Bacteria are grown in bioreactors (e.g., Haemophilus influenzae type b). Likewise, a recombinant protein derived from the viruses or bacteria can be generated in yeast, bacteria, or cell cultures. After the antigen is generated, it is isolated from the cells used to generate it. A virus may need to be inactivated, possibly with no further purification required. Recombinant proteins need many operations involving ultrafiltration and column chromatography. Finally, the vaccine is formulated by adding adjuvant, stabilizers, and preservatives as needed. The adjuvant enhances the immune response to the antigen, stabilizers increase the storage life, and preservatives allow the use of multidose vials. Combination vaccines are harder to develop and produce, because of potential incompatibilities and interactions among the antigens and other ingredients involved. The final stage in vaccine manufacture before distribution is fill and finish, which is the process of filling vials with vaccines and packaging them for distribution. Although this is a conceptually simple part of the vaccine manufacture process, it is often a bottleneck in the process of distributing and administering vaccines. Vaccine production techniques are evolving. Cultured mammalian cells are expected to become increasingly important, compared to conventional options such as chicken eggs, due to greater productivity and low incidence of problems with contamination. Recombination technology that produces genetically detoxified vaccines is expected to grow in popularity for the production of bacterial vaccines that use toxoids. Combination vaccines are expected to reduce the quantities of antigens they contain, and thereby decrease undesirable interactions, by using pathogen-associated molecular patterns. Vaccine manufacturers The companies with the highest market share in vaccine production are Merck, Sanofi, GlaxoSmithKline, Pfizer and Novartis, with 70% of vaccine sales concentrated in the EU or US (2013). Vaccine manufacturing plants require large capital investments ($50 million up to $300 million) and may take between 4 and 6 years to construct, with the full process of vaccine development taking between 10 and 15 years. Manufacturing in developing countries is playing an increasing role in supplying these countries, specifically with regards to older vaccines and in Brazil, India and China. The manufacturers in India are the most advanced in the developing world and include the Serum Institute of India, one of the largest producers of vaccines by number of doses and an innovator in processes, recently improving efficiency of producing the measles vaccine by 10 to 20-fold, due to switching to a MRC-5 cell culture instead of