 352 000 3 520 000 2 930 29 300 D 3 520 000 Not pre-defined  ( 11 ) 29 300 Not pre-defined  ( 11 ) III.4.5. For classification of the cleanroom, the minimum number of sampling locations and their positioning as set out in ISO 14644 Part 1 shall be followed. For the aseptic processing area and the background environment (the grade A and grade B areas, respectively), additional sample locations shall be considered as appropriate having regard to the risks, and all critical processing areas such as the point of fill and container closure feeder bowls shall be evaluated. Critical processing locations shall be determined on the basis of a documented risk assessment and knowledge of the process and operations to be performed in the area. III.4.6. The speed of air supplied by unidirectional airflow systems shall be clearly justified in the qualification protocol including the location for air speed measurement. Air speed shall be designed, measured and maintained to ensure that appropriate unidirectional air movement provides protection for the product and open components at the working position (e.g. where high-risk operations occur and where product and/or components are exposed). Unidirectional airflow systems shall provide a homogeneous air speed in a range of 0,36–0,54 m/s (guidance value) at the working position, unless otherwise scientifically justified in the contamination control strategy. Airflow visualisation studies shall correlate with the air speed measurement. III.4.7. The microbial contamination level of the cleanrooms shall be determined as part of the cleanroom qualification. The number of sampling locations shall be based on a documented risk assessment and the results obtained from room classification, air visualisation studies and knowledge of the process and operations to be performed in the area. The maximum limits for microbial contamination during qualification for each grade are given in Table 2. Qualification shall include both ‘at rest’ and ‘in operation’ states. Table 2 Maximum permitted microbial contamination level during qualification Grade Air sample CFU  ( 13 ) /m 3 Settle plates (diameter 90 mm) CFU/4 hours  ( 13 ) Contact plates (diameter 55 mm) CFU/plate A No growth B 10 5 5 C 100 50 25 D 200 100 50 Note 1 : All methods indicated for a specific grade in the table shall be used for qualifying the area of that specific grade. If one of the methods tabulated is not used, or alternative methods are used, the approach taken shall be appropriately justified. Note 2: Limits are applied using CFU throughout the document. If different or new technologies are used that present results in a manner different from CFU, the manufacturer shall scientifically justify the limits applied and where possible correlate them to CFU. Note 3: For the qualification of personnel gowning, the limits given for contact plates and glove prints in Table 6 shall apply. Note 4: Sampling methods shall not pose a risk of contamination to the manufacturing operations. III.4.8. The requalification of cleanrooms and clean air equipment shall be carried out periodically following defined procedures. The requalification shall include at least the following: — cleanroom classification (total particle concentration); — integrity test of final filters; — airflow volume measurement; — verification of air pressure difference between rooms; — air velocity test: this test is required for filling zones supplied with unidirectional airflow (e.g. when filling terminally sterilised products or background to grade A and RABS). In the case of grade B, C and D areas, the conduct of the air velocity test shall be based on a risk assessment, which shall be documented as part of the contamination control strategy. Finally, for grades with non-unidirectional airflow, the air velocity test shall be replaced by a measurement of recovery testing. III.4.9. The maximum time interval for requalification of grade A and B areas is 6 months, while for grade C and D areas the maximum time interval for requalification is 12 months. In addition, appropriate requalification consisting of at least the above tests shall also be carried out following completion of a remedial action implemented to rectify an out of compliance in the equipment or premises or, as appropriate, after changes to equipment, premises or processes. Examples of changes requiring requalification include the interruption of air movement which affects the operation of the installation, a change in the design of the cleanroom or of the operational setting parameters of the HVAC system, or maintenance activities affecting the operation of the installation (e.g. change of final filters). III.5.    Disinfection III.5.1. Particular attention shall be paid to the disinfection of cleanrooms. Specifically, cleanrooms shall be cleaned and disinfected thoroughly in accordance with a written programme. More than one type of disinfecting agent shall be used to ensure that, where they have different modes of action, their combined usage is effective against bacteria and fungi. Disinfection shall include the periodic use of a sporicidal agent. Monitoring to assess the effectiveness of the disinfection programme and to detect changes in types of microbial flora (e.g. organisms resistant to the disinfection regime currently in use) shall be undertaken regularly. For disinfection to be effective, it is necessary to previously clean to remove surface contamination. Additionally, in some cases, a cleaning process shall be implemented to effectively remove disinfectant residues. III.5.2. The disinfection process shall be validated. Validation studies shall demonstrate the suitability and effectiveness of the disinfectants in the specific manner in which they are used and on the type of surface material, or representative material if justified, and shall support the in-use expiry periods of prepared solutions. III.5.3. Disinfectants and detergents used in grade A and grade B areas shall be sterile prior to use. Disinfectants used in grade C and D may also have to be sterile when this is considered appropriate in the contamination control strategy. Where the disinfectants and detergents are diluted/prepared by the sterile product manufacturer, this shall be done in a manner to prevent contamination and there shall be monitoring for microbial contamination. Dilutions shall be kept in previously cleaned containers (and sterilised where applicable) and shall only be stored for the relevant defined period. If the disinfectants and detergents are supplied ‘ready-made’, the results from certificates of analysis or conformance may be accepted subject to successful completion of the appropriate vendor qualification. III.5.4. Where fumigation or vapour disinfection (e.g. Vapour-phase Hydrogen Peroxide) of cleanrooms and associated surfaces are used, the effectiveness of the fumigation agent and of the dispersion system used shall be understood and validated. SECTION IV EQUIPMENT IV.1. A written, detailed description of the equipment shall be available (including process and instrumentation diagrams as appropriate). This shall form part of the initial qualification package and shall be kept up to date. IV.2 Equipment monitoring requirements shall be established as part of the qualification. Process and equipment alarm events shall be acknowledged and evaluated for trends. The frequency at which alarms are assessed shall be based on their criticality (critical alarms shall be reviewed immediately). IV.3. As far as possible, equipment, fittings and services shall be designed and installed so that operations, maintenance and repairs can be performed outside the cleanroom. If maintenance has to be performed in the cleanroom and the required standards of cleanliness and/or asepsis cannot be maintained, precautions such as restricting access to the work area to specified personnel or the generation of clearly defined work protocols and maintenance procedures shall be considered. Additional cleaning, disinfection and environmental monitoring shall also be considered. If sterilisation of equipment is required, it shall be carried out, wherever possible, after complete reassembly. IV.4. The cleaning process shall be validated as being able to remove any residue or debris that would detrimentally impact the effectiveness of the disinfecting agent used, and to minimise the chemical, microbial and particulate contamination of the product during the process and prior to disinfection. IV.5. For aseptic processes, direct and indirect product contact parts shall be sterilised. For the purpose of complying with this requirement, ‘direct product contact parts’ are those parts of the equipment that the product passes through, such as filling needles or pump, while ‘indirect product contact parts’ are those parts of the equipment that are not in contact with the product but may come into contact with other sterilised surfaces that are critical to the overall product sterility (e.g. sterilised items such as stopper bowls and guides, and sterilised components). IV.6. All equipment such as sterilisers, air handling systems (including air filtration) and water systems shall be subject to qualification, monitoring and planned maintenance. Upon completion of maintenance, their return to use shall be approved. IV.7. Where unplanned maintenance of equipment critical to the sterility of the product is to be carried out, an assessment of the potential impact to the sterility of the product shall be performed and recorded. IV.8. A conveyor belt shall not pass through a partition between a grade A or B area and a processing area of lower air cleanliness, unless the belt itself is continually sterilised (e.g. in a sterilising tunnel). IV.9. Particle counters, including sampling tubing, shall be qualified. The manufacturer’s recommended specifications shall be considered for tube diameter and bend radii. Tube length shall typically be no longer than 1 m unless justified and the number of bends shall be minimised. Portable particle counters with a short length of sample tubing shall be used for classification purposes. Isokinetic sampling heads  ( 14 ) shall be used in unidirectional airflow systems. They shall be oriented appropriately and positioned as close as possible to the critical location to ensure that samples are representative. SECTION V UTILITIES V.1.    General requirements V.1.1. The nature and extent of controls applied to utility systems shall be commensurate with the risk to the quality of the product associated with the utility. The impact of the utility on the quality of the product is to be determined via a risk assessment and documented as part of the contamination control strategy. The following utilities can generally be considered associated with a higher risk: — utilities that are in direct contact with the product, e.g. water for washing and rinsing, gases and steam for sterilisation; — contact materials that will ultimately become part of the product; — contact surfaces that come into contact with the product; — utilities that otherwise directly impact the product. V.1.2. Utilities shall be designed, installed, qualified, operated, maintained and monitored in a manner that ensures that the utility system functions as expected. V.1.3. Results for critical parameters and critical quality attributes of high-risk utilities shall be subject to regular trend analysis to ensure that the system capabilities remain appropriate. V.1.4. Records of the utility system installation shall be kept throughout the utility system’s life-cycle, including drawings and schematic diagrams, construction materials and system specifications. Important information that shall be kept includes: — pipeline flow direction, slopes, diameter and length; — tank and vessel details; — valves, filters, drains, sampling and user points. V.1.5. Pipes, ducts and other utilities shall not be present in cleanrooms. If unavoidable, then they shall be installed so that they do not create recesses, unsealed openings or surfaces that are difficult to clean. In addition, the installation shall allow the cleaning and disinfection of the outer surface of the pipes. V.2.    Water systems   ( 15 ) V.2.1. Water treatment plants and distribution systems shall be designed, constructed, installed, commissioned, qualified, monitored and maintained so as to prevent microbiological contamination and ensure a reliable source of water of an appropriate quality. In particular, measures shall be taken to minimise the risk of presence of particulates, microbial contamination/proliferation and endotoxin/pyrogen (e.g. sloping of piping to provide complete drainage and the avoidance of dead legs  ( 16 ) ). Where filters are included in the system, special attention shall be paid to their monitoring and maintenance. V.2.2. Water systems shall be qualified and validated to maintain the appropriate levels of physical, chemical and microbial control, taking the effect of seasonal variations into account. V.2.3. Water flow shall remain turbulent through the pipes in water distribution systems to minimise the risk of microbial adhesion, and subsequent biofilm formation. The flow rate shall be established during the qualification and be routinely monitored. V.2.4. Water for injections shall be produced from water that meets the specifications defined during the qualification process and it shall be stored and distributed in a manner that minimises the risk of microbial growth (e.g. by constant circulation at a temperature above 70 °C). Moreover, water for injections shall be produced by distillation or by a purification process that is equivalent to distillation, such as reverse osmosis coupled with other appropriate techniques such as electrodeionisation (EDI), ultrafiltration or nanofiltration. V.2.5. Where water for injection storage tanks are equipped with hydrophobic bacteria retentive vent filters, the filters shall not be a source of contamination and the integrity of the filter shall be tested before installation and after use. Controls shall be put in place to prevent condensation formation on the filter (e.g. by heating). V.2.6. To minimise the risk of biofilm formation, sterilisation, disinfection or regeneration of water systems shall be carried out according to a predetermined schedule and also as a remedial action following out-of-limit or specification results. When chemicals are used to disinfect a water system, a validated rinsing/flushing procedure shall be subsequently performed. Additionally, water shall be tested after disinfection/regeneration. Chemical testing results shall be checked before the water system is returned to use and it shall be verified that microbiological/endotoxin results are within specification before batches manufactured using water from the system are considered for certification/release. V.2.7. Regular ongoing chemical and microbial monitoring of water systems shall be performed to ensure that the water continues to meet compendial requirements. Alert levels shall be set on the basis of the initial qualification data and thereafter be periodically reassessed on the basis of data obtained during subsequent re-qualifications, routine monitoring and investigations. Review of ongoing monitoring data shall be carried out to identify any adverse trend in the performance of the system. Sampling programmes shall be based on the qualification data and shall consider the potential worst case sampling locations ensuring that at least one representative sample of the water that is used for manufacturing processes is included every day as well as any other additional requirement that may be necessary in accordance with the contamination control strategy. To ensure that representative water samples are obtained for analysis on a regular basis, sampling programmes shall address all outlets and points of use at a specified interval. V.2.8. Alert level excursions shall be documented and reviewed and include an investigation to determine whether the excursion is a single (isolated) event or if the results are indicative of an adverse trend or of the deterioration of the system. Each action limit excursion shall be investigated to determine the probable root cause(s) and any potential impact on the product quality and manufacturing processes. V.2.9. Water for injection systems shall include continuous monitoring systems such as Total Organic Carbon (TOC) and conductivity, as these may give a better indication of overall system performance than discrete sampling. Sensor locations shall be based on risk. V.2.10. Water used in production shall comply with the current monograph of the relevant Pharmacopeia. V.3.    Steam used as a direct sterilising agent V.3.1. Feed water to a pure steam (clean steam) generator shall be appropriately purified. Pure steam generators shall be designed, qualified and operated in a manner to ensure that the quality of the steam produced meets the defined chemical and endotoxin levels. V.3.2. Steam used as a direct sterilising agent shall be of a suitable quality and shall not contain additives at a level that could cause contamination to the product or the equipment. In the case of generators supplying pure steam for the direct sterilisation of materials or product-contact surfaces (e.g. porous hard-good autoclave loads), the steam condensate shall meet the requirements of the current monograph for water for injections of the relevant Pharmacopeia (microbial testing is not mandatory for steam condensate). A suitable sampling schedule shall also be in place to ensure that representative pure steam is obtained for analysis on a regular basis. Other aspects of the quality of the pure steam used for sterilisation shall be assessed periodically against validated parameters, including – unless otherwise justified – non-condensable gases, dryness value (dryness fraction) and superheat. V.4.    Gases and vacuum systems V.4.1. Gases that come in direct contact with the product or primary container surfaces shall be of appropriate chemical, particulate and microbial quality. All relevant parameters, including oil and water content, shall be specified taking into account the use and type of the gas, the design of the gas generation system and, where applicable, comply with the current monograph of the relevant Pharmacopeia or the product quality requirement. V.4.2. Gases used in aseptic processes shall be filtered through a sterilising grade filter  ( 17 ) (with a nominal pore size of a maximum of 0,22 μm) at the point of use. Where the filter is used on a batch basis (e.g. for filtration of gas used for overlay of aseptically filled products) or as product vessel vent filter, the results of the integrity test shall be reviewed as part of the batch certification/release process. Any transfer pipework or tubing that is located after the final sterilising grade filter shall be sterilised. When gases are used in the process, microbial monitoring of the gas shall be performed periodically at the point of use. V.4.3. Where backflow from vacuum or pressure systems poses a potential risk to the product, mechanism(s) shall be put in place to prevent backflow when the vacuum or pressure system is shut off. V.5.    Heating and cooling and hydraulic systems V.5.1. Major items of equipment associated with hydraulic, heating and cooling systems shall, where possible, be located outside the filling room. Appropriate controls shall be implemented to contain any spillage or cross contamination associated with the system fluids. V.5.2. Appropriate systems shall be put in place to ensure that any leak from these systems that could present a risk to the product are detected (e.g. an indication system for leakage). SECTION VI PERSONNEL VI.1. The manufacturer shall ensure that there are sufficient personnel, suitably qualified, trained and experienced in the manufacture and testing of sterile products and any of the specific manufacturing technologies used in the site’s manufacturing operations. VI.2. Only the minimum number of personnel required shall be present in cleanrooms. The maximum number of operators in cleanrooms shall be determined and documented. During activities such as initial qualification and the aseptic process simulation the maximum number of operators that can be present in the cleanroom shall be duly considered so as not to compromise sterility assurance. VI.3. All personnel including those performing cleaning, maintenance, monitoring and those that access cleanrooms shall receive regular training on aspects relevant to the manufacture of sterile products/aseptic manufacturing, including on gowning, the basic elements of microbiology and hygiene, with a specific focus on cleanroom practices, contamination control, aseptic techniques and the protection of sterile products (for those operators entering the grade B cleanrooms and/or intervening into grade A) and the potential consequences to the treated animals if the product is not sterile / fails to meet the required quality specifications. The level of training shall be based on the criticality of the function and the area where the personnel are working. VI.4. Personnel accessing grade A and B areas shall be trained for aseptic gowning and aseptic behaviour. Compliance with aseptic gowning procedures is to be confirmed by means of an assessment prior to starting their functions and shall be periodically reassessed (at least annually). The assessment process shall involve both visual and microbial assessment (using monitoring locations such as gloved fingers, forearms, chest and hood (facemask/forehead). VI.5. Unsupervised access to the grade A and grade B areas where aseptic operations are or will be conducted shall be restricted to appropriately qualified personnel, who have passed the gowning assessment and have participated in a successful aseptic process simulation. Unqualified personnel shall not enter grade B cleanrooms or grade A in operation. If needed in exceptional cases, manufacturers shall establish written procedures outlining the process by which unqualified personnel can be brought into the grade B and A areas. An authorised person from the manufacturer shall supervise the unqualified personnel during their activities and assess the impact of these activities on the cleanliness of the area. Access by these persons shall be assessed and recorded. VI.6. A process shall be put in place for the disqualification of personnel based on aspects of ongoing assessment and/or identification of an adverse trend from the personnel monitoring programme and/or after being implicated in a failed aseptic process simulation. Once disqualified, retraining and requalification shall be completed before permitting the operator to have any further involvement in aseptic practices. For operators entering grade B cleanrooms or performing intervention into grade A, it is advised that the requalification includes participation in a successful aseptic process simulation. VI.7. High standards of personal hygiene and cleanness are essential. When a heath condition that may introduce an undue microbial hazard is declared by the relevant personnel or otherwise becomes apparent, access to the cleanroom shall be barred. Health conditions and actions to be taken with regard to personnel that can introduce an undue microbial hazard shall be documented in relevant procedures. VI.8. Personnel involved in the handling/processing of materials of human/animal origin or of cultures of micro-organisms, other than those used in the current manufacturing process, or in other activities that may have a negative impact to quality (e.g. microbial contamination), shall not enter clean areas unless clearly defined and effective decontamination and entry procedures have been followed and documented. VI.9. Wristwatches, make-up, jewellery, other personal items such as mobile phones and any other non-essential items shall not be allowed in clean areas. Electronic devices used in cleanrooms, e.g. mobile phones and tablets, that are supplied by the manufacturer solely for use in the cleanrooms, may be acceptable if suitably designed to permit cleaning and disinfection commensurate with the grade in which they are used. The use and disinfection of such equipment shall be included in the contamination control strategy. VI.10. Cleanroom gowning and hand washing shall be done in accordance with written procedures designed to minimise the contamination of cleanroom clothing and/or the transfer of contaminants to the clean areas. VI.11. The clothing and its quality shall be appropriate for the process and the grade of the working area. It shall be worn in such a way as to protect the product from contamination. When the required type of clothing needs to protect the operator from the product, it shall also be ensured that the protection of the product from contamination is not compromised. Garments shall be visually checked for cleanliness and integrity immediately prior to and after gowning. Gown integrity shall also be checked upon exit. Prior to the use of sterilised garments and eye coverings, it shall be checked that they have been subject to the sterilisation process, that they are within their specified hold time and that the packaging has not been tampered. Reusable garments (including eye coverings) are to be replaced if damage is identified, or at a set frequency that is determined during qualification studies. The qualification of garments shall consider any necessary garment testing requirements, including damage to garments that may not be identified by visual inspection alone. VI.12. A description of clothing typically required for each cleanliness grade is given below: (a) Grade B (including access/interventions into grade A): — appropriate garments that are dedicated for use under a sterilised suit shall be worn before gowning; — appropriately sterilised, non-powdered, rubber or plastic gloves shall be worn while donning the sterilised garments; — sterile headgear shall enclose all hair (including facial hair) and, where separate from the rest of the gown, it shall be tucked into the neck of the sterile suit; — a sterile facemask and sterile eye coverings (e.g. goggles) shall be worn to cover and enclose all facial skin and prevent the shedding of droplets and particles; — appropriate sterilised footwear (e.g. over-boots) shall be worn; — trouser legs shall be tucked inside the footwear and garment sleeves shall be tucked into a second pair of sterile gloves worn over the pair worn while donning the gown; — the protective clothing shall minimise shedding of fibres or particles and retain particles shed by the body. The particle shedding and the particle retention efficiencies of the garments is to be assessed during the garment qualification; — garments shall be packed and folded in such a way as to allow operators to don the gown without contacting the outer surface of the garment and to prevent the garment from touching the floor. (b) Grade C: — hair, beards and moustaches shall be covered; — a single or two-piece trouser suit gathered at the wrists and with high neck and appropriately disinfected shoes or overshoes shall be worn; they shall minimise the shedding of fibres and particles; — additional gowning, including gloves and facemask, may be required in grade C areas when performing activities that pose a risk of contamination. (c) Grade D: — hair, beards and moustaches shall be covered; — a general protective suit and appropriately disinfected shoes or overshoes shall be worn; — appropriate measures shall be taken to avoid any ingress of contaminants from outside the clean area; — additional gowning including gloves and facemask may be required in grade D areas when performing activities that pose a risk of contamination. VI.13. Cleanroom gowning shall take place in change rooms of an appropriate cleanliness grade to ensure that gown cleanliness is maintained. Outdoor clothing including socks (other than personal underwear) shall not be brought into changing rooms leading directly to grade B and C areas. In addition, a single or two-piece facility trouser suit, covering the full length of the arms and the legs, and facility socks covering the feet, shall be worn before entry to change rooms for grades B and C. Facility suits and socks shall not present a risk of contamination to the gowning area or processes. VI.14. Every operator entering grade B or A areas shall gown into clean, sterilised protective garments (including eye coverings and masks) of an appropriate size at each entry. The maximum period for which the sterilised gown may be worn before replacement during a shift shall be defined as part of the garment qualification. VI.15. Gloves shall be regularly disinfected during operations. Garments and gloves shall be changed immediately if they become damaged and present any risk of product contamination. VI.16. Reusable clean area clothing shall be cleaned in a laundry facility adequately segregated from production operations, using a qualified process ensuring that the clothing is not damaged or contaminated by fibres or particles during the repeated laundry process. Laundry facilities used shall not introduce a risk of contamination or cross-contamination. After washing and before packing, garments shall be visually inspected for damage and visual cleanliness. The garment management processes shall be established as part of the garment qualification programme and shall include a maximum number of laundry and sterilisation cycles. VI.17. Activities in clean areas that are not critical to the production processes shall be kept to a minimum, especially when aseptic operations are in progress. With a view to avoid excessive shedding of particles and organisms, movement of personnel shall be slow, controlled and methodical. Operators performing aseptic operations shall adhere to aseptic technique at all times to prevent changes in air currents that may introduce air of lower quality into the critical zone. In addition, movement adjacent to the critical zone shall be restricted and the obstruction of the path of the unidirectional (first air) airflow shall be avoided. SECTION VII PRODUCTION AND SPECIFIC TECHNOLOGIES VII.1.    Terminally sterilised products   ( 18 ) VII.1.1. Preparation of components and materials shall be performed in at least a grade D cleanroom in order to limit the risk of microbial, endotoxin/pyrogen and particle contamination, so that the product is suitable for sterilisation. However, where the product is at a high or unusual risk of microbial contamination (e.g. the product actively supports microbial growth, the product must be held for long periods before filling or the product is not processed mostly in closed vessels), then preparation shall be carried out in at least a grade C environment. Preparation of ointments, creams, suspensions and emulsions shall also be carried out in at least a grade C environment before terminal sterilisation. By way of derogation from the C grade environment as foreseen above, in exceptional cases, e.g. where the manufacturing process involves the generation of powder/dust that cannot be prevented by reasonable means, preparation of products to be terminally sterilised may be performed in a grade D environment. For the implementation of grade D in this exceptional case, the manufacturer shall be required to perform a risk assessment and apply suitable measures to ensure that there is no negative impact on the quality of the product. This shall be documented as part of the contamination control strategy. VII.1.2. Primary packaging containers and components shall be cleaned using validated processes to ensure that particle, endotoxin/pyrogen and bioburden contamination is appropriately controlled. VII.1.3. Filling of products for terminal sterilisation shall be carried out in at least a grade C environment. However, if the product is at an unusual risk of contamination from the environment (for example, the filling operation is slow, the containers are wide necked or are necessarily exposed for more than a few seconds before closing), the product shall be filled in grade A with at least a grade C background, unless additional measures to ensure the absence of a negative impact to the quality of the product are implemented, in which case the filling operation shall take place – as a minimum – in a grade D environment. VII.1.4. To reduce the level of bioburden and particles prior to filling into the final product container, processing of the bulk solution shall include, where possible, a filtration step with a microorganism retaining filter and a maximum time between preparation and filling shall be set. VII.1.5. Examples of operations to be carried out in the various grades are given in Table 3. Table 3 Examples of operations and grades for terminally sterilised preparation and processing operations Grade A Filling of products when unusual/high risk of microbial contamination, unless a lower grade can be justified in accordance with Section VII.1.3. Grade C Preparation of solutions when unusual/high risk of microbial contamination, unless grade D can be justified in accordance with the second subparagraph of Section VII.1.1. Filling of products (other than when grade A is required), unless grade D can be justified in accordance with Section VII.1.3. Grade D Preparation of solutions and components for subsequent filling. VII.2.    Aseptic preparation and processing VII.2.1. The aseptic process shall be documented as part of the contamination control strategy. Specifically, the risks associated with the aseptic process, and any associated requirements, shall be identified, assessed and appropriate controls shall be identified including the acceptance criteria for these controls, requirements for monitoring and the review of their effectiveness. Methods and procedures to control those risks shall be clearly described and implemented. Accepted residual risks shall be formally documented. VII.2.2. Precautions to minimise microbial, endotoxin/pyrogenic and particle contamination in the site shall be described in the contamination control strategy and shall be implemented during the preparation of the aseptic environment, during all processing stages (including the stages before and after bulk product sterilisation), and until the product is sealed in its final container. The presence of materials liable to generate particles and fibres shall be minimised in cleanrooms. VII.2.3. Where possible, the use of equipment such as RABS, isolators or other systems shall be used in order to reduce the need for critical interventions  ( 19 ) into grade A and to minimise the risk of contamination. Robotics and automation of processes may also be considered to eliminate direct human critical interventions (e.g. dry heat tunnel, automated lyophilizer loading, sterilisation in place). VII.2.4. Examples of operations to be carried out in the various environmental grades are given in Table 4. Table 4 Examples of operations and grades for aseptic preparation and processing operations Grade A — Aseptic assembly of filling equipment. — Connections made under aseptic conditions (where sterilised product contact surfaces are exposed) that are post the final sterilising grade filter. These connections shall be sterilised by steam-in-place whenever possible. — Aseptic compounding and mixing. — Replenishment of sterile bulk product, containers and closures. — Removal and cooling of unprotected (e.g. with no packaging) items from sterilisers. — Staging and conveying of sterile primary packaging components in the aseptic filling line while not wrapped. — Aseptic filling, sealing of containers such as ampoules, vial closure, transfer of open or partially stoppered vials. — Loading of a lyophilizer. Grade B — Background support for grade A (when not in an isolator). — Conveying or staging, while protected from the surrounding environment, of equipment, components and ancillary items for introduction into grade A. Grade C — Preparation of solutions to be filtered including sampling and dispensing. Grade D — Cleaning of equipment. — Handling of components, equipment and accessories after cleaning. — Assembly under HEPA filtered airflow of cleaned components, equipment and accessories prior to sterilisation. — Assembly of closed and sterilised single use systems using intrinsic sterile connection devices  ( 20 ) VII.2.5. For products where the final formulation cannot be filtered, the following measures shall be considered as appropriate: — all product and component contact equipment shall be sterilised prior to use; — all raw materials or intermediates shall be sterilised and aseptically added; — bulk solutions or intermediates shall be sterilised. VII.2.6. The unwrapping, assembly and preparation of sterilised equipment, components and ancillary items with direct or indirect product contact shall be treated as an aseptic process and performed in grade A with a grade B background. The filling line set-up and filling of the product shall be treated as an aseptic process and performed in grade A with a grade B background. Where an isolator is used, the background shall be in accordance with Section III.3.3 of this Annex. VII.2.7. Preparation and filling of products such as ointments, creams, suspensions and emulsions shall be performed in grade A with a grade B background when the product and components are exposed to the environment and the product is not subsequently filtered (via a sterilising grade filter) or terminally sterilised. Where an isolator or RABS is used, the background shall be in accordance with Section III.3.3 of this Annex. VII.2.8. Aseptic connections shall be performed in grade A with a grade B background unless subsequently sterilised in place or conducted with intrinsic sterile connection devices that minimise any potential contamination from the immediate environment. Intrinsic sterile connection devices shall be designed to mitigate the risk of contamination. Where an isolator is used, the background shall be in accordance with Section III.3.3 of this Annex. Aseptic connections shall be appropriately assessed and their effectiveness verified. VII.2.9. Aseptic manipulations (including non-intrinsic sterile connection devices) shall be minimised through the use of engineering design solutions such as preassembled and sterilised equipment. Whenever feasible, product contact piping and equipment shall be pre-assembled and sterilised in place. VII.2.10. A list of allowed and qualified interventions, both inherent  ( 21 ) and corrective, that may occur during production shall be set. The type of inherent and corrective interventions, and how to perform them, shall be first evaluated in accordance with quality risk management principles and the outcome of the aseptic process simulation and be kept up to date. Interventions shall be carefully designed to ensure that the risk of contamination of the environment, process and product is effectively minimised, including consideration of any impact on air-flows and critical surfaces  ( 22 ) and products. Engineering solutions shall be used whenever possible to minimise incursion by operators during the intervention. Aseptic technique shall be observed at all times, including the use of sterile tools for manipulations. Non-authorised/non-qualified interventions shall only be performed in exceptional circumstances, with due consideration of the risks associated with the intervention and with the authorisation of the quality unit. Moreover, the details of the intervention conducted shall be recorded, be thoroughly assessed by the quality department and be duly considered during batch release. VII.2.11. Interventions and stoppages shall be recorded in the batch record. Each line stoppage or intervention shall be sufficiently documented in batch records with the associated time, duration of the event, and operators involved. VII.2.12. The duration of each aspect of aseptic preparation and processing shall be minimised as far as possible and validated maximum times shall be set including: — the holding time between equipment, component, and container cleaning, drying and sterilisation; — the holding time for the sterilised equipment, components, and containers before use and during filling/assembly; — the holding time for a decontaminated environment, such as the RABS or isolator before use; — the time between the start of the preparation of a product and its sterilisation or filtration through a microorganism-retaining filter (if applicable), through to the end of the aseptic filling process. A maximum permissible time for each product shall be set taking into account its composition and the method of storage; — the holding time for the sterilised product prior to filling; — the aseptic processing time; and — the filling time. VII.2.13. Aseptic operations (including aseptic process simulation) shall be monitored on a regular basis by personnel with specific expertise in aseptic processing to verify the correct performance of operations, including operator’s behaviour in the cleanroom, and to address inappropriate practices if detected. VII.3.    Finishing activities VII.3.1. Open primary packaging containers shall be maintained under grade A conditions with the appropriate background for the technology as described in Section III.3.3. For vials that are partially stoppered or prefilled syringes, the additional considerations as set forth in Section VII.7.6 apply also. VII.3.2. Final containers shall be closed by appropriately validated methods. VII.3.3. Where final containers are closed by fusion, e.g. Blow-Fill-Seal, Form-Fill-Seal, small and large volume parenteral bags, glass or plastic ampoules, the critical parameters and variables that affect seal integrity shall be set and be effectively controlled and monitored during operations. Glass ampoules, Blow-Fill-Seal units and small volume containers (≤ 100 ml) closed by fusion shall be subject to 100 % integrity testing using validated methods. For large volume containers (> 100 ml) closed by fusion, reduced sampling may be acceptable where scientifically justified and based on data demonstrating the consistency of the existing process and a high level of process control. Visual inspection is not an acceptable integrity test method. VII.3.4. Samples of products using systems other than fusion shall be taken and checked for integrity using validated methods. The frequency of testing shall be based on the knowledge and experience of the container and closure systems being used. The sampling plan shall be scientifically justified and be based on information such as the supplier’s management, the packaging component specifications and the process knowledge. VII.3.5. Containers sealed under vacuum shall be tested for maintenance of vacuum after an appropriate pre-determined period prior to certification/release and during shelf life. VII.3.6. The container closure integrity validation shall take into consideration any transportation or shipping requirements that may negatively impact the integrity of the container (e.g. by decompression or extreme temperatures). VII.3.7. Where the equipment used to crimp vial caps can generate large quantities of non-viable particle, measures shall be taken to prevent particle contamination, such as locating the equipment at a physically separate station equipped with adequate air extraction. VII.3.8. Vial capping of aseptically filled products may be undertaken as an aseptic process using sterilised caps or as a clean process outside the aseptic processing area. Where the latter approach is adopted, vials shall be protected by grade A conditions up to the point of leaving the aseptic processing area, and thereafter stoppered vials shall be protected with a grade A air supply  ( 23 ) until the cap has been crimped. The supporting background environment of grade A air supply shall meet at least grade D requirements. Where capping is a manual process, it shall be performed under grade A conditions either in an appropriately designed isolator or in grade A with a grade B background. VII.3.9. Where capping of an aseptically filled product is conducted as a clean process with grade A air supply protection, vials with missing or displaced stoppers shall be rejected prior to capping. Appropriately qualified, automated methods for stopper height detection shall be in place. VII.3.10. Where human intervention is required at the capping station, appropriate technological and organisational measures shall be used to prevent direct contact with the vials and to minimise contamination. RABS and isolators may be beneficial in assuring the required conditions. VII.3.11. All filled containers of parenteral products shall be inspected individually for extraneous contamination or other defects. A defect classification including the criticality thereof shall be established during qualification and based on risk and historical knowledge. Factors to consider include, but are not limited to, the potential impact of the defect to the treated animal and the route of administration. A defect library capturing all known types of defects shall be established and be used for the training of production and quality assurance personnel. Critical defects shall be identified upfront and not during subsequent sampling and inspection of acceptable containers. Any critical defect identified subsequently shall trigger an investigation as it indicates a possible failure of the original inspection process. Batches with unusual levels of defects, when compared with routine defect numbers for the process (based on routine and trend data) shall be investigated. VII.3.12. When inspections are performed manually, suitable and controlled conditions of illumination and background shall be ensured. Inspection rates shall be appropriately controlled and qualified. Operators performing the inspection shall undergo visual inspection qualification (whilst wearing corrective lenses, if these are normally worn) at least annually. The qualification shall be performed using appropriate samples from the manufacturer's defect library sets and taking into consideration worst case scenarios (e.g. inspection time, line speed where the product is transferred to the operator by a conveyor system, container size or fatigue) and shall also include eyesight checks. Work conditions shall be adequate to reduce elements of distraction and, in order to minimise operator fatigue, frequent breaks of an appropriate duration shall be taken. VII.3.13. Where automated methods of inspection are used, the process shall be validated to detect known defects (which may impact product quality or safety). The performance of the automated methods shall be equal to, or better than, manual inspection methods. The performance of the equipment shall be challenged using representative defects prior to start up and at regular intervals throughout the batch. VII.3.14. Results of the inspection shall be recorded and defect types and numbers trended. Reject levels for the various defect types shall also be trended based on statistical principles. When adverse trends are observed, the impact on batches on the market shall be assessed. VII.4.    Sterilisation VII.4.1.    General requirements VII.4.1.1. Where possible, finished products shall be terminally sterilised, using a validated and controlled sterilisation process, as this provides a greater assurance of sterility than a validated and controlled sterile filtration process and/or aseptic processing. Where it is not possible for a product to undergo terminal sterilisation, consideration shall be given to using post-aseptic processing terminal heat treatment  ( 24 ) , combined with aseptic process to give improved sterility assurance. VII.4.1.2. The selection, design and location of the equipment and cycle/programme used for the sterilisation shall be based on scientific principles and data which demonstrate repeatability and reliability of the sterilisation process. All parameters shall be defined and critical parameters shall be controlled, monitored and recorded. VII.4.1.3. All sterilisation processes shall be validated. Validation studies shall take into account the product composition, the storage conditions and the maximum time between the start of the preparation of a product or material to be sterilised and its sterilisation. Before any sterilisation process is implemented, its suitability for the product and the equipment, and its efficacy in consistently achieving the desired sterilising conditions in all parts of each type of load to be processed shall be validated by physical measurements and, where appropriate, by biological indicators  ( 25 ) . For an effective sterilisation, the process shall be designed to ensure that the whole of the product, as well as the surfaces of the equipment and components are subject to the required treatment. VII.4.1.4. Particular attention shall be paid when the adopted product sterilisation method is not described in the current edition of the Pharmacopoeia, or when it is used for a product that is not a simple aqueous solution. Where possible, heat sterilisation shall be the method of choice. VII.4.1.5. Validated loading patterns shall be established for all sterilisation processes and load patterns shall be subject to periodic revalidation. Maximum and minimum loads shall also be addressed as part of the overall load validation strategy. VII.4.1.6. The validity of the sterilising process shall be reviewed at scheduled intervals based on risk. Heat sterilisation cycles shall be revalidated at least annually for load patterns that are considered worst case. Other load patterns shall be validated at an appropriate frequency that shall be justified as part of the contamination control strategy. VII.4.1.7. Routine operating parameters shall be established and adhered to for all sterilisation processes, e.g. physical parameters and loading patterns. VII.4.1.8. Mechanisms shall be put in place to detect a sterilisation cycle that does not conform to the validated parameters. Any failed sterilisation or any sterilisation that deviated from the validated process (e.g. have longer or shorter phases such as heating cycles) shall be investigated. VII.4.1.9. Suitable biological indicators placed at appropriate locations shall be considered as an additional method to support the validation of the sterilisation process. Biological indicators shall be stored and used according to the manufacturer’s instructions. Where biological indicators are used to support the validation and/or to monitor a sterilisation process (e.g. with ethylene oxide), positive controls shall be tested for each sterilisation cycle. Moreover, if biological indicators are used, strict precautions shall be taken to avoid transferring microbial contamination to the manufacturing or other testing processes. Biological indicator results in isolation cannot be used to override other critical parameters and process design elements. VII.4.1.10. The reliability of biological indicators is important. Therefore, suppliers shall be qualified and transportation and storage conditions shall be controlled to ensure that the quality thereof is not compromised. Prior to the use of a new batch/lot of biological indicators, the population, purity and identity of the indicator organism of the batch/lot shall be verified. For other critical parameters, e.g. D-value  ( 26 ) , Z-value  ( 27 ) , the batch certificate provided by the qualified supplier may normally be used. VII.4.1.11. Products, equipment and components that have not been subject to the sterilisation process shall be clearly distinguished from those that have through appropriate means. Equipment such as baskets or trays used to carry products, other items of equipment and/or components shall be clearly labelled (or electronically tracked) with the product name and batch number and an indication of whether or not it has been sterilised. Indicators such as autoclave tape or irradiation indicators may be used, where appropriate, to indicate whether or not a batch (or sub-batch material, component, equipment) has passed through a sterilisation process. It is noted that these indicators show only that the sterilisation process has occurred but are not indicative of product sterility or achievement of the required sterility assurance level. VII.4.1.12. Sterilisation records shall be available for each sterilisation run. Each cycle shall have a unique identifier. These records shall be reviewed and considered as part of the batch certification/release procedure. VII.4.1.13. Where required, materials, equipment and components shall be sterilised by validated methods appropriate to the specific material. Suitable protection after sterilisation shall be provided to prevent recontamination. If sterilised items are not used immediately after sterilisation, these shall be stored using appropriately sealed packaging and a maximum hold time shall be established. Where justified, components that have been packaged with multiple sterile packaging layers need not be stored in a cleanroom if the integrity and configuration of the sterile pack allows the items to be readily disinfected during transfer by operators into grade A (e.g. by the use of multiple sterile coverings that can be removed at each transfer from lower to higher grade). Where protection is achieved by containment in sealed packaging, that packaging process shall take place prior to sterilisation. VII.4.1.14. The transfer into grade A of sterilised materials, equipment, components and ancillary items in sealed packaging shall be done using appropriate validated methods (for example, airlocks or pass-through hatches) with accompanying disinfection of the exterior of the sealed packaging. The use of rapid transfer port technology  ( 28 ) may also be considered. The methods used shall be demonstrated to effectively control the potential risk of contamination of the grade A and grade B areas and, likewise, the disinfection procedure shall be demonstrated to be effective in reducing any contamination on the packaging to acceptable levels for entry of the item into the grade B and grade A areas. VII.4.1.15. Where materials, equipment, components and ancillary items are sterilised in sealed packaging or containers, the packaging shall be qualified for minimizing the risk of particulate, microbial, endotoxin/pyrogen or chemical contamination, and for compatibility with the selected sterilisation method. The packaging sealing process shall be validated. The validation shall consider the integrity of the sterile protective barrier system, the maximum hold time before sterilisation and the maximum shelf life assigned to the sterilised items. The integrity of the sterile protective barrier system for each of the sterilised items shall be checked prior to use. VII.4.1.16. For materials, equipment, components and ancillary items that are not a direct or indirect product contact part and are necessary for aseptic processing but cannot be sterilised, an effective and validated disinfection and transfer process shall be put in place. These items, once disinfected, shall be protected to prevent recontamination. These items, as well as other items that are potential routes of contamination, shall be included in the environmental monitoring programme. VII.4.2.    Sterilisation by heat VII.4.2.1. Each heat sterilisation cycle shall be recorded either electronically or by hardcopy, using equipment with suitable accuracy and precision. The system used shall have safeguards and/or redundancy in its control and monitoring instrumentation to detect a cycle that is not conforming to the validated cycle parameter requirements and to abort or fail such cycle (e.g. by the use of duplex/double probes connected to independent control and monitoring systems). VII.4.2.2. The position of the temperature probes used for controlling and/or recording shall be determined during the validation having regard to the system’s design and with a view to correctly record and represent routine cycle conditions. Validation studies shall demonstrate the suitability of the system’s control and recording probe locations, and shall include the verification of the function and location of these probes by the use of an independent monitoring probe located at the same position during validation. VII.4.2.3. The entire load shall reach the required temperature before the measurement of the sterilising time-period starts. For sterilisation cycles controlled by using a reference probe within the load, specific consideration shall be given to ensuring that the load probe temperature is controlled within a defined temperature range prior to the start of the cycle. VII.4.2.4. After completion of the high temperature phase of a heat sterilisation cycle, precautions shall be taken against contamination of a sterilised load during cooling. Any cooling liquid or gas that comes into contact with the product or sterilised material shall be sterilised. Additional requirements applicable where parametric release has been authorised are laid down in Annex IX. VII.4.3.    Moist heat sterilisation VII.4.3.1. Moist heat sterilisation can be achieved using steam (direct or indirect contact) or with other systems such as superheated water systems (cascade or immersion cycles) that can be used for containers that may be damaged by other cycle designs (e.g. Blow-Fill-Seal containers, plastic bags). VII.4.3.2. The items to be sterilised, other than products in sealed containers, shall be dry and packaged in a protective barrier system that allows removal of air and penetration of steam and prevents recontamination after sterilisation. All loaded items shall be dry upon removal from the steriliser. Load dryness shall be confirmed by visual inspection as a part of the sterilisation process acceptance. VII.4.3.3. For porous cycles (hard goods), time, temperature and pressure shall be used to monitor the process and be recorded. Each sterilised item shall be inspected for damage, packaging material integrity and moisture upon removal from the autoclave. Any item found not to be fit for purpose shall be removed from the manufacturing area and an investigation shall be performed. VII.4.3.4. For autoclaves capable of performing prevacuum sterilisation cycles, the temperature shall be recorded at the chamber drain throughout the sterilisation period. Load probes may also be used where appropriate, but the controlling system shall remain related to the load validation. For steam in place systems, the temperature shall be recorded at appropriate condensate drain locations throughout the sterilisation period. Validation of porous cycles shall include a calculation of equilibration time  ( 29 ) , exposure time, correlation of pressure and temperature and the minimum/maximum temperature range during the exposure. Validation of fluid cycles shall include temperature, time and/or F 0 value  ( 30 ) . Critical processing parameters shall be subject to defined limits (including appropriate tolerances) and be confirmed as part of the sterilisation validation and of the routine cycle acceptance criteria. VII.4.3.5. Leak tests on the steriliser shall be carried out periodically (normally weekly) when a vacuum phase is part of the cycle, and when the system is returned – post-sterilisation – to a pressure lower than the environment surrounding the steriliser. VII.4.3.6. When the sterilisation process includes air purging (e.g. porous autoclave loads, lyophilizer chambers), there shall be adequate assurance of air removal prior to and during sterilisation. For autoclaves, this shall include an air removal test cycle (normally performed on a daily basis) or the use of an air detector system. Loads to be sterilised shall be designed to support effective air removal and be free draining to prevent the build-up of condensate. VII.4.3.7. Distortion and damage of non-rigid containers that are terminally sterilised, such as containers produced by Blow-Fill-Seal or Form-Fill-Seal technologies, shall be prevented by appropriate cycle design and control (for instance setting correct pressure, heating and cooling rates and loading patterns). VII.4.3.8. Where steam in place systems are used for sterilisation (e.g. for fixed pipework, vessels and lyophilizer chambers), the system shall be appropriately designed and validated to ensure that all parts of the system are subject to the required treatment. The system shall be monitored for temperature, pressure and time at appropriate locations during routine use to ensure all areas are effectively and reproducibly sterilised. These locations shall be demonstrated as being representative of, and correlated with, the slowest to heat locations during initial and routine validation. Once a system has been sterilised by steam in place, it shall remain integral and, where required by the relevant operations, maintained under positive pressure or otherwise equipped with a sterilising vent filter prior to use. VII.4.3.9. In fluids load cycles where superheated water is used as the heat transfer medium, the heated water shall consistently reach all of the required contact points. Initial qualification studies shall include temperature mapping of the entire load. There shall be routine checks on the equipment to ensure that nozzles (where the water is introduced) are not blocked and drains remain free from debris. VII.4.3.10. Validation of the sterilisation of fluids loads in a superheated water autoclave shall include temperature mapping of the entire load and heat penetration and reproducibility studies. All parts of the load shall heat up uniformly and achieve the desired temperature for the specified time. Routine temperature monitoring probes shall be correlated to the worst case positions identified during the qualification process. VII.4.4.    Dry heat sterilisation VII.4.4.1. Dry heat sterilisation utilizes high temperatures of air or gas to sterilise a product or an article. It is of particular use in the thermal removal of difficult-to-eliminate thermally robust contaminants such as endotoxin/pyrogen. The combination of time and temperature to which the product, components or equipment are exposed shall produce an adequate and reproducible level of lethality and/or endotoxin/pyrogen inactivation/removal when operated routinely within the established limits. The process may be operated in an oven or in a continuous tunnel process, e.g. for sterilisation and depyrogenation of glass containers. VII.4.4.2. Dry heat sterilisation/depyrogenation tunnels shall be configured to ensure that airflow protects the integrity and performance of the grade A sterilising zone by maintaining appropriate pressure differentials and airflow through the tunnel. Air pressure difference profiles shall be assessed. The impact of any airflow change shall be assessed to ensure that the heating profile is maintained. All air supplied to the tunnel shall pass through at least a HEPA filter and periodic tests (at least biannually) shall be performed to demonstrate air filter integrity. In addition, any tunnel parts that come into contact with sterilised components shall be appropriately sterilised or disinfected. Critical process parameters that shall be addressed during validation and/or routine processing include, but are not limited to: — belt speed or dwell time within the sterilising zone; — temperature – minimum and maximum temperatures; — heat penetration of the material/article; — heat distribution/uniformity; — airflows determined by air pressure difference profiles correlated with the heat distribution and penetration studies. VII.4.4.3. When a thermal process is used as part of the depyrogenation process for any component or product contact equipment/material, validation studies shall be performed to demonstrate that the process provides a suitable F h value  ( 31 ) and results in a minimum 3 log 10 reduction in endotoxin concentration. When this is attained, there is no additional requirement to demonstrate sterilisation. VII.4.4.4. During validation, containers spiked with endotoxin shall be used and a full reconciliation performed. Containers shall be representative of the materials normally processed (in respect to composition of the packaging materials, porosity, dimensions, nominal volume). Endotoxin quantification and recovery efficiency shall also be demonstrated. VII.4.4.5. Dry heat ovens are typically employed to sterilise or depyrogenate primary packaging components, starting materials or active substances but may be used for other processes. They shall be maintained at a positive pressure relative to lower grade clean areas throughout the sterilisation and post sterilisation hold process unless the integrity of the packaging is maintained. All air entering the oven shall pass through a HEPA filter. Critical process parameters that shall be considered in qualification and/or routine processing include, but are not limited to: — temperature; — exposure period/time; — chamber pressure (for maintenance of over pressure); — air speed; — air quality within the oven; — heat penetration of material/article (slow to heat spots); — heat distribution/uniformity; — load pattern and configuration of articles to be sterilised/depyrogenated including minimum and maximum loads. VII.4.5.    Sterilisation by radiation VII.4.5.1. Sterilisation by radiation is used mainly for the sterilisation of heat sensitive materials and products. Ultraviolet irradiation is not an acceptable method of sterilisation. Specific requirements related to the use of ionising radiation sterilisation are laid down in Annex VII. VII.4.5.2. Validation procedures shall ensure that the effects of variation in density of the product and packages are considered. VII.4.6.    Sterilisation with ethylene oxide VII.4.6.1. This method shall only be used when no other method is practicable. During process validation, it shall be shown that there is no damaging effect on the product and that the conditions and time allowed for degassing are suitable to attain a reduction of any residual ethylene oxide gas and reaction products to defined acceptable limits for the given product or material. VII.4.6.2. Direct contact between the gas and microbial cells is essential. Therefore, precautions shall be taken to avoid the presence of organisms likely to be enclosed in material such as crystals or dried protein. The nature, porosity and quantity of packaging materials can also significantly affect the process. VII.4.6.3. Before exposure to the gas, materials shall be brought into equilibrium with the humidity and temperature required by the process. Where steam is used to condition the load for sterilisation, it shall be of an appropriate quality. The time required for this operation shall be balanced against the need to minimise the time before sterilisation. VII.4.6.4. Each sterilisation cycle shall be monitored with suitable biological indicators, using the appropriate number of test units distributed throughout the load at defined locations that have been shown to be worst case locations during validation. VII.4.6.5. Critical process parameters to be considered as part of the sterilisation process validation and routine monitoring include, but are not limited to: — ethylene oxide gas concentration; — pressure; — amount of ethylene oxide gas used; — relative humidity; — temperature; — exposure time. VII.4.6.6. After sterilisation, the load shall be aerated to allow ethylene oxide gas and/or its reaction products to desorb from the packaged product to predetermined levels. Aeration can occur within a steriliser chamber and/or in a separate aeration chamber or aeration room. The aeration phase shall be validated as part of the overall ethylene oxide sterilisation process validation. VII.4.7.    Filter sterilisation of products that cannot be sterilised in their final container VII.4.7.1 Solutions or liquids that cannot be sterilised in their final container shall be sterilised by filtration through a sterile sterilising grade filter (with a nominal pore size of a maximum of 0,22 μm that has been appropriately validated to obtain a sterile filtrate) and subsequently aseptically filled into a previously sterilised container. The selection of the filter used shall ensure that it is compatible with the product and in compliance with the marketing authorisation. VII.4.7.2. Suitable bioburden reduction prefilters and/or sterilising grade filters may be used at multiple points during the manufacturing process to ensure a low and controlled bioburden of the liquid prior to the final sterilising filter. Due to the potential additional risks of a sterile filtration process, as compared with other sterilisation processes, an additional filtration through a sterile sterilising grade filter, as close to the point of fill as possible, shall be considered as part of an overall contamination control strategy. VII.4.7.3. The selection of components for the filtration system and their interconnection and arrangement within the filtration system, including pre-filters, shall be based on the critical quality attributes of the product, justified and documented. The filtration system shall minimise the generation of fibres and particles, not cause or contribute to unacceptable levels of impurities, or possess characteristics that otherwise alter the quality or the efficacy of the product. Similarly, the filter characteristics shall be compatible with the fluid and not be adversely affected by the product to be filtered. Adsorption of product components and extraction/leaching of filter components shall be evaluated. VII.4.7.4. The filtration system shall be designed to: — allow operation within validated process parameters; — maintain the sterility of the filtrate; — minimise the number of aseptic connections required between the final sterilising grade filter and the final filling of the product; — allow cleaning procedures to be conducted as necessary; — allow sterilisation procedures, including sterilisation in place, to be conducted as necessary; — permit in-place integrity testing of the 0,22 μm final sterilising grade filter, preferably as a closed system, both prior to and following filtration as necessary. In-place integrity testing methods shall be preferably used to avoid any adverse impact on the quality of the product. VII.4.7.5. Sterile filtration of liquids shall be validated in accordance with relevant Pharmacopeia requirements. Validation may be grouped by different strengths or variations of a product but shall be done under worst-case conditions. The rationale for grouping shall be justified and documented. VII.4.7.6. Wherever possible during filter validation, the product to be filtered shall be used for bacterial retention testing  ( 32 ) of the sterilising grade filter. Where the product to be filtered is not suitable for use in bacterial retention testing, a suitable surrogate product shall be justified for use in the test. The challenge organism used in the bacterial retention test shall also be justified. VII.4.7.7. Filtration parameters that shall be considered and established during validation include, but are not limited to: (a) The wetting fluid used for filter integrity testing: — it shall be based on the filter manufacturer’s recommendation or the fluid to be filtered. The appropriate integrity test value specification shall be established; — if the system is flushed or integrity tested in situ with a fluid other than the product, appropriate actions shall be taken to avoid any deleterious effect on product quality. (b) Filtration process conditions including: — fluid pre-filtration holding time and effect on bioburden; — filter conditioning, with fluid if necessary; — maximum filtration time/total time that the filter is in contact with the fluid; — maximum operating pressure; — flow rate; — maximum filtration volume; — temperature; — the time taken to filter a known volume of bulk solution and the pressure difference to be used across the filter. VII.4.7.8. Routine process controls shall be implemented to ensure adherence to validated filtration parameters. Results of critical process parameters shall be included in the batch record, including – but not limited to – the minimum time taken to filter a known volume of bulk solution and pressure difference across the filter. Any significant difference from critical parameters during manufacturing shall be documented and investigated. VII.4.7.9. The integrity of the sterilised filter assembly shall be verified by integrity testing before use (pre-use post sterilisation integrity test or PUPSIT), to check for damage and loss of integrity caused by the filter preparation prior to use. However, it is recognised that PUPSIT may not always be possible after sterilisation due to process constraints (e.g. the filtration of very small volumes of solution). In these cases, an alternative approach may be taken providing that a thorough risk assessment has been performed and compliance is achieved by the implementation of appropriate controls to mitigate any risk of a non-integral filtration system. Points to consider in such a risk assessment shall include but are not limited to: — in-depth knowledge and control of the filter sterilisation process to ensure that the potential for damage to the filter is minimised; — in-depth knowledge and control of the supply chain including contract sterilisation facilities, defined transport conditions and packaging of the sterilised filter (to prevent damage to the filter during transportation and storage); — in-depth process knowledge such as the specific product type, including particle burden and whether there exists any risk of impact on filter integrity values (such as the potential to alter integrity-testing values and therefore prevent the detection of a non-integral filter during a post-use filter integrity test), and the implementation of pre-filtration or processing steps prior to the final sterilising grade filter that would remove particle burden prior to the sterile filtration. In addition, a sterilising grade filter that is used to sterilise a fluid shall be subject to a non-destructive integrity test post-use prior to removal of the filter from its housing. The integrity test process shall be validated and test results shall correlate to the microbial retention capability of the filter established during validation. Examples of tests that are used include bubble point, diffusive flow, water intrusion or pressure hold test. VII.4.7.10. The integrity of critical sterile gas and air vent filters (that are directly linked to the sterility of the product) shall be verified by testing after use, with the filter remaining in the filter assembly or housing. VII.4.7.11. The integrity of non-critical air or gas vent filters shall be confirmed and recorded at appropriate intervals. Where gas filters are in place for extended periods, integrity testing shall be carried out at installation and prior to replacement. The maximum duration of use shall be specified and monitored based on risk (e.g. considering the maximum number of uses and heat treatment/sterilisation cycles permitted as applicable). VII.4.7.12. For gas filtration, unintended moistening or wetting of the filter or filter equipment shall be avoided. VII.4.7.13. If the sterilising filtration process has been validated as a system consisting of multiple filters to achieve the sterility for a given fluid, the filtration system is considered to be a single sterilising unit and all filters within the system shall satisfactorily pass integrity testing after use. VII.4.7.14. In a redundant filtration system (where a second redundant sterilising grade filter is present as a backup but the sterilising process is validated as only requiring one filter), post-use integrity test of the primary sterilising grade filter shall be performed and, if demonstrated to be integral, a post-use integrity test of the redundant (backup) filter is not necessary. However, in the event of a failure of the post-use integrity test on the primary filter, post-use integrity test on the secondary (redundant) filter shall be performed, in conjunction with an investigation and risk assessment to determine the reason for the primary filter test failure. VII.4.7.15. Bioburden samples shall be taken from the bulk product and immediately prior to the final sterile filtration. In case where a redundant filtration set-up is used, the samples shall be taken prior to the first filter. Procedures for taking samples shall be designed so as not to introduce contamination. VII.4.7.16. Liquid sterilising grade filters shall be discarded after the processing of a single batch and the same filter shall not be used continuously for more than one working day, unless such use has been validated. VII.4.7.17. Where campaign manufacture of a product has been appropriately justified in the contamination control strategy and validated, the manufacturer shall: (a) assess and document the risks associated with the duration of filter use for the sterile filtration process for a given fluid; (b) conduct and document effective validation and qualification studies to demonstrate that the duration of filter use for a given sterile filtration process and for a given fluid does not compromise the performance of the final sterilising grade filter or the filtrate quality; (c) document the maximum validated duration of use for the filter and implement controls to ensure that filters are not used beyond the validated maximum duration. Records of these controls shall be maintained; (d) implement controls to ensure that filters contaminated with fluid or cleaning agent residues or otherwise considered defective, are removed from use. VII.5.    Form-Fill-Seal   ( 33 ) VII.5.1. Form-Fill-Seal machines used for terminally sterilised products shall comply with the environmental requirements set out in Section VII.1.3 of this Annex, while Form-Fill-Seal machines used in aseptic manufacture shall comply with the environmental requirements set out in table 4 of this Annex. VII.5.2. Contamination of the packaging films used during the Form-Fill-Seal process shall be minimised by the implementation of appropriate controls regarding components, supply and handling. Due to the criticality of packaging films, procedures shall be implemented to ensure that the films supplied meet defined specifications and are of the appropriate quality, including material thickness and strength, microbial and particulate contamination, integrity of printed information and packaging design, as relevant. The sampling frequency, the bioburden and, where applicable, endotoxin/pyrogen levels of packaging films and associated components shall be addressed as part of the contamination control strategy. VII.5.3. The operation of the equipment, including set-up, filling, sealing and cutting processes shall be assessed so that critical process parameters can be identified, validated, controlled and monitored appropriately. VII.5.4. Any product contact gases (e.g. those used to inflate the container or used as a product overlay) shall be appropriately filtered, as close to the point of use as possible. The quality of gases used and the effectiveness of the gas filtration systems shall also be verified periodically in accordance with Section V.4 of this Annex. VII.5.5. The controls to be identified during the qualification of Form-Fill-Seal processes, which shall be part of the contamination control strategy, include but are not limited to: — determination of the boundaries of the critical zone; — environmental control and monitoring, both of the machine and of the background in which it is placed; — personnel gowning requirements; — integrity testing of the product filling lines and filtration systems (as relevant); — duration of the batch or filling campaign; — control of the packaging films, including any requirements for film decontamination or sterilisation; — cleaning-in-place and sterilisation-in-place of the equipment as necessary; — machine operation, settings and alarm management (as relevant). VII.5.6. Critical process parameters for Form-Fill-Seal shall be established during the equipment qualification and shall include, but are not limited to: — settings for uniform package dimensions and cutting in accordance with validated parameters; — setting, maintenance and monitoring of validated forming temperatures (including preheating and cooling), forming times and pressures as relevant; — setting, maintenance and monitoring of validated sealing temperatures, sealing temperature uniformity across the seal, sealing times and pressures as relevant; — environmental and product temperature; — batch-specific testing of package seal strength and uniformity; — settings for correct filling volumes, speeds and uniformity; — settings for any additional printing (batch coding), embossing or debossing to ensure that unit integrity is not compromised; — methods and parameters for integrity testing of filled containers. VII.5.7. Appropriate procedures for the verification, monitoring and recording of Form-Fill-Seal critical process parameters and equipment operation shall be implemented during production. VII.5.8. Operational procedures shall describe how forming and sealing issues are detected and rectified. Rejected units or sealing issues shall be recorded and investigated. VII.5.9. Appropriate maintenance procedures shall be established based on risks, and shall include maintenance and inspection plans for tooling critical to the effectiveness of unit sealing. Any issues identified that indicate a potential product quality concern shall be documented and investigated. VII.6.    Blow-Fill-Seal   ( 34 ) VII.6.1. Blow-Fill-Seal equipment used for the manufacture of products that are terminally sterilised shall be installed in at least a grade D environment. The conditions at the point of fill shall comply with the environmental requirements set out in Section VII.1.3 of this Annex. VII.6.2. Where Blow-Fill-Seal equipment is used for aseptic processing, the following requirements shall apply: (a) For shuttle type equipment used for aseptic filling, the parison  ( 35 ) is open to the environment and therefore the areas where parison extrusion, blow-moulding and sealing take place shall meet grade A conditions at the critical zones. In addition, the filling environment shall be designed and maintained to meet grade A conditions for viable and total particle limits both at rest and when in operation. (b) For rotary-type equipment used for aseptic filling, the parison is generally closed to the environment once formed and therefore the filling environment within the parison shall be designed and maintained to meet grade A conditions for viable and total particle limits both at rest and when in operation. (c) The equipment shall be installed in at least a grade C environment, provided that grade A/B clothing is used. The microbiological monitoring (including setting of limits and frequencies applied) of operators wearing grade A/B clothing in a grade C area shall be performed in accordance with risk management principles. VII.6.3. Due to the generation of particles from polymer extrusion and cutting during operation and the restrictive size of critical filling zones of Blow-Fill-Seal equipment, in operation monitoring of total particle for the equipment is not required. However, data shall be available to demonstrate that the design of the equipment ensures that critical zones of the filling process environment meet grade A conditions in operation. VII.6.4. Viable environmental monitoring of Blow-Fill-Seal processes shall be risk-based and in accordance with Section VIII of this Annex. In operation viable monitoring shall be performed for the full duration of critical processing, including during equipment assembly, with the exception of rotary-type equipment where monitoring of the critical filling zone is not possible. VII.6.5. The environmental control and monitoring programme shall take into consideration the moving parts and complex airflow paths generated by the Blow-Fill-Seal process and the effect of the high heat outputs of the process (e.g. through the use of airflow visualisation studies and/or other equivalent studies). Environmental monitoring programmes shall also consider factors such as air-filter configuration, air-filter integrity, cooling systems integrity, equipment design and qualification. VII.6.6. Air or other gases in contact with critical surfaces of the container during extrusion, formation or sealing of the moulded container shall undergo appropriate filtration. The quality of the gas used and the effectiveness of the gas filtration systems shall be verified periodically in accordance with Section V.4 of this Annex. VII.6.7. Particulate and microbial contamination of the polymer granulate shall be prevented by appropriate design, control and maintenance of the polymer granulate storage, sampling and distribution systems. VII.6.8. The capability of the extrusion system to provide appropriate sterility assurance for the moulded container shall be validated. The sampling frequency, the bioburden and, where applicable, endotoxin/pyrogen levels of the raw polymer shall be defined and controlled. VII.6.9. Interventions requiring cessation of filling and/or extrusion, moulding and sealing and, where required, re-sterilisation of the filling machine shall be clearly defined and described in the filling procedure, and included in the aseptic process simulation as relevant. VII.6.10. The controls identified during qualification of Blow-Fill-Seal equipment shall be in alignment with the site’s contamination control strategy. Aspects to be considered include but are not limited to: — determination of the boundaries of the critical zone; — environmental control and monitoring, both of the machine and of the background in which it is placed; — personnel gowning requirements; — integrity testing of the product filling lines and filtration systems (as relevant); — duration of the batch or filling campaign; — control of polymer granulate, including distribution systems and critical extrusion temperatures; — cleaning-in-place and sterilisation-in-place of equipment as necessary; — machine operation, settings and alarm management (as relevant). VII.6.11. Critical process parameters for Blow-Fill-Seal equipment shall be determined during equipment qualification and shall include, but are not limited to: — clean-in-place and sterilisation-in-place of product pipelines and filling needles (mandrels); — setting, maintenance and monitoring of extrusion parameters, including the temperature, speed and extruder throat settings for parison thickness; — setting, maintenance and monitoring of mould temperatures, including the rate of cooling where necessary for product stability; — preparation and sterilisation of ancillary components added to the moulded unit, e.g. bottle caps; — environmental control, cleaning, sterilisation and monitoring of the critical extrusion, transfer and filling areas as relevant; — batch-specific testing of the package wall-thickness at critical points of the container; — settings for correct filling volumes, speeds and uniformity; — settings for any additional printing (batch coding), embossing or debossing to ensure that unit integrity and quality is not compromised; — methods and parameters for integrity testing of 100 % of all filled containers; — settings for cutters or punches used to remove waste plastic surrounding filled units (flash removal). VII.6.12. Appropriate procedures for the verification, monitoring and recording of Blow-Fill-Seal critical process parameters and equipment operation shall be implemented during production. VII.6.13. Operational procedures shall describe how blowing, forming and sealing issues are detected and rectified. Rejected units or sealing issues shall be recorded and investigated. VII.6.14. Where the Blow-Fill-Seal process includes the addition of components to moulded containers (e.g. addition of caps to large volume parenteral bottles), these components shall be appropriately decontaminated and added to the process using a clean, controlled process. The following shall apply: (a) For aseptic processes, the addition of components shall be performed under grade A conditions, to ensure the sterility of critical surfaces, using pre-sterilised components. (b) For terminally sterilised products, the validation of terminal sterilisation processes shall ensure the sterility of all critical product pathways between the component and moulded container, including areas that are not wetted during sterilisation. (c) Testing procedures shall be established and validated to ensure the effective sealing of components and moulded containers. VII.6.15. Appropriate maintenance procedures shall be established based on risk, including maintenance and inspection plans for items critical to unit sealing, integrity and sterility. VII.6.16. The moulds used to form containers are considered critical equipment. Therefore, any changes or modification to moulds requires an assessment of finished product container integrity, and where appropriate having regard to the outcome of the assessment, shall be supported by validation. Any issues identified that indicate a potential product quality concern shall be documented and investigated. VII.7.    Lyophilisation   ( 36 ) VII.7.1. Lyophilisation is a critical process step and all activities that can affect the sterility of the product or material shall be regarded as extensions of the aseptic processing. In particular, the lyophilisation equipment and its processes shall be designed to ensure that product or material sterility is maintained during lyophilisation by preventing microbial and particle contamination between the filling of products for lyophilisation and the completion of lyophilisation process. The control measures shall form part of the contamination control strategy. VII.7.2. The sterilisation of the lyophilizer and any associated equipment (e.g. trays, vial support rings) shall be validated and the holding time between the sterilisation cycle and use shall be appropriately challenged during the aseptic process simulation. The lyophilizer shall be sterilised regularly, based on system design. In addition, re-sterilisation shall be performed after maintenance or cleaning. Sterilised lyophilizers and any associated equipment shall be protected from contamination after sterilisation. VII.7.3. Lyophilizers and any associated product transfer and loading/unloading areas shall be designed to minimise operator intervention as far as possible. The frequency of the lyophilizer sterilisation shall be determined based on the design and risks related to system contamination during use. Lyophilizers that are manually loaded or unloaded with no barrier technology separation shall be sterilised before each load. For lyophilizers loaded and unloaded by automated systems or protected by closed barrier systems, the frequency of sterilisation shall be justified and documented as part of the contamination control strategy. VII.7.4. The integrity of the lyophilizer shall be maintained following sterilisation and during lyophilisation. The filter used to maintain the lyophilizer’s integrity shall be sterilised before each use of the system and the integrity testing results shall be part of the batch certification/release. In addition, the frequency of vacuum/leak integrity testing of the chamber shall be documented and the maximum permitted leakage of air into the lyophilizer shall be specified and checked at the start of every cycle. VII.7.5. Lyophilisation trays shall be checked regularly to ensure that they are not misshapen or damaged. VII.7.6. Points to consider for the design of loading (and unloading, where the lyophilised material is still unsealed and exposed), include but are not limited to: — the loading pattern within the lyophilizer shall be specified and documented; — the transfer of partially closed containers to a lyophilizer shall take place under grade A conditions at all times and handled in a manner designed to minimise direct operator intervention. Technologies such as conveyor systems or portable transfer systems (e.g. clean air transfer carts, portable unidirectional airflow workstations) shall be used to ensure that the cleanliness of the system used to transfer the partially closed containers is maintained. Alternatively, where supported by validation, trays closed in grade A and not reopened whilst in the grade B area may be used to protect partially stoppered vials (e.g. appropriately closed boxes); — airflow patterns shall not be adversely affected by transport devices and venting of the loading zone; — unsealed containers (such as partially stoppered vials) shall be maintained under grade A conditions and shall normally be separated from operators by means of a physical barrier technology or any other appropriate measures; — where the seating of the stoppers is not completed prior to the opening of the lyophilizer chamber, the product removed from the lyophilizer shall remain under grade A conditions during subsequent handling; — tools used during loading and unloading of the lyophilizer (e.g. trays, bags, placing devices, tweezers) shall be sterile. VII.8.    Closed systems VII.8.1. The use of closed systems can reduce the risk of microbial, particle and chemical contamination from the adjacent environment. Closed systems shall be designed to reduce the need for manual manipulations and the associated risks. VII.8.2. It is critical to ensure the sterility of all product contact surfaces of closed systems used for aseptic processing. Therefore, the design and selection of any closed system used for aseptic processing shall ensure maintenance of sterility. Connection of sterile equipment (e.g. tubing/pipework) used after the final sterilising grade filter shall be connected aseptically (e.g. by intrinsic sterile connection devices). VII.8.3. Appropriate measures shall be put in place to ensure the integrity of components used in aseptic connections. The means by which this is achieved shall be determined and addressed in the contamination control strategy. In particular, appropriate system integrity tests shall be considered when there is a risk of compromising product sterility. Supplier assessment shall include the collation of data in relation to potential failure modes that may lead to a loss of system sterility. VII.8.4. The background environment in which closed systems are located shall be determined having regard to the system’s design and the processes undertaken. For aseptic processing and where there is a risk that the system’s integrity may be compromised, the system shall be located in grade A. If the system can be shown to remain integral at every usage (e.g. via pressure testing and/or monitoring) then a lower classified area may be used. Any transfer between classified areas shall be thoroughly assessed in accordance with Section III.2 of this Annex. When the closed system is opened (e.g. for maintenance of a bulk manufacturing line), this shall be performed in a classified area appropriate to the materials (e.g. grade C for terminal sterilisation processes, or grade A for aseptic processing) or be subject to further cleaning and disinfection (and sterilisation in case of aseptic processes). VII.9.    Single use systems   ( 37 ) VII.9.1. Single use systems may be used in the manufacture of sterile products as an alternative to reusable equipment. Single use systems can be individual components or be made up of multiple components such as bags, filters, tubing, connectors, valves, storage bottles and sensors. Single use systems shall be designed to reduce the need for manipulations and complexity of manual interventions. VII.9.2. There are some specific risks associated with single use systems that shall be assessed as part of the contamination control strategy, including but not limited to: — the interaction between the product and product contact surface (such as adsorption, or leachables  ( 38 ) and extractables  ( 39 ) ); — the fragile nature of the system compared with fixed reusable systems; — the increase in the number and complexity of manual operations (including inspection and handling of the system) and connections made; — the complexity of the assembly; — the performance of the pre- and post-use integrity testing for sterilising grade filters; — the risk of holes and leakage; — the potential for compromising the system at the point of opening the outer packaging; — the risk of particle contamination. VII.9.3. Sterilisation processes for single use systems shall be validated and shown to have no adverse impact on the system’s performance. VII.9.4. Assessment of suppliers of disposable systems including sterilisation is critical to the selection and use of these systems. Therefore, for sterile single use systems, verification of sterility assurance shall be performed as part of the supplier qualification and evidence of sterilisation of each unit shall be checked on receipt. VII.9.5. The adsorption and reactivity of the product with product contact surfaces shall be evaluated under process conditions. VII.9.6. The extractable and leachable profiles of the single use systems and any impact on the quality of the product – especially where the system is made from polymer-based materials – shall be evaluated. An assessment shall be carried out for each component to evaluate the extractable profile data. For components considered to be at high risk from leachables, including those that may absorb processed materials or those with extended material contact times, an assessment of leachable profile studies, including safety concerns, shall be taken into consideration. When applying simulated processing conditions, these shall accurately reflect the actual processing conditions and be based on a scientific rationale. VII.9.7. Single use systems shall be designed to maintain integrity throughout processing under the intended operational conditions. Attention to the structural integrity of the single use components is necessary where these may be exposed to extreme conditions (e.g. freezing and thawing processes) during routine processing or transportation, including verification that intrinsic sterile connection devices (both heat sealed and mechanically sealed) remain integral under these conditions. VII.9.8. Acceptance criteria shall be established and implemented for single use systems corresponding to the risks or criticality of the products and its processes. On receipt, each piece of single use systems shall be checked to ensure that they have been manufactured, supplied and delivered in accordance with the approved specification. A visual inspection of the outer packaging (e.g. appearance of exterior carton, product pouches), label printing, and review of attached documents (e.g. certificate of conformance and proof of sterilisation) shall be carried out and documented prior to use. VII.9.9. Critical manual handling operations of single use systems such as assembly and connections shall be subject to appropriate controls and verified during aseptic process simulation. SECTION VIII ENVIRONMENTAL AND PROCESS MONITORING VIII.1.    General requirements VIII.1.1. Each site shall have an environmental and process monitoring programme to monitor the controls designed to minimise the risk of microbial and particle contamination. The programme, which shall form part of the overall contamination control strategy, shall typically consist of the following elements: — environmental monitoring – total particle; — environmental and personnel monitoring – viable particle; — temperature, relative humidity and other specific characteristics; — aseptic process simulation (only for aseptically manufactured products). VIII.1.2. The reliability of each of the elements of the monitoring system when taken in isolation is limited. Therefore, the outcome from the each of the elements above-described cannot be considered – on its own – as an indicator of asepsis. However, the results from all the elements of the programme help confirm the reliability of the design, validation and operation of the monitored system. VIII.1.3. The information from the programme shall be used for routine batch certification/release and for periodic assessment during process review or investigation. While this applies to both terminal sterilisation and aseptic processes, it is acknowledged that the criticality of the impact may differ depending upon the product and process type. VIII.2.    Environmental and process monitoring VIII.2.1. The purpose of the environmental monitoring programme is twofold: — to provide assurance that cleanrooms and clean air equipment continue to provide an environment of appropriate air cleanliness, in accordance with design and regulatory requirements; — to effectively detect excursions from environmental limits, which -in turn- shall trigger an investigation and an assessment of the risks to product quality. Risk assessments shall be performed in order to establish a comprehensive environmental monitoring programme, including sampling locations, frequency of monitoring, monitoring methods and incubation conditions (e.g. time, temperature(s), aerobic and/or anaerobic conditions). In particular, the risk assessment shall include the determination of critical monitoring locations, those locations where the presence of microorganisms during processing may have an impact on product quality (e.g. grade A, aseptic processing areas and the grade B areas that directly interface with the grade A area). The risk assessments shall be performed on the basis of the specific characteristics of the process inputs and the final product, the facility, the equipment, the criticality of specific processes and steps, the operations involved, the routine monitoring data, the monitoring data obtained during qualification and the knowledge of typical microbial flora isolated from the environment. Detailed knowledge of those aspects is therefore required for the establishment of the environmental monitoring program. Other relevant information such as air visualisation studies shall also be considered. The risk assessments shall be reviewed regularly to confirm the effectiveness of the site’s environmental monitoring programme. VIII.2.2. Routine monitoring of cleanrooms, clean air equipment and personnel shall be performed in operation throughout all critical stages of processing, including equipment set-up. VIII.2.3. Other characteristics, such as temperature and relative humidity, shall be controlled within ranges that align with product/processing/personnel requirements and support the maintenance of the defined cleanliness standards (e.g. grade A or B). VIII.2.4. The monitoring of grade A shall demonstrate the maintenance of aseptic processing conditions during critical operations. Monitoring shall be performed at locations posing the highest risk of contamination to the sterile equipment surfaces, the containers, the closures and the product. The selection of monitoring locations and the orientation and positioning of sampling devices shall be appropriate to obtain reliable data from the critical zones. VIII.2.5. Sampling methods shall not pose a risk of contamination to the manufacturing operations. VIII.2.6. Appropriate alert levels and action limits shall be set for the results of viable and total particle monitoring. The maximum total particle action limits are described in Table 5 and the maximum viable particle action limits are described in Table 6. However, more stringent action limits may be required based on data trending, the nature of the process or as determined in the contamination control strategy. Both viable and total particle alert levels shall be established based on results of cleanroom qualification tests and periodically reviewed based on ongoing trend data. VIII.2.7. Alert levels for grade A (total particle only) grade B, grade C and grade D shall be set such that adverse trends (e.g. a number of events or individual events that indicate a deterioration of environmental control) are detected and addressed. VIII.2.8. Monitoring procedures shall define the approach to trending. Trends shall include, but are not limited to: — increasing numbers of excursions from action limits or alert levels; — consecutive excursions from alert levels; — regular but isolated excursion from action limits that may have a common cause (e.g. single excursions that always follow planned preventative maintenance); — changes in microbial flora type and numbers and predominance of specific organisms. Particular attention shall be paid to organisms recovered that may indicate a loss of control, deterioration in cleanliness or organisms that may be difficult to control such as spore-forming microorganisms and moulds. VIII.2.9. The monitoring of grade C and D cleanrooms in operation shall be performed on the basis on data collected during qualification and routine data to allow effective trend analysis. The requirements of alert levels and action limits will depend on the nature of the operations carried out. Action limits may be more stringent than those listed in Table 5 and Table 6. VIII.2.10. If action limits are exceeded, a root cause investigation, an assessment of the potential impact to the product (including batches produced between the monitoring and the reporting) and implementation of corrective and preventive actions (as appropriate) shall be required. If alert levels are exceeded, an assessment and follow-up is mandatory, including consideration of an investigation and/or corrective actions to avoid any further deterioration of the environment. The above shall be reflected in operating procedures. VIII.3.    Environmental monitoring – total particle VIII.3.1. A total particle monitoring programme shall be established to obtain data for assessing the potential contamination risks and to ensure the maintenance of the environment for sterile/aseptic operations in a qualified state. VIII.3.2. The limits for environmental monitoring of airborne particle concentration for each graded area are given in Table 5. Table 5 Maximum permitted total particle concentration for monitoring Grade Maximum limits for total particle ≥ 0,5 μm/m 3 Maximum limits for total particle ≥ 5 μm/m 3 at rest in operation at rest in operation A 3 520 3 520 29 29 B 3 520 352 000 29 2 930 C 352 000 3 520 000 2 930 29 300 D 3 520 000 not pre-defined  ( 40 ) 29 300 not pre-defined  ( 40 ) Note 1: The particle limits given in the table for the ‘at rest’ state shall be achieved after a short ‘clean up’ period defined during qualification (guidance value of less than 20 minutes) in an unmanned state, after the completion of operations. Note 2: The occasional indication of macro particle counts, especially ≥ 5 μm, within grade A may be considered to be false counts due to electronic noise, stray light, coincidence loss etc. However, a consecutive or regular counting of low levels may be indicative of a possible contamination event and shall therefore be investigated. Such events may be indicative of an early failure of the room air supply filtration system, an equipment failure, or may also be a signal of poor practices during machine set-up and routine operation. VIII.3.3. For grade A, particle monitoring shall be undertaken for the full duration of the critical processing, including equipment assembly. VIII.3.4. The grade A area shall be monitored continuously (for particles ≥ 0,5 and ≥ 5 μm) and with a suitable sample flow rate (at least 28 litres (1 ft 3 ) per minute) so that all interventions, transient events and any system deterioration is captured. The system shall frequently correlate each individual sample result with alert levels and action limits at such a frequency that any potential excursion can be identified and responded to in a timely manner. Alarms shall be triggered if alert levels are exceeded. Procedures shall define the actions to be taken in response to alarms including the consideration of additional microbial monitoring. VIII.3.5. It is recommended that a similar system be used for the grade B area although the sample frequency may be decreased. The grade B area shall be monitored at such a frequency and with a suitable sample size to ensure that the programme captures any increase in levels of contamination and a system deterioration. If alert levels are exceeded, alarms shall be triggered. VIII.3.6. The selection of the monitoring system shall take into account any risk presented by the materials used in the manufacturing operation (e.g. those involving live organisms, powdery products or radiopharmaceuticals) that may give rise to biological, chemical or radiation hazards. VIII.3.7. In case where contaminants are present due to the processes involved and can potentially damage the particle counter or present a hazard (e.g. live organisms, powdery products and radiation hazards), the frequency and strategy employed shall be adequate to ensure the environmental classification, both prior to and post exposure to the risk. An increase in the monitoring of viable particle shall be considered to ensure a comprehensive monitoring of the process where appropriate. Additionally, monitoring shall be performed during simulated operations at appropriate intervals. The defined approach is part of the contamination control strategy. VIII.3.8. The size of the monitoring samples taken using automated systems will usually depend on the sampling rate of the system used. It is not necessary for the sample volume to be the same as that used for formal classification of the cleanrooms and of the clean air equipment. The monitoring sample volumes shall be justified. VIII.4.    Environmental and personnel monitoring – viable particle VIII.4.1. Frequent microbial monitoring using a combination of methods such as settle plates, volumetric air sampling, glove, gown and surface sampling (e.g. swabs and contact plates) shall be required where aseptic operations are performed. Specifically: — Viable particle monitoring shall be performed within the cleanrooms when normal manufacturing operations are not occurring (e.g. post disinfection, prior to start of manufacturing, on completion of the batch and after a shutdown period) and in associated rooms that have not been used, in order to detect potential incidents of contamination that may affect the controls within the cleanrooms. In case of an incident, additional sample locations may be used as a verification of the effectiveness of a corrective action (e.g. cleaning and disinfection). — Continuous viable air monitoring in grade A (e.g. air sampling or settle plates) shall be performed for the full duration of critical processing, including equipment assembly (aseptic set-up) and critical processing. A similar approach shall be considered for grade B cleanrooms based on the risk of impact on the aseptic processing. The monitoring shall be performed in such a way that all interventions, transient events and any system deterioration are captured and any risk caused by interventions of the monitoring operations is avoided. The method of sampling used shall be justified as part of the contamination control strategy and be demonstrated not to have a detrimental impact on grade A and B airflow patterns. Cleanroom and equipment surfaces shall be monitored at the end of an operation. VIII.4.2. Monitoring of personnel shall be conducted on the basis of a risk assessment, which shall evaluate the locations, type and frequency of monitoring based on the activities performed and the proximity to critical zones. Microbial monitoring of personnel in the grade A and grade B areas is essential. Where operations are manual in nature (e.g. aseptic compounding or filling), an enhanced emphasis shall be placed on microbial monitoring of gowns and the implemented monitoring measures shall be justified within the contamination control strategy. VIII.4.3. Monitoring shall include the sampling of personnel at periodic intervals during the process. Sampling of personnel shall be performed in such a way that it does not compromise the process. Particular consideration shall be paid to the monitoring of personnel following involvement in critical interventions (as a minimum gloves, but other parts of gown may also need to be monitored as applicable to the process) and on each exit from the grade B cleanroom (gloves and gown). VIII.4.4. Where monitoring of gloves is performed after critical interventions, the outer gloves shall be replaced prior to continuation of activity. Where monitoring of gowns is required after critical interventions, the gown shall be replaced before further activity in the cleanroom. VIII.4.5. Regular oversight by the quality unit is required if monitoring is routinely performed by manufacturing personnel. VIII.4.6. The adoption of suitable alternative monitoring systems such as rapid methods may be considered by manufacturers in order to expedite the detection of microbiological contamination issues and to reduce the risks to the product. These rapid and automated microbial monitoring methods may be adopted after validation has demonstrated their equivalency or superiority to the established methods. VIII.4.7. Procedures shall be in place for the assessment and interpretation of appropriate actions (where required) in light of the results obtained from the sampling. Supporting data for the recovery efficiency of the sampling methods chosen shall be available. Action limits for viable particle contamination are shown in Table 6. Table 6 Maximum action limits for viable particle contamination Grade Air sample CFU/m 3 Settle plates (diam. 90 mm) CFU/4 hours  ( 41 ) Contact plates (diam. 55mm), CFU/plate  ( 42 ) Glove print, Including 5 fingers on both hands CFU/glove A No growth  ( 43 ) B 10 5 5 5 C 100 50 25 — D 200 100 50 — Note 1: The types of monitoring methods listed in the table above are examples and other methods may be used provided they are capable of providing information across the entire critical process where the product may be contaminated (e.g. aseptic line set-up, aseptic processing, filling and lyophilizer loading). Note 2: Limits are applied using CFU throughout the document. If different or new technologies are used that present results in a manner different from CFU, the manufacturer shall scientifically justify the limits applied and where possible correlate them to CFU. VIII.4.8. Microorganisms detected in the grade A and grade B areas shall be identified to the species level and the potential impact of such microorganisms on product quality (for each batch implicated) and overall state of control shall be evaluated. The identification of microorganisms detected in grade C and D areas shall be considered, as appropriate, as part of the contamination control strategy (for example where action limits or alert levels are exceeded) or following the isolation of organisms that may indicate a loss of control or deterioration in cleanliness, or the isolation of organisms that may be difficult to control such as spore-forming microorganisms and moulds and at a sufficient frequency to maintain a current understanding of the typical flora of these areas. VIII.5.    Aseptic process simulation (also known as media fill)   ( 44 ) VIII.5.1. The periodic verification of the effectiveness of the controls in place for aseptic processing shall include an aseptic process simulation using a sterile nutrient media and/or a surrogate in place of the product. The selection of the nutrient media and/or the surrogate shall be made based on the ability of the media and/or the surrogate to imitate physical product characteristics posing a risk to the product sterility during the aseptic process. Where processing stages may indirectly impact the viability of any introduced microbial contamination, (e.g. aseptically produced semi-solids, powders, solid materials, microspheres, liposomes and other formulations where product is cooled, heated or lyophilised), alternative procedures that represent the operations as closely as possible shall be developed. Where surrogate materials, such as buffers, are used in parts of the aseptic process simulation, the surrogate material shall not inhibit the growth of any potential contamination. The aseptic process simulation is not the primary means to validate the aseptic process or aspects of the aseptic process. The effectiveness of the aseptic process shall be determined through process design and process controls, training, and evaluation of monitoring data. VIII.5.2. The aseptic process simulation shall imitate as closely as possible the routine aseptic manufacturing process and include all the critical manufacturing steps, specifically: (a) the aseptic process simulation shall assess all aseptic operations performed subsequent to the sterilisation and decontamination cycles of materials utilised in the process to the point where the container is sealed; (b) for non-filterable formulations, any additional aseptic steps shall be assessed; (c) where aseptic manufacturing is performed under an inert atmosphere, the inert gas shall be substituted with air in the process simulation unless anaerobic simulation is intended; (d) processes requiring the addition of sterile powders shall use an acceptable surrogate material in the same containers as those used in the process under evaluation; (e) separate simulations of individual unit operations (e.g. processes involving drying, blending, milling and subdivision of a sterile powder) shall be avoided. Any use of individual simulations shall be supported by a documented justification and ensure that the sum total of the individual simulations continues to fully cover the whole process; (f) the process simulation procedure for lyophilised products shall represent the entire aseptic processing chain including filling, transport, loading, a representative duration of the chamber dwell, unloading and sealing under specified, documented and justified conditions representing worst case operating parameters; (g) the lyophilisation process simulation shall mimic all aspects of the process, except those that may affect the viability or recovery of contaminants. For instance, boiling-over or actual freezing of the solution shall be avoided. Factors to consider in determining aseptic process simulation design include, where applicable: — the use of air to break vacuum instead of nitrogen or other process gases; — replicating the maximum interval between sterilisation of the lyophilizer and its use; — replicating the maximum period of time between filtration and lyophilisation; — quantitative aspects of worst-case situations, e.g. loading the largest number of trays, replicating the longest duration of loading where the chamber is open to the environment. VIII.5.3. The aseptic process simulation shall take into account the aseptic manipulations and interventions known to occur during normal production as well as worst-case situations, as well as the following: (a) inherent and corrective interventions representative of the routine process shall be performed in a manner and frequency similar to that during the routine aseptic process; (b) the inclusion and frequency of interventions in the aseptic process simulation shall be based on the assessed risks posed to the product sterility. VIII.5.4. The aseptic process simulation shall not be used to justify practices that pose unnecessary contamination risks. VIII.5.5. The following elements are relevant for the development of the aseptic process simulation plan: (a) identification of worst-case conditions covering the relevant variables, such as container size and line speed, and their impact on the process. The outcome of the assessment shall justify the variables selected; (b) identification of the representative sizes of container/closure combinations to be used for validation. A bracketing or matrix approach may be considered for validation of the same container/closure configuration for different products where the process equivalence is scientifically justified; (c) identification of the maximum permitted holding times for the product and for the equipment exposed during the aseptic process; (d) identification of the volume filled per container, which shall be sufficient to ensure that the media contacts all the equipment and component surfaces that may directly contaminate the product. The volume used shall also provide sufficient headspace to support potential microbial growth and ensure that turbidity can be detected during inspection; (e) substitution of any inert gas used in the routine aseptic manufacturing process by air, unless anaerobic simulation is intended. In this case, the inclusion of occasional anaerobic simulations as part of the overall validation strategy shall be considered as appropriate; (f) the selected nutrient media shall be capable of growing a designated group of reference microorganisms as described by the relevant pharmacopeia and suitably representative local isolates  ( 45 ) ; (g) the method of detection of microbial contamination shall be scientifically justified to ensure that contamination is reliably detected; (h) the process simulation shall be of sufficient duration to challenge the process, the operators that perform interventions, shift changes and the suitability of the processing environment; (i) where the manufacturer operates different or extended shifts, the aseptic process simulation shall be designed to capture factors specific to those shifts that may pose a risk to product sterility, for example the maximum duration for which an operator may be present in the cleanroom; (j) simulating normal aseptic manufacturing interruptions where the process is idle (e.g. shift changeovers, recharging dispensing vessels, introduction of additional equipment); (k) ensuring that the environmental monitoring is conducted as required for routine production and throughout the entire duration of the process simulation; (l) where campaign manufacturing occurs, such as in the use of barrier technologies or manufacture of sterile active substances, consideration shall be given to designing and performing the process simulation so that it simulates the risks associated with both the beginning and the end of the campaign and demonstrating that the campaign duration does not pose any risk; (m) the performance of ‘end of production or campaign aseptic process simulation’ may be used as additional assurance; however, such approach cannot replace routine aseptic process simulation. VIII.5.6. For sterile active substances, the batch size shall be large enough to represent the routine operation, simulate operation at the worst case, and cover all surfaces that may come into contact with the sterile product. In addition, all the simulated materials (surrogates or growth medium) shall be subject to microbial evaluation. The simulation materials shall be sufficient to ensure the robustness of the evaluation of the process being simulated and shall not compromise the recovery of microorganisms. VIII.5.7. Aseptic process simulation shall be performed as part of the initial validation, with at least three consecutive satisfactory simulation tests that cover all working shifts that the aseptic process may occur in. In addition, an aseptic process simulation is also mandatory after any significant modification to operational practices, facilities, services or equipment that may have an impact on the sterility assurance of the product (e.g. modification to the HVAC system, the equipment, changes to process, the number of shifts or number of personnel, or after a major facility shut down). Moreover, an aseptic process simulation (periodic revalidation) shall usually be repeated twice a year (approximately every six months) for each aseptic process, each filling line and each shift. Each operator shall participate in at least one successful aseptic process simulation annually. Consideration shall be given to performing an aseptic process simulation after the last batch prior to shut down, before long periods of inactivity or before the decommissioning (i.e. definitively removing from the manufacturing process) or the relocation of a line. VIII.5.8. Where manual operation occurs (e.g. aseptic compounding or filling), each type of container, container closure and equipment train shall be initially validated with each operator participating in at least 3 consecutive successful aseptic process simulation and revalidated with one aseptic process simulation approximately every 6 months for each operator. The aseptic process simulation batch size shall mimic the one used in the routine aseptic manufacturing process. VIII.5.9. The number of units processed (filled) for aseptic process simulation shall be sufficient to effectively simulate all the activities that are representative of the aseptic manufacturing process. Justification for the number of units to be filled shall be addressed as part of the contamination control strategy. Typically, a minimum of 5 000 to 10 000 units shall be filled. For small batches (e.g. those under 5 000 units), the number of containers for aseptic process simulation shall at least equal the size of the production batch. VIII.5.10. Filled aseptic process simulation units shall be agitated, swirled or inverted before incubation to ensure contact of the media with all interior surfaces in the container. All integral units from the aseptic process simulation shall be incubated and evaluated, including units with defects without a critical impact on the integrity of the container (e.g. those with cosmetic defects) or those which have gone through non-destructive in-process control checks. If units are discarded during the process simulation and not incubated, those shall be comparable to the units discarded during a routine fill, and only if the standard operating procedures applicable to production specify that units must be removed under the same circumstances (i.e. type of intervention, line location, specific number of units removed). Under no circumstances may more units be removed during a media fill intervention than during a production run. Examples may include those that shall be discarded during routine production after the set-up process or following a specific type of intervention. VIII.5.11. Where the manufacturing process includes materials that are in contact with the product surface but are then discarded (e.g. product flushes), the discarded material shall be simulated with nutrient media and be incubated as part of the aseptic process simulation, unless it can be clearly demonstrated that this waste process does not have an impact on the sterility of the product. VIII.5.12. Filled aseptic process simulation units shall be incubated in a clear container to ensure visual detection of microbial growth. Where the product container is not clear (e.g. amber glass, opaque plastic), clear containers of identical configuration may be substituted to aid in the detection of contamination. When a clear container of identical configuration cannot be substituted, a suitable method for the detection of microbial growth shall be developed and validated. Microorganisms isolated from contaminated units shall be identified to the species level where possible, to assist in the determination of the likely source of the contaminant. VIII.5.13. Filled aseptic process simulation units shall be incubated without unnecessary delay to achieve the best possible recovery of potential contamination. The selection of the incubation conditions and duration shall be scientifically justified and validated to provide an appropriate level of sensitivity for the detection of microbial contamination. VIII.5.14. Upon completion of incubation, filled aseptic process simulation units shall be inspected by personnel who have been appropriately trained and qualified for the detection of microbiological contamination. Such inspection shall be conducted under conditions that facilitate the identification of any microbial contamination. In addition, samples of the filled units shall undergo a positive control by inoculation with a suitable range of reference organisms and suitably representative local isolates. VIII.5.15. The target is zero growth. Any contaminated unit shall be considered as a failed aseptic process simulation and the following actions shall be taken: (a) investigation to determine the most probable root cause(s); (b) determination and implementation of appropriate corrective measures; (c) a sufficient number of successful, consecutive aseptic process simulations (normally a minimum of 3) shall be conducted in order to demonstrate that the process has been returned to a state of control; (d) a prompt review of all appropriate records relating to aseptic production since the last successful aseptic process simulation shall be made. The outcome of the review shall include a risk assessment of the potential breaches in batches manufactured since the last successful aseptic process simulation. In addition, all other batches not released to the market shall be included in the scope of the investigation. Any decision regarding their release status shall take into account the investigation outcome; (e) all products that have been manufactured on a line subsequent to a process simulation failure shall be quarantined until a successful resolution of the process simulation failure has occurred; (f) where the root cause investigation indicates that the failure was related to operator activity, actions to limit the involved operator’s activities, until retrained and requalified, shall be taken; (g) production shall resume only after completion of successful revalidation. VIII.5.16. All aseptic process simulation runs shall be fully documented and include a reconciliation of the units processed (e.g. units filled, incubated and not incubated). Justification for the filled and the non-incubated units shall be included in the documentation. All interventions performed during the aseptic process simulation shall be recorded, including the start and the end time of each intervention and the involved person(s). All microbial monitoring data as well as other testing data shall be recorded in the aseptic process simulation batch record. VIII.5.17. An aseptic process simulation run shall only be aborted under circumstances in which written procedures require commercial lots to be equally handled. An investigation shall be performed and documented in such cases. VIII.5.18. The validation of the aseptic process shall be repeated when the specific aseptic process has not been in operation for an extended period of time or when there is a change to the process, the equipment, the procedures or the environment that has the potential to affect the aseptic process, or when new product containers or container closure combinations are added. SECTION IX QUALITY CONTROL IX.1. There shall be personnel available with appropriate training and experience in microbiology, sterility assurance and knowledge of the processes to support the design of the manufacturing activities, environmental monitoring regime and any investigation assessing the impact of microbiologically linked events to the safety of the sterile product. IX.2. Specifications for raw materials, components and products shall include requirements for microbial, particulate and endotoxin/pyrogen limits when considered necessary having regard to the monitoring data and the overall contamination control strategy. IX.3. The bioburden assay shall be performed on each batch for both aseptically filled product and terminally sterilised products and the results shall be taken into consideration as part of the final batch review. Limits for bioburden immediately before the final sterilising grade filter or the terminal sterilisation process shall be set having regard to the efficiency of the method to be used. Samples shall be taken to be representative of the worst-case scenario (e.g. at the end of hold time). Where overkill sterilisation  ( 46 ) parameters are set for terminally sterilised products, bioburden shall be monitored at suitable scheduled intervals. IX.4. For products authorised for parametric release, a supporting pre-sterilisation bioburden monitoring programme for the filled product prior to initiating the sterilisation cycle shall be developed and the bioburden assay shall be performed for each batch. The sampling locations of filled units before sterilisation shall be based on a worst-case scenario and be representative of the batch. Any organisms found during the bioburden testing shall be identified and their impact on the effectiveness of the sterilising process determined. Where appropriate, the level of endotoxin/pyrogen shall also be monitored. IX.5. The sterility test applied to the finished product shall be validated for the product concerned. This test is only the last in a series of critical control measures by which sterility is assured and it may not be used to ensure sterility of a product that does not meet the relevant design, procedural or validation parameters. IX.6. The sterility test shall be performed under aseptic conditions. In addition, samples taken for sterility testing shall be representative of the whole batch but shall in particular include samples taken from parts of the batch considered to be most at risk of contamination, for example: — for products which have been filled aseptically, samples shall include containers filled at the beginning and end of the batch. The taking of additional samples shall be considered based on risk (e.g. after critical interventions); — for products that have been heat sterilised in their final containers, samples taken shall be representative of the worst case locations (e.g. the potentially coolest or slowest to heat part of each load); — for products that have been lyophilised, samples shall be taken from different lyophilisation loads. Note: Where the manufacturing process results in sub-batches (e.g. for terminally sterilised products), samples from each sub-batch shall be taken and a sterility test performed for each sub-batch. Where appropriate, consideration shall be given to performing separate testing for other finished product tests. IX.7. When it is not possible to have the sterility test result prior to release because the shelf life of the product is too short, additional process controls and monitoring and/or alternative test methods implemented to mitigate the identified risks shall be scientifically justified and documented. IX.8. Any process (e.g. vaporised hydrogen peroxide, ultra violet) used to decontaminate the external surfaces of the sterility samples prior to testing shall not negatively impact the sensitivity of the test method or the reliability of the sample. IX.9. Media used for product testing shall be quality control tested according to the Pharmacopeia before use. Media used for environmental monitoring and aseptic process simulation shall be tested for growth promotion before use, using a scientifically justified and designated group of reference microorganisms and including suitably representative local isolates. Media quality control testing shall usually be performed by the end user. Reliance on outsourced testing or supplier testing of media shall be justified and transportation and shipping conditions be duly considered. IX.10. Environmental monitoring data and trend data generated for classified areas shall be reviewed as part of the product batch certification/release. A written procedure shall be available describing the actions to be taken when data from environmental monitoring are found out of trend or exceeding the established limits. For products with a short shelf life, where the environmental data for the time of manufacture is not available, a review of the most recent available data is required. In addition, the use of rapid/alternative methods may be considered. IX.11. Where rapid and automated microbial methods are used in manufacturing, those methods shall be validated for the product(s) or processes concerned. ( 1 )   For the purposes of this Annex, ‘restricted access barrier system’ means a system that provides an enclosed, but not fully sealed, environment meeting defined air quality conditions, and using a rigid-wall enclosure and integrated gloves to separate its interior from the surrounding cleanroom environment. The inner surfaces of the RABS are disinfected and decontaminated with a sporicidal agent. Operators use gloves, half suits, rapid transfer system/ports and other integrated transfer ports to perform manipulations or convey materials to the interior of the RABS. Depending on the design, doors are rarely opened, and only under strictly pre-defined conditions. ( 2 )   For the purposes of this Annex, ‘critical zone’ means a space within the aseptic processing area in which product and critical surfaces are exposed to the environment. ( 3 )   For the purposes of this Annex, ‘first air’ means filtered air that has not been interrupted prior to contacting the exposed product and product contact surfaces. ( 4 )   For the purposes of this Annex, ‘unidirectional airflow’ means an airflow moving in a single direction, in a robust and uniform manner, and at sufficient speed, to reproducibly sweep particles away from the critical processing or testing area. ( 5 )   For the purposes of this Annex, ‘personnel airlock’ means an area of increasing cleanliness used for entry of personnel (e.g. from the grade D area to the grade C area, or from the C area to the grade B area). ( 6 )   For the purposes of this Annex, ‘material airlock’ means an area used for transfer of materials and equipment. ( 7 )   For the purposes of this Annex, ‘turbulent airflow’ means air that is not unidirectional. Turbulent air in cleanrooms shall flush the cleanroom via mixed flow distribution and ensure maintenance of an acceptable air quality. ( 8 )   For the purposes of this Annex, ‘unidirectional airflow unit’ means a cabinet supplied with filtered unidirectional airflow. The concept is interchangeable with ‘laminar airflow unit’. ( 9 )   It is noted that qualification of cleanrooms is a different process than environmental monitoring. ( 10 )   Classification including 5μm particles may be considered where relevant in accordance with the contamination control strategy or historical trends. ( 11 )   For grade D, in operation limits are not pre-defined. The manufacturer shall establish relevant in operation limits based on a risk assessment and routine data where applicable. ( 12 )   For the purposes of this Annex, ‘colony forming unit’ or ‘CFU’ means a single detectable colony that originates from one or more microorganisms. Colony forming units are typically expressed as CFU per ml for liquid samples, CFU per m 3 for air sample and CFU per sample for samples captured on solid medium such as settle or contact plates. ( 13 )   Settle plates shall be exposed for the duration of operations and changed as required after a maximum of 4 hours. Exposure time shall be based on recovery studies and shall not allow desiccation of the media used. ( 14 )   For the purposes of this Annex, ‘isokinetic sampling head’ means a sampling head designed to disturb the air as little as possible so that the same particles go into the nozzle as would have passed the area if the nozzle had not been there (i.e. the sampling condition in which the mean velocity of the air entering the sample probe inlet is nearly the same (± 20 percent) as the mean velocity of the airflow at that location). ( 15 )   For the purposes of this Annex, ‘water system’ means a system for producing, storing and distributing water, usually compliant to a specific pharmacopeia grade (e.g. purified water and water for injection). ( 16 )   For the purposes of this Annex, ‘dead leg’ means a length of a non-circulating pipe (where fluid may remain static) that is greater than 3 internal pipe diameters. ( 17 )   For the purposes of this Annex, ‘sterilising grade filter’ means a filter that, when appropriately validated, is able to remove a defined microbial challenge from a fluid or gas producing a sterile effluent. Usually, such filters have a pore size equal or less than 0,22 μm. ( 18 )   For the purposes of this Annex, ‘terminal sterilisation’ means the application of a lethal sterilising agent or conditions to a product in its final container to achieve a predetermined sterility assurance level of 10 –6 or below (e.g. the theoretical probability of there being a single viable microorganism present on or in a sterilised unit is equal to or less than 1 × 10 -6 ). ( 19 )   For the purposes of this Annex ‘critical intervention’ means an intervention into the critical zone. ( 20 )   For the purposes of this Annex, ‘intrinsic sterile connection device’ means a device that reduces the risk of contamination during the connection process; it can be mechanical or fusion sealing. ( 21 )   For the purposes of this Annex, ‘inherent interventions’ means interventions that are an integral part of the aseptic process and required for either set-up, routine operation or monitoring (e.g. aseptic assembly, container replenishment, environmental sampling) and which are foreseen in relevant standard operating procedures/work instructions. ( 22 )   For the purposes of this Annex, ‘critical surface’ means a surface that may come directly in contact with, or otherwise directly affect the sterility/absence of contamination of, a product or its containers or closures. ( 23 )   For the purposes of this Annex, ‘grade A air supply’ means air that has passed through a filter qualified as capable of producing grade A total particle quality air, but where there is no requirement to perform continuous total particle monitoring or meet grade A viable monitoring limits. ( 24 )   For the purposes of this Annex, ‘post-aseptic processing terminal heat treatment’ means a terminal moist heat process employed after aseptic processing which has been demonstrated to provide a sterility assurance level ≤ 10 -6 but where the requirements of steam sterilisation (for example, F 0  ≥ 8 min) are not fulfilled. This may also be beneficial in the destruction of viruses that may not be removed through filtration. ( 25 )   For the purposes of this Annex, ‘biological indicators’ means a population of microorganisms inoculated onto a suitable medium (e.g. solution, container or closure) and placed within a steriliser or load or room locations to determine the sterilisation or disinfection cycle efficacy of a physical or chemical process. The challenge microorganism shall be selected and validated based upon its resistance to the given process. Incoming lot D-value, microbiological count and purity define the quality of the biological indicator. ( 26 )   For the purposes of this Annex, ‘D value’ means the value of a parameter of sterilisation (duration or absorbed dose) required to reduce the number of viable organisms to 10 percent of the original number. ( 27 )   For the purposes of this Annex, ‘Z value’ means the temperature difference that leads to a 10-fold change in the D-value of the biological indicators. ( 28 )   For the purposes of this Annex, ‘rapid transfer system/port’ means a system used for the transfer of items into RABS or isolators that minimises the risk to the critical zone. An example would be a rapid transfer container with an alpha/beta port. ( 29 )   For the purposes of this Annex, ‘equilibration time’ means the time that elapses between the attainment of the sterilisation temperature at the refence measurement point and the attainment of the sterilisation temperature at all points within the load. ( 30 )   For the purposes of this Annex, ‘F 0 value’ means the lethality expressed in terms of the equivalent time in minutes at the reference temperature delivered by the process to the sterilisation load, with reference to micro-organisms possessing the relevant theoretical z-value. ( 31 )   For the purposes of this Annex, ‘F h value’ means the lethality expressed in terms of the equivalent time in minutes at the reference temperature delivered by the process to the sterilisation load, with reference to micro-organisms possessing the relevant theoretical z-value. ( 32 )   For the purposes of this Annex, ‘bacterial retention testing’ means a test performed to validate that a filter can remove bacteria from a gas or liquid. The test is usually performed using a standard organism, such as Brevundimonas diminuta at a minimum concentration of 10 7 Colony Forming Units/cm 2 . ( 33 )   For the purposes of this Annex, ‘Form-Fill-Seal’ means an automated filling process, typically used for terminally sterilised products, which constructs the primary container out of a continuous flat roll of packaging film while simultaneously filling the formed container with product and sealing the filled containers in a continuous process. Form-Fill-Seal processes may utilize a single web system (where a single flat roll of film is wrapped around itself to form a cavity), or a dual web system (where two flat rolls of film are brought together to form a cavity), often with the aid of vacuum moulds or pressurised gases. The formed cavity is filled, sealed and cut into sections. Films typically consist of a polymeric material, polymeric coated foil or other suitable material. ( 34 )   For the purposes of this Annex, ‘Blow-Fill-Seal’ means a technology in which containers are formed from a thermoplastic granulate, filled with product, and then sealed in a continuous, integrated, automatic operation. The two most common types of Blow-Fill-Seal machines are the Shuttle type (with Parison cut) and the Rotary type (Closed Parison). ( 35 )   For the purposes of this Annex, ‘parison’ means the tube of polymer extruded by the Blow-Fill-Seal machine from which containers are formed. ( 36 )   For the purposes of this Annex, ‘lyophilisation’ means a physical-chemical drying process designed to remove solvents, by way of sublimation, from both aqueous and non-aqueous systems, primarily to achieve product or material stability. Lyophilisation is synonymous to the term freeze-drying. ( 37 )   For the purposes of this Annex, ‘single use systems’ means systems in which product contact components are used only once to replace reusable equipment such as stainless-steel transfer lines or bulk containers. ( 38 )   For the purposes of this Annex, ‘leachables’ means chemical entities that, under normal conditions of use or storage, migrate from the product contact surface of the process equipment or containers into the product or material being processed. ( 39 )   For the purposes of this Annex, ‘extractables’ means chemical entities that migrate from the surface of the process equipment, when exposed to an appropriate solvent at extreme conditions, into the product or material being processed. ( 40 )   For grade D, in operation limits are not predetermined. The manufacturer shall establish in operation limits based on a risk assessment and on routine data, where applicable. ( 41 )    — Settle plates shall be exposed in grade A and B areas for the duration of operations (including equipment set-up) and changed as required after a maximum of 4 hours (exposure time shall be based on validation including recovery studies and it shall not have any negative effect on the suitability of the media used). — For grade C and D areas, exposure time (with a maximum of 4 hours) and frequency shall be based on quality risk management principles. — Individual settle plates may be exposed for less than 4 hours. ( 42 )   Contact plate limits apply to equipment, room and gown surfaces within the grade A and grade B areas. Routine gown monitoring is not normally required for grade C and D areas, depending on their function. ( 43 )   For grade A, any growth shall trigger an investigation. ( 44 )   For the purposes of this Annex, ‘aseptic process simulation’ means the simulation of the entire aseptic manufacturing process to verify whether the process is adequate to ensure sterility/prevent contamination during production. It includes all operations associated with routine manufacturing, such as equipment assembly, formulation, filling, lyophilisation and sealing process as necessary. ( 45 )   For the purposes of this Annex, ‘local isolates’ means suitably representative microorganisms of the site that are frequently recovered through environmental monitoring within the classified zone/areas especially grade A and B areas, personnel monitoring or positive sterility test results. ( 46 )   For the purposes of this Annex, ‘overkill sterilisation’ means a process that is sufficient to provide at least a 12 log 10 reduction of microorganisms having a minimum D-value of 1 minute.