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Influenza vaccine production technologies 20 December 2006 production. It is technically possible and necessary to secure the flocks against avian influenza, to ensure that egg-based production is possible, even in the worst-case scenario that the pandemic virus infects both mammals and birds. • Waste disposal. Up to 80% of the mass of eggs used (potentially 4,000 kg per day) will be solid infectious waste, which will require inactivation and disposal. • Selection of variants Growth of human influenza virus in eggs can lead to the selection of variants that differ antigenically from the original seed virus (Katz and Webster, 1989). It is therefore necessary to adapt the strains to egggrowth. • Yield in eggs. Many years of experience producing reassortant viruses as vaccine strains have made this technology robust and reproducible. However, individual virus isolates may exhibit different growth characteristics in eggs, thereby affecting the yield of the manufacturing process. This might particularly be the case with an avian-derived pandemic strain. Experience to date with H5N1 viruses generated by reverse genetics has shown that these reassortant viruses produce only 30-40% of the yield of HA of seasonal vaccine strains, despite growing to normal titres in eggs. 5.4.4 Outstanding questions and issues Due to the decades of experience with this process, many issues have been resolved. However, the process for producing influenza vaccine in eggs is high specialized and to some extent, is specific to each vaccine strain. The procedure can still be complicated by the need to introduce changes in the process steps that arise when novel strains are incorporated into the vaccine. When considering establishing a new facility, three of the key outstanding issues are: • Egg availability. How many eggs would be required to support the process? What grade of eggs is needed? How secure, dependable and rapid would the supply be in the face of pandemic influenza? • Economic viability. Can the process be made economically viable for seasonal vaccine production for a single region? Will this require manufacturing for both Northern and Southern hemispheres, and can suitable markets be identified? • Specific experience. Due to the highly-specialized nature of manufacture of IIV at large scale, it would be necessary for a new manufacturer to seek advice on the process in order to avoid the time and cost of acquiring the expertise. This expertise currently resides within the large vaccine multinational manufacturers. 6 Inactivated influenza vaccines: cell-culture-based manufacture 6.1 Introduction Growing influenza virus in tissue culture is being developed in order to overcome some of the limitations of egg-based production. The potential advantages of this approach compared with egg-based manufacture include: • No requirement for a supply of large numbers of eggs • A more-rapidly scaleable process (using technologies that are used in other vaccine production plants) • Improved aseptic handling during manufacture • Avoidance of the risk of embryonated eggs containing avian retroviruses (Ghendon et al., 2005). • Elimination of egg components in the vaccine and the resultant risk of potential allergic reactions. Page 16 of 34
Influenza vaccine production technologies 20 December 2006 6.1.1 Cell lines Three cell lines are currently in late-stage development as substrates for the growth of influenza virus. Some manufacturers have filed for the approval of their influenza vaccines produced in cell lines and authorization is expected in 2007. Vero cells Vero cells are a well-characterized and widely used African green monkey kidney-cell line. They have been used for the production of polio vaccine for more than 20 years. They are an adherent cell line and some strains can be cultured in serum-free medium in stationary culture or on microcarriers. MDCK Madin Darby canine kidney cells were originally isolated in 1958 and have been widely used for the production of veterinary vaccines. They can produce high quantities of influenza virus (Tree et al., 2001). Like Vero cells, they are adherent and can be cultured in serum-free medium. PER.C6 PER.C6 is a relatively new, immortalised human retinal-cell line. It can be cultured in serum-free medium, but only grows in suspension cultures. Others Several other cell lines that may be particularly suitable for production of influenza virus are under investigation, including a chicken-derived cell line from Vivalis (Nantes, France). These cell lines have not yet been fully characterized and several years of research and characterization will be required before these can be approved for the production of human vaccines. An potential alternative is the use of chicken embryo fibroblasts (see section 9) which are used in the production of measles vaccine. How well the yield of growth of influenza virus on CEF compares to immortalised cell lines needs to be established. 6.2 Laboratory-scale 6.2.1 Manufacturing process A laboratory-scale cell-culture-based process should be capable of producing a few thousand doses using relatively straightforward equipment such as roller bottles or cell factories and standard incubators. Roller-bottle cultures of MDCK cells can produce up to 1x10 9 pfu/ml of influenza virus (Tree et al., 2001). The cells need to be expanded to the desired quantity from working cell banks. They are then infected with influenza virus and incubated for three to five days. Parameters such as multiplicity of infection (moi), incubation time and temperature need to be optimized for each cell line and each strain of virus. After the incubation period, the virus is harvested by removing the tissue-culture supernatant. The harvested supernatant is concentrated using diafiltration or centrifugation. Further downstream processing is then required to remove contaminating host-cell DNA. Typically, this involves ion-exchange chromatography to reduce the level of DNA, combined with the addition of benzonase (or a similar reagent) to reduce the size of the DNA fragments that are still present. Procedures for splitting and inactivating the virus are similar to those as described for egg-based manufacture (section 5.2.1). Inactivated whole-virion vaccines are currently not produced by cell culture. Page 17 of 34
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