The vaccine production: From the egg to the cell

Flu vaccines are produced using the same fundamental technique as the first industrially manufactured vaccines: in live chicken eggs. However, there are changes afoot in the industry: The pharma concern Novartis has produced a flu vaccine using a cell culture and has now applied for European approval for its new product. And Novartis is not alone; pharma players such as Solvay, Baxter, GlaxoSmithKline and Sanofi Pasteur are also presently hard at work developing vaccines using cell cultures.

Established method: Production in chickens’ eggs

To manufacture a vaccine, a self-contained system in which pathogens can be safely produced is required. In 1931, it was discovered that fertilized chicken’s eggs could be used as virus cultivation machines, and they have been employed ever since in the mass production of vaccines. However, many viruses, for instance avian flu viruses, are so aggressive that they cannot be easily grown in eggs. For this reason, pathogen strains must be altered beforehand, which, in earlier times, was an extremely complicated process; researchers would mix together pathogens that multiplied well with their more aggressive cousins, until a suitable version for production emerged. Today, this lengthy process can be achieved more deliberately and effectively thanks to sophisticated gene-technological procedures. Much like a stack of cartons, genetic material can be combined in the laboratory to produce the exactly desired results. In this process, the desired genes are mixed with genes from potent vaccination strains. At the same time, gene sequences that are responsible stunting the eggs’ growth can be removed. The basis of this ‘reverse genetics’ methodology was developed in Germany by Gabriele Neumann and Erik Hoffmann in the Giessen University laboratory of the Virologist Gerd Hobom. Today, Hoffmann works alongside Robert Webster at the Children's Research hospital St. Judes in Memphis, USA. The 74-year-old Webster is one of the most famous virologists in the field of flu research and advises many governments, as well as the WHO, on influenza-related matters.

Lengthy vaccine production in eggs

After the virus has been designed, the fertilized eggs must be prepared and incubated for 11 days. At this point, the flu viruses are injected into the egg. So that the viruses are able to optimally multiply, the eggs are kept for a two further days at a temperature of 36°C. Then the viruses are collected and cleaned over several complex stages, as well as being inactivated chemically and with the use of low temperatures, since most vaccines are non-live. These steps from the chicken egg to the finished vaccine are carried out separately for each of the three strains selected by the WHO. Finally, the three vaccines are merged into a single vaccine and can be placed in individual syringes. The entire process takes at least six months, as well as the months of preparation required before manufacturing begins. The final volume must be well planned beforehand, as the process of vaccine production requires a huge quantity of specially certified, fertilized chicken eggs up to one year in advance.In the Northern Hemisphere, production for the winter usually begins in April at the earliest, whereas preparation for the Southern Hemisphere begins in the Autumn. As a single vaccine dose requires one egg, the 90 million doses manufactured in Europe each year, and approximately 300 million produced worldwide require just as many chicken eggs be delivered. In Germany, over 23 million vaccine doses have already been produced for the vaccination season 2006-2007. In order to guarantee the supply, a number of plants are based locally. For example, the British company GlaxoSmithKline has a plant in Dresden, which manufactures flu vaccines in chicken eggs.

Chicken eggs – their services to vaccine production will soon end.Source: Pixelquelle.de
Chicken eggs – their services to vaccine production will soon end.

Source: Pixelquelle.de

What would happen during a pandemic?

So far, the process of vaccine production has met demands. But what would happen if an extremely speedy production of vaccines is required? Exactly this would be of vital importance during a pandemic - a border-hopping and extremely fast-spreading virus - and virologists think the danger of such a scenario is increasing daily. Some devastating pandemics have occurred surprisingly recently. The 20th Century alone saw three major occurrences: The Spanish Flu (1918/19), the Asiatic Flu (1957/1958) and the Hong Kong Flu (1968-70). The most destructive of these was the Spanish Flu, which was triggered by Influenza type A (H1N1) and took more than 30 million lives.

Today, the avian flu virus subtype H5N1 is regarded as posing the greatest threat to public health. This grim status was secured in 1997 in Hong Kong, where it claimed the lives of six of the eighteen infected individuals. Before this incident, experts had not thought it possible that avian flu viruses could be transmitted to humans and, consequently, pharma companies did not see the production of vaccines as an area worthy of major investment.

This attitude has now changed significantly, and H5N1 is currently seen as the number one pandemic-triggering candidate, and the danger of a pandemic is apparently growing. Initially, H5N1 could only be found in chicken farms, but it eventually found its way into wild animals. To date, over one hundred humans worldwide have now died following an infection with the avian flu virus, and in February 2006 the virus was identified for the first time in wild animals in Germany, in this case in swans in the North of the country. However, there have been no recorded transmissions from human to human and most victims have lived and worked in close proximity to the infected animals.

Financial support for infection research
Nevertheless, politicians have recognised the danger of a pandemic and are investing more intensively than ever before in infection research. In March 2006, the German government, amongst other measures, decided on a research agreement backed by 60 million euros of public funding, which is intended to ease cooperation between the fields of animal and human medicine in Germany. At the focus of attention will be diseases that can be transmitted from animals to humans (zoonoses). Taking part in this initiative is the German Federal Ministry for Nutrition, Agriculture and Consumer Protection (BMELV), the German Federal Ministry for Education and Research (BMBF) and the German Federal Ministry for Health (BMG). Beyond this, ‘Influenza’, a special emergency research program, will be attempting to close the knowledge gaps relating to the H5N1-virus and to the danger of a pandemic, and will lay the groundwork for future responses to the danger, and further necessary studies.

New approach: Vaccines from cells

To be able to produce the suitable vaccines quickly in vast quantities which will be demanded during a pandemic, the use of cell cultures in the manufacturing process has been taken more seriously in recent years. A cell culture is the growth and multiplication of cells in a growth medium outside of an organism. Descendants of a specific cell that are able to grow for an unlimited period in the culture medium are referred to as a cell line.

Growing viruses in cell cultures is actually nothing new, as viruses normally multiply in the cells of their host organisms. In 1949, American researchers were able to repeat this process for the first time in the Petri dish, and succeeded in nurturing viruses in a laboratory cell culture. In 1954, John Enders, Thomas Weller and Frederick Robbins received the Nobel Prize for this achievement. However, if the entire flu vaccine production is to be moved over to cell cultures, it will not be sufficient to find a cell culture in which viruses grow well - it will also have to be guaranteed that industrial production is possible with that particular cell line. For example, it will have to be proven that the cells do not only grow when in contact with a surface (e.g. with the container base), but will also be able to multiply when in suspension. Only then they can be cultured in enormous fermenters. Then, if the production with a suitable cell line has developed scientifically and technically, the pharma companies must test the finished vaccine in clinical studies and prove that it is at least just as reliable, effective and safe as flu vaccines manufactured in eggs.

Enormous timesavings: 16 weeks for a finished vaccine

The pharma concern Novartis has now succeeded in achieving all of this with it’s cell-culture vaccine for a ‘normal’ seasonal flu vaccine and has already applied to the European Medicines Agency (EMEA ) for market approval in Europe. For production, Novartis uses cells from the kidneys of dogs - so-called MDCK cells (Madin-Darby Canine Kidney Cells). In contrast with the use of eggs, the production of a cell-based vaccine takes only 16 weeks. What is more, the capacity can be adapted more easily to the actual demand. Since cell cultures are also suitable for aggressive flu viruses, a great deal of time can be saved: The time-consuming genetic changes that need to be made to pathogens to make them less aggressive are rendered redundant by the use of cell cultures. This new method is also safe and cleaner: There is less risk of contamination by foreign bacteria or viruses when shielded in a cell culture than in chicken eggs. And last but not least, sufferers of a certain chicken protein allergy will greatly benefit from this new approach, as they were previously unable to receive the vaccine at all.


Production plant for vaccines on the basis of cell cultures.

Quelle: Novartis Behring

Cell cultures an advantage in a pandemic

The cell culture also offers substantial advantages for the development of a vaccine in the case of a pandemic, as, in principle, a much faster production is possible. Ultimately, 60 million doses alone would be required to be able to vaccinate everybody in Germany twice with one dose each within six weeks - eight times more than to the normal flu season for Germany. Such short-term capacity increases are more feasible with cell-culture-based vaccines than with chicken eggs. In order to receive quick permission in the case of emergency, experts from the regulatory body Paul Ehrlich Institute, as well as the pharma giants Novartis and GlaxoSmithKline, are working on vaccine prototypes that will serve as a basic structure for later vaccines. In such a way, the manufacturers can carry out the necessary clinical studies ahead of time and submit the so-called core dossiers to the EMEA to quicken the later approval process. These contain data on the quality, security and effectiveness of the prototypes. Then, if a pandemic occurs and a vaccine with the actual pathogen is produced, the manufacturer is only required to deliver a small amount of data. The vaccine manufacturers would thus not have to begin from scratch and would be able to save valuable time. The German Federal Government of Germany is promoting the development of such prototypes with 20 million euros.

Before a pandemic vaccine: Antiviral medicines

As long as the development of pandemic vaccine is unfinished, experts think the best response to a pandemic is the use of antiviral medicines such as Tamiflu (Roche) or Relenza (GlaxoSmithKline). These can bridge the time between the outbreak and the subsequent development of the vaccine and can be used as a prophylaxis, preventing the virus from spreading further.

The principle of such medicines as Tamiflu or Relenza is based on restraining certain protein structures (Neuraminidase) on the surface of cells from the flu viruses. Thus, the host cell cannot produce new viruses. However, the viruses are not permanently eliminated. This means that such medicines do not offer a long-term protection from a pandemic virus and the first reports that avian flu strains have developed resistance to Tamiflu have recently emerged. Nevertheless, many countries have already decided to stock up on Tamiflu camp in the case of an emergency. In 2005, Germany ordered 6 million doses of Tamiflu. The WHO has advised all countries to stockpile enough of the drug to be able to protect 25% of the population.