BIOLOGICAL AND IMMUNOLOGICAL PRODUCTS
ANNEX IISupplementary provisions
ANNEX II BIOLOGICAL AND IMMUNOLOGICAL PRODUCTS I. SCOPE I.1. The additional requirements set out in this Annex shall apply to the manufacture, control and testing of biological and immunological veterinary medicinal products, with the exception of inactivated immunological veterinary medicinal products which are manufactured from pathogens and antigens obtained from an animal or animals in an epidemiological unit and used for the treatment of that animal or those animals in the same epidemiological unit or for the treatment of an animal or animals in a unit having a confirmed epidemiological link. Throughout the Annex, reference to ‘biological veterinary medicinal products’ or ‘biologicals’ is to be understood as encompassing also immunologicals. I.2. Antibiotics are generally not considered biologicals. Nevertheless, manufacturers are advised to follow the requirements set out in this Annex with regard to manufacturing procedures described in this document that are used in the manufacture of such veterinary medicinal products. I.3. Table 1 illustrates the manufacturing activities which are generally within the scope of this Annex. Table 1 Type and source of material Example product Manufacturing steps covered by this Annex shown in grey GMP requirements shall increase from the earlier steps (e.g. collection) to the final manufacturing steps (formulation, filling). For earlier stages of manufacturing, GMP principles shall at least be adhered to. Human source Urine derived enzymes, hormones Collection of material ( 1 ) Mixing, and/or initial processing Isolation and purification Formulation, filling Animal or plant sources (not genetically modified/edited) Heparins, insulin, enzymes, proteins, allergen extract, immunosera Collection of plant or animal material ( 2 ) Cutting, mixing, and/or initial processing Isolation and purification Formulation, filling Virus or bacteria /fermentation/cell culture, etc. Viral or bacterial vaccines; enzymes, proteins Establishment & maintenance of MCB, WCB, MSL, WSL ( 3 ) Cell culture and/or fermentation Inactivation when applicable, isolation and purification Formulation, filling Biotechnology- fermentation/cell culture Recombinant products, MAb, allergens, vaccines Establishment & maintenance of MCB and WCB, MSL, WSL ( 4 ) Cell culture and/or fermentation Isolation, purification, modification Formulation, filling Animal sources, (genetically modified/edited) Recombinant proteins Master and working (genetically modified/edited) bank Collection, cutting, mixing, and/or initial processing Isolation, purification, modification Formulation, filling Plant sources: (genetically modified/edited) Recombinant proteins, vaccines, allergen Master and working (genetically modified/edited) bank Growing, harvesting ( 5 ) Initial extraction, isolation, purification, modification Formulation, filling I.4. In cases where there is a continuous process from the sourcing/isolation of the active substance from a biological source to the manufacturing of the finished product (e.g. veterinary medicinal products that consists of cells, viral-based vaccines, phages), the requirements of this Regulation shall apply to the entire manufacturing process. II. PERSONNEL II.1. Personnel (including those concerned with cleaning and maintenance) employed in areas where biological products are manufactured and tested shall receive initial and periodic retraining specific to the products manufactured and their respective tasks, including measures to protect the product, personnel and the environment, as well as – where appropriate – training on microbiology. II.2. Personnel shall be protected against a possible infection with the biological agents used in the manufacture. In the case of biological agents known to cause disease in humans, adequate measures shall be taken to prevent infection of personnel working with the agent or with experimental animals. Where appropriate, relevant vaccination and health monitoring shall be offered having regard to the specific characteristics of the manufactured product (e.g. BCG vaccine ( 6 ) , rabies, brucella, leptospira, tuberculin products) and the tasks of the staff. II.3. When a heath condition that may have a negative impact on the quality of the product is declared by the relevant personnel or otherwise becomes apparent, access to the production or control area shall be barred. II.4. Where required to minimise the opportunity for cross-contamination, restrictions on the movement of all personnel (including quality control, maintenance and cleaning staff) shall be applied on the basis of quality risk management principles ( 7 ) . In general, personnel shall not pass from areas where exposure to live micro-organisms, genetically modified/genome edited organisms, toxins or animals occurs to areas where other products, or different organisms are handled. If such passage is unavoidable, appropriate contamination control measures commensurate to the risks shall be implemented. II.5. Adequate measures shall be implemented to prevent biological agents being taken outside the manufacturing plant by personnel acting as a carrier. Dependent on the type of biological agent, such measures may include a complete change of clothes and compulsory showering before leaving the production area. III. PREMISES AND EQUIPMENT III.1. Premises III.1.1. Premises shall be designed in such a way as to control the risks to the product and the risks to the environment. This may be achieved by the use of contained, clean, or controlled areas. In particular: (a) Live biological agents that are highly pathogenic shall be handled in contained areas (including quality control operations, research and diagnostic services, etc). The level of containment shall be adapted to the pathogenicity of the agent. (b) Without prejudice to the containment measures that may be required for certain biological organisms as provided for under (a), production of biological agents may generally take place in controlled areas provided it is carried out in totally enclosed and sterilised equipment and all relevant connections are made in accordance with the requirements set out in Annex I. (c) Inactivated biological agents shall be handled in clean areas. Clean areas shall also be used when handling non-infected cells isolated from multicellular organisms and, where appropriate, filtration-sterilised media. (d) Open circuit operations involving products or components not subsequently sterilised shall be carried in accordance with the requirements set out in Annex I, where relevant. (e) With the exception of blending and subsequent filling operations (or when closed systems are used), only one biological agent shall be handled at a given time within an area. (f) Production operations such as cell maintenance, media preparation, virus culture, etc. which are likely to cause contamination shall be performed in separate areas, unless appropriate organisational and technical measures may be put in place to prevent contamination. (g) Production areas where biological agents particularly resistant to disinfection (e.g. spore-forming bacteria) are handled shall be separated and dedicated to that particular purpose until the biological agents have been inactivated, unless appropriate organisational and technical measures may be put in place to prevent contamination. III.1.2. As part of the contamination control strategy, the degree of environmental control of particulate and microbial contamination of the production premises shall be adapted to the active substance, intermediate or finished product and the production step, bearing in mind the potential level of contamination of the starting materials and the risks to the product. Where relevant in accordance with quality risk management principles, the environmental monitoring programme shall be supplemented by the inclusion of methods to detect the presence of specific microorganisms (i.e. host organism, yeast, moulds, anaerobes, etc.). III.1.3. Manufacturing and storage facilities, processes and environmental classifications shall be designed to prevent the extraneous contamination of products. Where processes are not closed and there is therefore exposure of the product to the immediate room environment (e.g. during additions of supplements, media, buffers, gases) adequate control measures shall be put in place, including engineering and environmental controls. Relevant aspects addressed in Annex I shall be applied, including aspects related to the required environmental grades and associated controls. III.1.4. Air handling units shall be designed, constructed and maintained to minimise the risk of cross-contamination between different manufacturing areas, to provide relevant containment where applicable, and may need to be specific for an area. Consideration, based on quality risk management principles, shall be given to the use of single pass air systems. III.1.5. Positive pressure areas shall be used to process sterile products but negative pressure in specific areas at the point of exposure of pathogens is acceptable for containment reasons. Where negative pressure areas or safety cabinets are used for aseptic processing of materials with particular risks (e.g. pathogens) they shall be surrounded by a positive pressure clean zone of appropriate grade. These pressure cascades shall be clearly defined and continuously monitored with appropriate alarm settings. III.1.6. Air vent filters shall be hydrophobic and validated for their scheduled life span with integrity testing at appropriate intervals based on appropriate quality risk management principles. III.1.7. Where necessary to address the contamination risks, equipment passes and changing rooms shall have an interlock mechanism or other appropriate system to prevent the opening of more than one door at a time. Changing rooms shall be supplied with air filtered to the same standard as that for the work area and equipped with air extraction facilities to produce an adequate air circulation independent of that of the work area. Equipment passes shall normally be ventilated in the same way, but unventilated passes, or those equipped with supply air only, may be acceptable. III.1.8. Drainage systems shall be designed so that effluents can be effectively neutralised or decontaminated to minimise the risk of cross-contamination. Local regulation shall be followed to minimise the risk of contamination of the external environment according to the risk associated with the biohazardous nature of waste materials. Containment III.1.9. Containment premises shall be designed so as to ensure that they can be easily disinfected and shall have the following characteristics: (a) Absence of direct venting to the outside. (b) Ventilation with air at negative pressure: Air shall be extracted through HEPA filters and not be re circulated except to the same area, and provided further HEPA filtration is used (normally this condition would be met by routing the recirculated air through the normal supply HEPAs for that area). Recycling of air between areas is acceptable only if it passes through two exhaust HEPAs, the first of which is continuously monitored for integrity, and there are adequate measures for safe venting of exhaust air should this filter fail. Where negative pressure areas are used for containment purposes of aseptic processing of materials with particular risks, they shall be combined with a positive pressure clean zone of appropriate grade (pressure bubble or sink airlock). These pressure cascades shall be clearly defined and continuously monitored with appropriate alarm settings. (c) By way of derogation from (b), air from manufacturing areas used for the handling of exotic organisms ( 8 ) shall be vented through two sets of HEPA filters in series, and air from production areas shall not be re-circulated. (d) A system shall be put in place for the collection and disinfection of liquid effluents including the contaminated condensate from sterilisers, biogenerators, etc. In addition, solid wastes, including animal carcasses, shall be disinfected, sterilised or incinerated as appropriate. Contaminated filters shall be removed using a safe method. (e) Changing rooms shall be designed and used as air locks and equipped with washing and showering facilities if appropriate. Air pressure differentials shall be such that there is no flow of air between the work area and the external environment or a risk of contamination of the outer clothing worn outside the area. (f) An airlock system is in place for the passage of equipment, which is constructed so that there is no flow of contaminated air between the work area and the external environment or a risk of contamination of the equipment within the lock. The airlock shall be of a suitable size to enable the effective surface decontamination of materials being passed through it. Consideration shall be given to having a timing device on the door interlock to allow sufficient time for the decontamination process to be effective. (g) Where appropriate, a barrier double-door autoclave shall be used for the secure removal of waste materials and introduction of sterile items. III.1.10. The measures and procedures implemented for containment to address operator and environmental safety shall not conflict with those required to ensure product quality. Multiproduct facility III.1.11. Manufacture of biologicals in a multi-product facility is acceptable where appropriate measures are implemented to prevent cross-contamination, such as: — measures to prevent cross-contamination to non-related areas or equipment (e.g. use of single use components and engineering measures such as closed systems); — validated cleaning and decontamination procedures before the subsequent manufacture of other products, covering also heating, ventilation and air conditioning (HVAC) system; — environmental monitoring in adjacent areas – during manufacture and after completion of cleaning and decontamination – specific for the micro-organism being manufactured. Risks arising from the use of certain monitoring equipment (e.g. airborne particle monitoring) in areas handling live and/or spore-forming organisms shall be duly considered; — controls on the movement/removal of products, equipment, ancillary equipment (e.g. for calibration and validation) and disposable items to prevent contamination of other areas, other products or different product stages (e.g. prevent contamination of inactivated or toxoided products with non-inactivated products); — campaign-based manufacturing. Where production involves the manufacture of multiple small batches from different starting materials, factors such as the health status of donors and the risk of total loss of product shall be taken into account when considering the acceptance of concurrent working. III.1.12. For finishing operations (formulation, filling and packaging), the assessment whether dedicated facilities are required shall also take into consideration the specific characteristics of the concerned biological product and the characteristics of other products, including any non-biological products, handled in the same facility. Other control measures for finishing operations may include the need for specific addition sequences, mixing speeds, time and temperature controls, limits on exposure to light, as well as containment and cleaning procedures in the event of spillages. III.2. Equipment III.2.1. Equipment used during handling of live organisms and cells, including equipment used for sampling, shall be suitable to prevent contamination during processing, facilitate decontamination and sterilisation (where applicable), and to avoid any mix-up between different organisms or products. Particular attention shall be paid to control measures to avoid cross-contamination from parts of equipment that are not fixed, such as pipes, valves and filters (e.g. appropriate identification as to their function). III.2.2. Equipment used for the storage of biological agents or products shall be suitable and used in such a manner as to prevent any possible mix-up. All stored items shall be clearly and unambiguously labelled and in leak-proof containers. III.2.3. Where relevant, equipment shall be fitted with recording and/or alarm systems (e.g. equipment requiring temperature control). To avoid breakdowns, a system of preventive maintenance, together with trend analysis of recorded data, shall be implemented. III.2.4. Close equipment used for primary containment shall be designed and periodically tested to ensure the prevention of escape of biological agents into the immediate working environment. Inlets and outlets for gases shall be protected so as to achieve adequate containment, e.g. by the use of sterilising hydrophobic filters. The introduction or removal of material shall take place using a sterilisable closed system, or possibly in an appropriate laminar air flow. III.2.5. Where necessary, equipment shall be properly sterilised by a validated method. Equipment used for purification, separation or concentration shall be sterilised or disinfected at least between each use for different products. The effect of the sterilisation methods on the effectiveness and validity of the equipment shall be studied in order to determine the life span of the equipment. III.2.6. The use of ‘clean in place’ ( 9 ) and ‘steam in place’ ( 10 ) (‘sterilisation in place’) systems shall be used where appropriate. Valves on fermentation vessels shall be completely steam sterilisable. III.2.7. The loading of freeze dryers requires an appropriate clean/contained area. Unloading freeze dryers contaminates the immediate environment. Therefore, for single-ended freeze dryers, the clean room shall be decontaminated before a further manufacturing batch is introduced into the area, unless that batch involves the same organism(s). Double door freeze dryers shall be sterilised after each cycle unless opened in a clean area. Sterilisation of freeze dryers shall be done in accordance with Annex I. In case of campaign working, they shall at least be sterilised after each campaign. IV. ANIMALS The use of animals or animal materials in the manufacture of veterinary medicinal products is subject to specific additional requirements: (a) Biological substances and products shall comply with the latest version of the Note for guidance on minimising the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products. (b) Specifications for materials of animal origin shall consider aspects such as age, weight and health status of the animals, as appropriate, and in accordance with the terms of the marketing authorisation. (c) Where applicable, pharmacopeia requirements shall be complied with, including the need for specific tests at defined stages. (d) A health programme shall be established to monitor adventitious agents that are of concern (zoonotic diseases, diseases of source animals). Specialist advice shall be obtained for the establishment of such programme. In particular, reports from trustworthy sources on national disease prevalence shall be considered when performing the assessment of risk and mitigation factors. Such sources include the World Organisation for Animal Health (WOAH) ( 11 ) , as well as information on health monitoring and control programme(s) at national and local level. (e) Instances of ill-health occurring in the source/donor animals shall be investigated with respect to their suitability and the suitability of in-contact animals for continued use (in manufacture, as sources of starting and raw materials, in quality control and safety testing). Decisions shall be documented. A look-back procedure shall be in place to inform the decision-making process on the continued suitability of the biological active substance or medicinal product in which the animal sourced starting or raw materials have been used or incorporated. This decision-making process may include the re-testing of retained samples from previous collections from the same donor animal (where applicable) to establish the last negative donation. The withdrawal period of therapeutic agents used to treat source/donor animals shall be documented and used to determine the removal of those animals from the programme for defined periods. (f) Particular care shall be paid to the prevention and monitoring of infections in the source/donor animals, including addressing aspects regarding the sourcing, the facilities, the husbandry, the biosecurity procedures, the testing regimes or the control of bedding and feed materials. This is of special relevance to specified pathogen free ( 12 ) animals where PhEur monograph requirements shall be met. Housing and health monitoring shall be defined for other categories of animals (e.g. healthy flocks or herds). (g) For products manufactured from animals that have been genetically modified or whose genome has been edited, traceability shall be maintained in the creation of such animals from the source animals. (h) Housing for animals used in production and control of biological active substances and medicinal products shall be separated from production and control areas. Such houses shall be provided with the appropriate containment and/or clean area measures and shall be separate from other animal accommodation. It is particularly important to ensure that animal houses where animals used for quality control involving the use of pathogenic biological agents are accommodated are adequately contained. (i) At the production site, animals, biological agents and tests carried out on them shall be duly identified to prevent any risk of confusion and to control all the identified hazards. (j) Suitable measures shall be implemented to ensure the quality and the traceability of materials of animal origin sourced from abattoirs. In particular, the contract/supply agreement with the abattoir shall address the required measures to ensure traceability of sourced materials, as well as the implementation of adequate hygiene and other necessary control measures at the abattoir. (k) Adequate controls (based on quality risk management principles) shall be applied over the supply chain and during the transport of animals or animal materials used in the manufacture of veterinary medicinal products, including detailed documentation to ensure traceability. Traceability shall be ensured, including the movement of material between sites of initial collection, partial and final purification(s), storage sites, hubs, consolidators and brokers. Details of such arrangements shall be recorded and any breaches recorded, investigated and actions taken. V. STARTING AND RAW MATERIALS V.1. The source, origin and suitability of biological starting and raw materials (e.g. cryoprotectants, feeder cells, reagents, culture media, buffers, serum, enzymes, cytokines, growth factors) shall be clearly defined in written specifications. Specifications shall include quality requirements necessary to ensure the suitability of the materials for the intended use and to minimise variability (covering relevant aspects of the production and control). Microbiological controls are particularly important. The specifications set shall be in compliance with the terms of the marketing authorisation. Starting and raw materials shall be appropriately identified through the various stages of production. V.2. Where the results from the test(s) required to release the starting materials take a long time (e.g. sterility test), it may be permissible to process the starting materials before the results of the test(s) are available, provided that the risk of using a potentially failed material and its potential impact on other batches is understood and assessed under quality risk management principles. In such cases, the release of a finished product shall be conditional on the satisfactory results of these tests. V.3. The risk of contamination of the starting and raw materials during their passage along the supply chain shall be assessed, with particular emphasis on TSE. Materials that come into direct contact with the manufacturing equipment or with the product (such as media used in media fill experiments and lubricants that may contact the product) shall also be taken into account. V.4. A control strategy to protect the product and the preparation of solutions, buffers and other additions based on the principles set out in Annex I shall be implemented. The controls required for the quality of starting and raw materials and on the aseptic manufacturing process are particularly important for products in respect of which final sterilisation is not possible. V.5. Where sterilisation of starting and raw materials is required, it shall be carried out – where possible – by heat. Where necessary, other appropriate methods may also be used for inactivation of biological materials (e.g. irradiation and filtration). V.6. The use of antibiotics at early manufacturing stages to reduce bioburden (e.g. bioburden associated with the procurement of living tissues and cells) shall generally be avoided. The use thereof shall be duly justified. In such cases, the presence of antibiotics shall be removed from the manufacturing process at the stage specified in the marketing authorisation. V.7. Adequate measures shall be implemented throughout the supply chain to ensure the traceability of substances of animal and human origin that are used in the manufacture of veterinary medicinal products. Donor (human or animal) health information having an impact on the quality of the veterinary medicinal product which becomes available after procurement shall be taken into account in recall procedures. VI. SEED LOT AND CELL BANK SYSTEM VI.1. In order to prevent the unwanted drift of properties that may result from repeated subcultures or multiple generations, the production of biological substances and products obtained by microbial culture, cell culture or propagation in embryos and animals shall be based on a system of master and working seed lots ( 13 ) and/or cell banks ( 14 ) . VI.2. The number of generations (doublings, passages) between the seed lot or cell bank, the active biological substance and the finished product shall be consistent with the specifications in the marketing authorisation. VI.3. As part of the product lifecycle management, the establishment of seed lots and cell banks, including master and working generations, shall be performed under circumstances which are demonstrably appropriate, including a controlled environment which is adequate to protect the seed lot and the cell bank and the personnel handling it. In addition, during the establishment of the seed lot and cell bank, no other living or infectious material (e.g. virus, cell lines or cell strains) shall be handled simultaneously in the same area or by the same persons. VI.4. For stages prior to the master seed or cell bank generation, where only the principles of GMP may be applied, documentation shall be available to support traceability, including regarding components used during the development with a potential impact on product safety (e.g. reagents of biological origin), from initial sourcing and genetic development if applicable. For vaccines, compliance with the requirements in Pharm. Eur. monograph on Vaccines for veterinary use 01/2023:0062 is required. VI.5. Following the establishment of master and working cell banks and master and working seed lots, quarantine and release procedures shall be followed, including adequate characterisation and testing for contaminants. Their on-going suitability for use shall be further demonstrated by the consistency of the characteristics and quality of the successive batches of product. Evidence of the stability and recovery of the seeds and banks shall be documented and records shall be kept in a manner permitting trend evaluation. VI.6. Seed lots and cell banks shall be stored and used in such a way as to minimise the risks of contamination (e.g. stored in the vapour phase of liquid nitrogen in sealed containers) or alteration. Control measures for the storage of different seeds and/or cells in the same area or equipment shall prevent mix-up and take into account the infectious nature of the materials to prevent cross contamination. VI.7. Storage containers shall be sealed, clearly labelled and kept at an appropriate temperature. A stock inventory shall be kept. The storage temperature shall be recorded continuously and, where used, the liquid nitrogen level monitored. Deviation from set limits and corrective and preventive action taken shall be recorded. VI.8. It is desirable to split stocks and to store the split stocks at different locations so as to minimise the risks of total loss. The controls at such locations shall provide the assurances outlined in the preceding paragraphs. VI.9. The storage and handling conditions for stocks shall be managed according to the same procedures and parameters. Once containers are removed from the seed lot/cell bank management system, the containers shall not be returned to stock. VII. PRODUCTION VII.1. Quality risk management principles shall be implemented across all the stages of the manufacture of biological veterinary medicinal products to minimise process variability and to enhance reproducibility. The effectiveness of the implemented measures shall be reassessed during product quality reviews. VII.2. Critical operational (process) parameters and other input parameters that affect product quality shall be identified, validated, documented and be shown to be maintained within required parameters. VII.3. Changes introduced in the manufacturing process shall comply with the requirements laid down in Article 26(3). In addition, the cumulative effects of changes introduced in the manufacturing process on the quality, safety and efficacy of the finished product shall be evaluated on periodic basis. VII.4. Where starting materials from different donors are used, adequate controls shall be implemented to minimise the risk of cross-contamination or mix-ups. VII.5. For biological materials that cannot be sterilised (e.g. by filtration), processing shall be conducted aseptically to minimise the introduction of contaminants. The requirements on aseptic manufacturing set forth in Annex I shall be implemented. VII.6. The terms of the marketing authorisation or – as appropriate – Pharmacopoeia monographs, shall determine whether, and up to what stage, substances and materials used in the manufacturing of biological veterinary medicinal products can have a defined level of bioburden or need to be sterile. Appropriate controls shall be implemented to ensure that the specified limits are respected. VII.7. Appropriate measures shall be implemented throughout all the production stages and controls to prevent or minimise the occurrence of unwanted bioburden and associated metabolites and endotoxins. VII.8. A control strategy for the entry of articles and materials into production areas shall be implemented based on quality risk management principles. The following shall be implemented where applicable: (a) For aseptic processes, heat stable articles and materials entering a clean/contained area ( 15 ) shall preferably do so through a double-ended autoclave or oven. Heat labile articles and materials shall enter through an airlock with interlocked doors where they shall be subject to effective surface sanitisation procedures. The sterilisation of articles and materials elsewhere is acceptable provided that multiple wrappings are used, as appropriate to the number of stages of entry to the clean area, and enter through an airlock with the appropriate surface sanitisation precautions. (b) Equipment, glassware, the external surfaces of product containers and other such materials shall be disinfected before being transferred from a contained area using a validated method. Only the absolute minimum of materials shall enter or leave the area. (c) Liquid or solid wastes such as the debris after harvesting eggs, disposable culture bottles, unwanted cultures or biological agents, shall preferably be sterilised or disinfected before being transferred from a contained area. However, alternatives such as the use of sealed containers or piping may be appropriate in some cases. (d) Where relevant/critical raw materials (such as culture media and buffers) have to be measured or weighed during the production process (e.g. due to variability concerns), small stocks of these raw materials may be kept in the production area for a specified duration based on defined criteria (e.g. for the duration of manufacture of the batch or of the campaign). VII.9. The growth promoting properties of culture media shall be demonstrated to be suitable for its intended use. If possible, media shall be sterilised in situ. VII.10. The addition of materials or cultures to fermenters and other vessels and sampling shall be carried out under carefully controlled conditions to prevent contamination and, in case of live micro-organism, egress. It shall be verified that vessels are correctly connected when the addition or the sampling takes place. Gases, media, acid or alkalis, defoaming agents and other materials introduced into sterile biogenerators shall be sterile where appropriate. VII.11. Continuous monitoring of some production processes, such as e.g. fermentation, may be necessary (e.g. continuous monitoring of parameters such as temperature, pH, pO 2 , CO 2 and the rate of feed or carbon source with respect to growth of cells) and such data shall form part of the batch record. Special consideration shall be given to the quality controls required when continuous culture is used. VII.12. The formation of droplets and the production of foam shall be avoided or minimised as far as possible during manufacturing. Centrifugation and blending of products can lead to aerosol formation. Therefore, such activities shall be adequately contained with a view to minimise the risk of cross-contamination or, where relevant, risks to operators or the environment. VII.13. Accidental spillages, especially of live organisms, shall be dealt with quickly and safely. Validated decontamination measures shall be available for each organism or groups of related organisms. Where different strains of single bacteria species or very similar viruses are involved, the decontamination process may be validated with one representative strain, unless there is reason to believe that they may vary significantly in their resistance to the agent(s) involved. VII.14. Production and control materials, including paperwork, that are obviously contaminated, such as by spills or aerosols, or if a potential hazardous organism is involved, shall be adequately decontaminated, or the information be transferred out by other means. VII.15. Precautions shall be taken to avoid contamination or confusion during incubation. Separate incubators shall be used for infected and non-infected containers and also generally for different organisms or cells. Incubators containing more than one organism or cell type are only acceptable if adequate steps are taken to seal, decontaminate the surface and segregate the containers. Culture vessels and any other container shall be carefully and clearly labelled. Specific cleaning/decontamination procedures for incubators shall be laid down. VII.16. In cases where a virus inactivation or a removal process is performed during manufacture, measures shall be taken to avoid the risk of recontamination of treated products by non-treated products. Vessels containing inactivated products shall not be opened or sampled in areas containing live biological agents. For sterile products and for aseptic manufacturing, handling shall be done in accordance with Annex I. VII.17. The inactivation process for live organisms shall be validated. For products that are inactivated by the addition of a reagent (e.g. micro-organisms in the course of vaccine manufacture) the process shall ensure the complete inactivation of the live organism and prevent any subsequent contamination from any equipment surface. VII.18. Where chromatography equipment is used in campaign manufacture and in multi-product environments, a suitable control strategy (based on risk management principles) for matrices, the housings and associated equipment shall be implemented. The re-use of the same matrix at different stages of processing is discouraged. When it occurs, such re-usage shall be supported by appropriate validation data. Acceptance criteria, operating conditions, regeneration methods, life span, and sanitisation or sterilisation methods of chromatography columns shall be defined. VII.19. Applicable requirements when irradiated equipment and materials are used are laid down in Annex VII. VII.20. Filling shall be carried out as soon as possible following production. Containers of bulk product prior to filling shall be sealed, appropriately labelled and stored under specified conditions of temperature. VII.21. When there is a delay between the filling of final containers and their labelling and packaging, procedures shall be laid down for the storage of unlabelled containers in order to prevent confusion and to ensure satisfactory storage conditions. Special attention shall be paid to the storage of heat labile or photosensitive products. Storage temperatures shall be specified. VII.22. A system to ensure the integrity and closure of containers after filling shall be implemented where the final products or intermediates pose specific risks and procedures shall be established to deal with any leaks or spillages. There shall be also procedures in place regarding filling and packaging operations so as to maintain the product within relevant specified limits, e.g. time and/or temperature. VII.23. The handling of vials (including the capping thereof) containing live biological agents shall be performed so as to prevent the contamination of other products or egress of the live agents into the work environment or the external environment. The viability of such organisms and their biological classification shall be taken into consideration as part of the management of such risks. VII.24. The suitability of primary packaging materials having regard to the characteristics of the product and the storage conditions ( e.g. products that should be stored at ultra-low temperature) shall be ensured. The compatibility of labels with ultra-low storage temperatures, where such temperatures are used, shall be verified. VIII. QUALITY CONTROL VIII.1. As controls for biological products usually involve biological analytical techniques, which typically have a greater variability than physico-chemical determinations, particular attention shall be paid to in-process controls. In-process control testing shall be performed at appropriate stages of production to control those conditions that are important for the quality of the finished product. Particular attention shall be paid to quality controls when continuous culture is used. VIII.2. Continuous monitoring of data during a production process may be required, for example monitoring of physical parameters during fermentation. VIII.3. It may be necessary to retain samples of intermediate products in sufficient amount and under appropriate storage conditions to allow repetition or confirmation of a batch control. VIII.4. Where intermediates can be stored for extended periods of time (days, weeks or longer), consideration shall be given to the inclusion of finished product batches made from materials held for their maximum in-process periods in the on-going stability programme. VIII.5. The ongoing stability monitoring may require animal testing. In such cases, where no alternative testing methods are available and with a view to reduce the use of animals for testing purposes, the frequency of testing may be adapted under a risk-based approach. Bracketing and matrix approaches may also be applied if scientifically justified in the stability protocol. VIII.6. For cellular products, sterility tests shall be conducted on antibiotic-free cultures of cells or cell banks to provide evidence for absence of bacterial and fungal contamination and to be able to detect fastidious organisms where appropriate. VIII.7. For biological medicinal products with a short shelf life (i.e. a period of 14 days or less) requiring batch certification before completion of all end product quality control tests (e.g. sterility tests) a suitable control strategy shall be put in place taking into account the specific characteristics of the product and manufacturing process and taking into account the controls and attributes of starting and raw materials. A detailed description of the release procedure, including the responsibilities of the different personnel involved in assessment of production and analytical data is required. A continuous assessment of the effectiveness of the quality assurance system shall be in place including records kept in a manner that permits trend evaluation. Where end product tests are not available due to their short shelf life, alternative methods of obtaining equivalent data to permit initial batch certification may be considered (e.g. rapid microbiological methods). The procedure for batch certification and release may be carried out in two or more stages: — assessment (by a designated person) of batch processing records, the results from environmental monitoring (where available), all deviations from standard procedures and the available analytical results; — assessment of the final analytical tests and other information available for final certification by the Qualified Person. A procedure shall be in place to describe the measures to be taken where out of specification test results are obtained. Such events shall be fully investigated and the relevant corrective and preventive actions taken to prevent recurrence documented. IX. SPECIFIC REQURIMENTS FOR SELECTED PRODUCT TYPES IX.1. Allergen products The following additional requirements are applicable for allergen products: (a) Source materials shall be described in sufficient detail to ensure consistency in their supply, e.g. common and scientific name, origin, nature, contaminant limits, method of collection. Materials derived from animals shall be from healthy sources. Appropriate biosecurity controls shall be in place for colonies (e.g. mites, animals) used for the extraction of allergens. Allergen products shall be stored under defined conditions to minimise deterioration. (b) The production process steps including pre-treatment, extraction, filtration, dialysis, concentration or freeze-drying steps shall be described in detail and validated. (c) The modification processes to manufacture modified allergen extracts (e.g. allergoids ( 16 ) , conjugates) shall be described. Intermediates in the manufacturing process shall also be identified and controlled. (d) Allergen extract mixtures shall be prepared from individual extracts from single source materials. Each individual extract shall be considered as one active substance. For recombinant allergens, the additional requirements in Section IV.4 also apply. IX.2. Animal immunosera products The following additional requirements are applicable for animal immunosera products: (a) Particular care shall be paid to the control of antigens of biological origin to assure their quality, consistency and absence of contamination from adventitious agents. The preparation of materials used to immunise the source animals (e.g. antigens, hapten ( 17 ) carriers, adjuvants, stabilising agents) as well as the storage conditions for such material immediately prior to immunisation shall be in accordance with documented procedures. (b) The immunisation, test bleed and harvest bleed schedules shall be in accordance with the terms of the marketing authorisation. (c) The manufacturing conditions for the preparation of antibody sub-fragments (e.g. Fab or F(ab’)2) and any further modifications shall be in accordance with validated parameters. Where such enzymes are made up of several components, their consistency shall be assured. IX.3. Vaccines The following additional requirements are applicable for vaccines: (a) Where eggs are used, the health status of all source flocks used in the production of eggs (whether specified pathogen free or healthy flocks) shall be assured. (b) The integrity of containers used to store intermediate products and the hold times shall be validated. (c) The sequence of addition of active ingredients, adjuvants and excipients during the formulation of an intermediate or final product shall be in compliance with specifications. (d) Where the manufacture or testing involves the handling of organisms with a higher biological safety level (e.g. panzootic vaccine strains), appropriate containment arrangements shall be in place in accordance with relevant national requirements. Relevant approvals shall be available for verification. IX.4. Recombinant products The following additional requirements are applicable for recombinant products: (a) Process conditions during cell growth, protein expression and purification shall be maintained within validated parameters to ensure consistent production with a defined range of impurities. Depending on the type of cell used in production, additional measures may be required to ensure viral safety. When the manufacturing process involves multiple harvests, the period of continuous cultivation shall be within specified limits. (b) The purification processes to remove unwanted host cell proteins, nucleic acids, carbohydrates, viruses and other impurities shall be within defined validated limits. IX.5. Monoclonal antibody products The following additional requirements are applicable for monoclonal antibody products: (a) Monoclonal antibodies may be manufactured from hybridomas or by recombinant DNA technology. Control measures appropriate to the different source cells (including feeder cells if used) and materials used to establish the hybridoma/cell line shall be in place to assure the safety and quality of the product. It shall be verified that these are within approved limits. Viral safety is particularly important. Data originating from products generated by the same manufacturing technology platform may be acceptable to demonstrate suitability. (b) Production and product parameters at the end of a production cycle (e.g. temperature, Ph, density, oxygen, cell viability, etc.) and for early termination of production cycles shall be defined and monitored. (c) The manufacturing conditions for the preparation of antibody sub-fragments (e.g. Fab, F(ab’)2, scFv) and any further modifications (e.g. radio labelling, conjugation, chemical linking) shall be in accordance with validated parameters. IX.6. Veterinary medicinal products derived from genetically modified/genome edited animals The following additional requirements are applicable for veterinary medicinal products derived from genetically modified/genome edited animals: (a) Animals used for production shall be clearly and uniquely identified and backup arrangements shall be put in place in the event of loss of the primary marker. (b) The genealogy of the founder animals through to production animals shall be documented. Since the genetically modified/genome edited line will be derived from a single genetic founder animal, materials from different genetically modified/genome edited lines shall not be mixed. (c) The conditions under which the product is harvested shall be in accordance with the terms of the marketing authorisation. The harvest schedule and conditions under which animals may be removed from production shall be performed according to approved procedures and acceptance limits. (d) Particular attention shall be made to demonstration of batch-to-batch consistency. IX.7. Veterinary medicinal products derived from genetically modified/genome edited plants The following additional requirements are applicable for veterinary medicinal products derived from genetically modified/genome edited plants. (a) Additional specific measures may be required to prevent contamination of the master and working genetically modified/genome edited banks by extraneous plant materials and relevant adventitious agents. The stability of the gene within the defined generation numbers shall be monitored. (b) Plants shall be clearly and uniquely identified, the presence of key plant features, including health status, across the crop shall be verified at defined intervals through the cultivation period to assure consistency of yield between crops. (c) Security arrangements for the protection of crops shall be defined, wherever possible, to minimise the exposure to contamination by microbiological agents and cross-contamination with non-related plants. Measures shall be put in place to prevent materials such as pesticides and fertilisers from contaminating the product. A monitoring programme shall be established and all results documented. Any incident shall be investigated and its impact on the continuation of the crop in the production programme shall be determined. (d) Conditions under which plants may be removed from production shall be defined. Acceptance limits shall be set for materials (e.g. host proteins) that may interfere with the purification process. It shall be verified that the results are within the approved limits. (e) Environmental conditions (temperature, rain) that may affect the quality attributes and yield of the recombinant protein from the time of planting, through cultivation to harvest and interim storage of harvested materials shall be documented. The principles in documents such as ‘Guideline on Good Agricultural and Collection Practice for Starting Materials of Herbal Origin’ ( 18 ) shall be taken into account when drawing up such conditions. (f) Particular attention shall be made to the demonstration of batch-to-batch consistency. ( 1 ) GMP principles shall be adhered to. ( 2 ) See Section IV for the extent to which GMP principles apply. ( 3 ) See Section VI for the extent to which GMP applies. ( 4 ) See Section VI for the extent to which GMP applies. Maintenance of working cell bank is expected to take place in a GMP environment. ( 5 ) The standards of good agricultural and collection practice for starting materials of herbal origin (GACP) is applicable. ( 6 ) Bacillus Calmette–Guérin (BCG) vaccine. ( 7 ) Personnel entering a contained area where organisms have not been handled in open circuit operations in the previous twelve hours are not regarded as being at risk of contamination, unless the organism involved is a biological agent where the corresponding disease does not exist in the relevant country or geographical area, or where the corresponding disease is subject to prophylactic measures or an eradication programme undertaken in the relevant country or geographical area. ( 8 ) For the purpose of this Annex, ‘exotic organism’ means a biological agent where the corresponding disease does not exist in a relevant country or geographical area, or where the disease is the subject of prophylactic measures or an eradication programme undertaken in the relevant country or geographical area. ( 9 ) For the purposes of this Annex, ‘clean in place’ means a method, usually automated, used to clean the internal surfaces of equipment, piping, vessels, and associated fittings without disassembling them. ( 10 ) For the purposes of this Annex, ‘steam in place’ means a method, usually automated, of sterilising the internal surfaces of equipment, piping, vessels, and associated fittings by using steam, without disassembling the equipment. ( 11 ) https://www.woah.org/en/home . ( 12 ) For the purposes of this Annex, ‘specified pathogen free’ means animals free from specified pathogens. Such flocks or herds share a common environment and have their own caretakers who have no contact with non-specified pathogen free groups. ( 13 ) For the purposes of this Annex, ‘master seed lot’ means a culture of a micro-organism distributed from a single bulk into containers and processed together in a single operation in such a manner as to ensure uniformity and stability and to prevent contamination. For the purposes of this Annex, ‘working seed lot’ means a culture of a micro-organism derived from the master seed lot and intended for use in production. ( 14 ) For the purposes of this Annex, ‘master cell bank’ means a culture of cells distributed into containers in a single operation, processed together and stored in such a manner as to ensure uniformity and stability and to prevent contamination. For the purposes of this Annex, ‘working cell bank’ means a culture of cells derived from the master cell bank and intended for use in the preparation of production cell cultures. Genetically modified/genome edited working bank is used with the same meaning but for plants or animals that have been genetically modified or whose genome has been edited. ( 15 ) For the purposes of this Annex, ‘clean/contained area’ means an area constructed and operated in such a manner that it achieves the aims of both a clean area and a contained area at the same time. ( 16 ) Allergens that are chemically modified to reduce IgE reactivity. ( 17 ) A low molecular weight molecule that is not in itself antigenic unless conjugated to a ‘carrier’ molecule. ( 18 ) EMEA/HMPC/246816/2005.