The choice of technology was based on its simple and robust production process,

and therefore its feasibility for transfer to developing countries to produce pandemic influenza vaccine. In addition, whole virus vaccines evoke the broadest immune responses, are largely exempt from intellectual property hurdles and can be produced without using licensed adjuvants [7]. This said, the ability to produce rapidly a pandemic vaccine invariably depends on the existence of annual seasonal influenza vaccine production; since split-virion vaccine is by far selleck compound the most widely used technology in seasonal influenza programmes, NVI has added a process for split vaccine to its curriculum. The process established at pilot scale (10,000 eggs) follows the international quality and safety regulations of WHO [8] and the European Pharmacopoeia [9] (Fig. 1). To determine robustness, we used one monovalent seasonal strain to set up and test a classical egg-based process in our facilities. The main steps outlined in Fig. 1 can be summarized as follows. The primary seed virus obtained from the National Institute for Biological Standards and Control (NYMC X-175C reassortant derived from A/Uruguay/716/2007) was processed to working seed on specific pathogen-free eggs before propagating the bulk

virus at pilot scale for 48–72 h in fertilized hen eggs at 35 °C. The virus-containing fluid was harvested semi-automatically and clarified by centrifugation and depth filtration. The virus was purified Alisertib and concentrated by sucrose gradient zonal ultracentrifugation until and then inactivated by ß-propriolactone, filtrated using depth filters and further purified by subsequent ultrafiltration/diafiltration. Finally, the product was formulated and filtrated at 0.22 μm to obtain monovalent vaccine. After producing 12 monovalent batches, the final production settings were defined and consistency runs performed. The average recovery

from zonal ultracentrifugation to monovalent vaccine was 53% and the average yield 1.1 dose/egg. The sucrose density gradient purification method – the international standard for influenza virus purification – resulted in the purification profile shown in Fig. 2. The performance per process step and the impurity profile for the consistency runs are shown in Table 2 and Table 3, respectively. The ovalbumin, total protein and endotoxin content meet the specifications set by WHO and the European Pharmacopoeia. Comparison with other industrial processes is difficult, as most international manufacturers do not publish their process results. We found one publication on density gradient yields [10] and another comparing six European influenza vaccines for impurities [11].