A comparison of Embryonated Egg and Cell Culture Vaccine Production Platforms in Response to Influenza Pandemics. How Best to use Current Technology. Ruzaiq Omar 5th May 2013
Abstract Influenza is a seasonally occurring disease and is a major course of morbidity and mortality worldwide, every few decades a novel strain causes a global pandemic. The human population is generally immunologically naive to pandemic strains allowing for higher levels of hospitalization and fatalities of those who are infected. In response to seasonal and pandemic influenza, vaccination has proven to be the most effective method of controlling the virus. Presently the embryonated egg production platform constitutes the main method for producing influenza vaccines despite the fact that its annual turnover is insufficient to meet global demands, a factor which contributes greatly to the â€˜pandemic influenza vaccine supply gapâ€™ between industrialized and developing countries. The embryonated egg platform also requires a long production lead-time from first isolation of a pandemic strain to production of the vaccine; this may prove problematic when rapid vaccine production is required during a pandemic outbreak. The more recent cell culture production platform promises production of vaccine at a higher capacity with a shorter production lead-time, however it may not be as cost effective or as easy to implement as the embryonated egg platform. This review is a discussion pertaining to the production of influenza vaccines in response to a pandemic. The discussion involves a comparison of using embryonated eggs or mammalian cell culture systems for production of either a live attenuated influenza vaccine (LAIV) or whole kill trivalent inactivated vaccine (TIV). A comparative overview of the two systems is needed in order to determine the best possible way to move forward using current influenza vaccine production technology, so that if a pandemic were to occur today, manufacturers would be able to produce affordable vaccines in a timely manner and at a capacity that can satisfy a global need.
Introduction Seasonal influenza causes about 250 000 to 500 000 deaths worldwide each year . Most seasonal influenza related deaths occur among persons above 65 years of age and persons
belonging to high risk groups (ie. children younger than 2 years, the elderly, pregnant women and health care workers) .
Pandemic influenza viruses are formed every few decades by genetic reassortment of a human strain with an avian or a swine strain or both avian and swine strains . In this way genetic reassortment can produced a human permissive strain which is completely novel to the human immune system. Pandemic influenza tends to cause death in all age groups and is associated with higher levels of hospitalization and fatalities of those who are infected . It is estimated that the next major pandemic outbreak may cause the deaths of up to 62 million people . In order to predict and prepare for such pandemics, the World Health Organization (WHO) conducts continual surveillance of possible future pandemic strains. Surveillance data has shown reoccurring outbreaks and zoonosis of a highly virulent avian influenza virus, H5N1, which poses a continuing pandemic threat [5,6]. These threats have brought to the forefront some important issues regarding current pandemic vaccine manufacturing such as; low production capacity, insufficient supply to developing countries and lengthy production leadtime . If a pandemic outbreak were to occur today, global spread could easily be achieved in a matter of months and an appropriate vaccination response would be required . Using current influenza vaccine manufacturing technology it would take several months to produce the first pandemic vaccine and production capacity would be inadequate to meet global demands . An initiative adopted by WHO, and various governmental organizations such as the Center for Disease Control, is that of producing and stockpiling vaccines in preparation for a potential pandemic . Such an initiative may easily become unfeasible if the pandemic strain is antigenically different to the vaccine strain  as this would greatly reduce the efficacy of the vaccine . Stockpiling of vaccines would also not provide protection against novel pandemic strains which may arise from zoonosis of an avian influenza A virus. Another approach to improving response to pandemic vaccine supply is through the development of new production platforms such as plant cells, insect cells, bacteria, yeast and recombinant DNA technology . Due to approval requirements, new production platforms may take decades before they are approved and implemented, even
though they may prove useful in the future, a more immediate plan of action is needed to tackle todayâ€™s issues regarding pandemic influenza vaccine production. A practical response would be to focus on more effective use of currently approved influenza vaccine production platforms, which in turn, may facilitate a better response to future pandemics. The two platforms discussed here, embryonated egg and cell culture, are both approved for influenza vaccine production and both produce commercial influenza vaccines licenced for human use . Each platform has its own set of advantages and disadvantages and both differ greatly in terms of production lead-time, production capacity, initial investment and technology required . Both platforms are capable of producing both whole kill trivalent influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV), the advantages and disadvantages of producing either type of vaccine also differs between cell culture and egg based platforms. This paper will provide a comparative overview of the two platforms in the context of pandemic influenza vaccine production. The aim of the paper is to determine the best way to move forward using currently approved influenza vaccine production technology to produce affordable pandemic vaccines in a timely manner and at a capacity that can satisfy a global need.
The Embryonated Egg Vaccine Production Platform Manufacturing of influenza vaccines using the embryonated egg platform involves the use of a high growth master strain which is genetically recombined with the relevant pandemic field strain, identified by the WHO, to produce a seed strain for growth in eggs . The seed strain virus is inoculated into the allantoic cavity of a 10 day old embryonated egg. The eggs are incubated to allow the virus to replicate, the allantoic fluid containing the virus is then harvested .
Advantages of the Embryonated Egg Platform The embryonated egg production platform has been used as the main method of influenza vaccine production since the 1950s when the first large scale egg-grown whole-kill influenza vaccine was made . During this period the platform has earned its place as a licenced, trusted and well-established method that has been optimized over 60 years. The process has become highly automated, needs little monitoring and makes use of an assembly line style of production, in these ways the production efficiency has been greatly improved. Further advantages of the egg platform involve the use of master influenza strains (A/PR8/34 or PR8) which are available for genetic reassortment with field influenza A strains . Reassortment of a field strain with a master strain will produce a recombinant with a higher replicative ability in eggs . The platform is especially attractive to start-up manufacturers, who generally prefer low risk investments involving little research and development . The platform requires a 5 fold lower initial investment than cell culture platforms and needs half the time to become fully operational . Its employment of standardized techniques and an already optimized process significantly reduces the amount of research and expertise needed by the production plant . On the administrative front; initial investment is further reduced by the absence of intellectual property rights for production of TIV in eggs as all basic methods are described in expired patents, although such legal rights do restrict propagation of LAIV . For both TIV and LAIV; an established regulatory pathway to licensure may be used which speeds up what is usually quiet a lengthy approval process . The establishment and standardization of the embryonated egg platform makes it easier and cheaper to adopt. Thus in terms of a business model, the platform promises the lowest risk return on investment.
Disadvantages of the Embryonated Egg Platform Even though there are many clear advantages of using embryonated eggs as a vaccine production platform, the technology falls short when attempting to respond to a sudden increase in vaccine demand as is observed during an influenza pandemic .
For one, production of influenza vaccines in eggs requires 6 months of advanced planning to ensure breeding of enough hens to produce the embryonated eggs . In this way dependence on egg availability becomes a limiting factor in production as eggs are not readily scalable and egg supply will not always meet vaccine production demand . Dependence on eggs can also be hampering when producing a vaccine to protect against a highly virulent form of avian influenza, such as H5N1, which can compromise the embryonated egg platform in two ways. Firstly it may reduce embryonated egg supply by killing hen livestock and secondly it may reduce vaccine turnover by killing the embryonated eggs the virus is propagated in . The latter raises the need for genetic modification of highly pathogenic avian influenza strains before it can be propagated in eggs, this will add to the production lead-time and may raise issues concerning intellectual property . As mentioned above, field strains of influenza A viruses are recombined with master strains to yield high egg growth recombinants, this recombination can cause a loss of specific antigenicity of the virus . A compromised specific antigenicity of the virus is known to reduce the effectiveness of the vaccine . The surface antigens of the recombinant strain therefore has to undergo sequence analysis by WHO in order to determine their similarity to the field isolate  adding to the production lead-time. Additionally, not all influenza stains, H5N1 for example, have a suitable master strain . The passaging of influenza virus through eggs has also been shown to alter the hemagglutinin (HA) viral surface antigen and thus its specific antigenicity . Due to the factors which add to embryonated egg production lead-time; it would take at least four months to produce the first pandemic vaccine, TIV and LAIV, after isolation of the wild type strain . Other disadvantages of the egg platform include potential biohazards for the production staff and the vaccine recipient. The virus harvest phase of TIV and LAIV egg production involves manually removing the virus containing allantoic fluid from the embryonated egg and poses a potential biohazard for production staff [15,16]. For the vaccinee of a TIV, the organic material contained within eggs, such as proteins, inherit viruses and other egg-derived components, can cause fever and potentially pathogenicity . TIV Egg-based vaccines cannot be used by people with egg allergies .
The use of eggs allows for a current vaccine production capacity of only 425 million doses per year . This number is largely insufficient to protect all high risk groups of the global population. The low production capacity in eggs also keeps the cost per dose high (US$ 3.007.00) . These factors contribute greatly to the ‘pandemic influenza vaccine supply gap’ between developed and developing countries, where regions such as Western Europe and North America receive 170 and 265 doses per 1000 people per year respectively and a developing region such as Africa receives 3.6 doses per 1000 people per year . An aim of WHO is to provide global protection from influenza in response to a pandemic. If these aims are to be achieved with the use of eggs, several billion embryonated eggs will have to be produced . From the numerous disadvantages mentioned, the platform appears to unable to respond well to influenza pandemics. It should however be noted that almost all influenza vaccines produced today are TIVs, therefore the issues of production capacity may be resolved, to a certain extent, by switching from production of TIVs in eggs to production of LAIVs in eggs.
Production of TIV vs LAIV Using Embryonated Eggs Both LAIV and TIV are clinically effective, licenced for production in eggs and constitute many of the influenza vaccines on the market today [28,13]. LAIVs are comprised of an attenuated cold adapted virus that it incapable of replication at human body temperature of 37⁰C . LAIVs are delivered intranasaly, simulating natural infection. TIVs are whole kill inactivated vaccines made by splitting or solubilizing the virus  and are delivered intravenously.
Production of LAIV confers certain advantages over TIV. It does not require large-scale equipment and high levels of atomisation which is a costly necessity for producing TIV . LAIV platforms can produce 20 times more vaccine doses per egg than TIV . This means that LAIV production may start earlier as fewer eggs are needed to produce the same quantity of vaccine. TIVs have been shown to be less effective against pandemic strains leading to the need of a possible two course vaccination schedule in future pandemics . A two dose requirement will reduce the number of people that can be fully vaccinated against a pandemic strain.
A drawback of the LAIV platform is that it requires the genetic engineering of a cold adapted recombinant; this recombinant is made via reassortment between a wild type strain and a cold adapted strain. This adds to lead-time of production. Furthermore, the engineering of a cold adapted recombinant, and many other aspects of LAIV manufacturing, are governed by patents . On the other hand, the process of growing TIV in eggs is described in expired patents, thus intellectual property does not have to be purchased . Setting up a LAIV egg production plant requires a US$2-3m initial investment and a 1-2 year period to become operational. This puts it in the lead when compared to TIV egg production which requires a US$20m initial investment and a 3-4 year period to become operational . A switch from TIV to LAIV production in eggs may increase overall production capacity and therefore partially resolve issues such as high cost per dose and the ‘pandemic influenza vaccine supply gap’. A drawback of LAIV production is its ties with intellectual property.
The Cell Culture Vaccine Production Platform The platform involves the propagation of the influenza virus in cell lines such as MBDK, Vero and PER.C6 (derived from duck kidney, monkey kidney and human retina cells respectively) . The cell lines are grown up in bioreactors followed by infection with influenza virus. The virus replicates until a sufficient titre is obtained, the virus is then extracted from cells for manufacturing of the vaccine .
Advantages of the Cell Culture Platform
Various studies have shown that large quantities of immunogenic influenza vaccine can be manufactured rapidly using cell culture systems [34,35,36]. Many clinical studies have found cell culture derived TIVs to be immunogenic, well tolerated and safe [33,37,38]. Cell culture derived TIVs have also exceeded criteria defined by regulatory authorities and some, such as Celvapan, Influvac® and Optaflu®, are already distributed annually and have proven to be as effective as egg derived TIVs .
When compared to embryonated egg platforms, cell culture systems are far simpler as they do not require the use of large scale automated equipment nor do they require copious amounts of labour . Virus propagation is done under sterile conditions in a closed bioreactor, avoiding bacterial and viral contamination, and is also less hazardous for staff and in terms of the vaccinee, cell culture derived vaccines are free of any biological components that may be present in eggderived vaccines, allowing persons with egg allergies to be vaccinated .
Cell culture systems also have a head start over egg based methods as cells are easily stockpiled and scalable upon demand, this permits a shorter lead-time and faster response to pandemic . Furthermore a high growth reassortant virus, a requirement for egg propagation, is not needed. This avoids many pre-propagation procedures, such as screening and selection of reassortant strains followed by assessment of seed strains for specific antigenicity . Additionally, the virus does not have to undergo antigenic specificity tests after propagation as antigenicity of the virus does not change after growth in cell culture [39,40]. Reverse genetics can be used in cell culture to produce vaccines against highly pathogenic avian influenza viruses which cannot always be grown in embryonated eggs [21,26].
Disadvantages of the Cell Culture Platform Some studies have reported that cell culture systems produce low titers of influenza virus, however these results may be strain specific or may be due to lack of optimization [13,41] as many other studies have obtained high titers using cell culture [42,24]. Lack of optimization and standardization are two major constraints in cell culture influenza vaccine manufacturing. Although MBDK cells are providing good results [35,37], Vero and PER.C6 are also under investigation and have proved compatible for growth of the virus . For a cell line to be suitable for production purposes; it needs to allow for high viral infectivity while maintaining high cell numbers. Researchers are not yet sure which cell line under what conditions would produce optimal results for all strains of influenza [41,43]. Due to novelty and lack of standardization of cell culture systems, the main barriers for startup manufactures are administrative. Many aspects of cell culture production such as new
technology, characterized cell lines and the use of reverse genetics are governed by intellectual property rights [21,18]. This adds immensely to the initial investment and time required to start using cell culture systems commercially . A start-up plant would have to produce its own technology and establish its own cell line or purchase an existing patent, when compared to egg based platforms, this can double the time it takes to become operational . A new cell line has to be fully characterized and tested before commercial use . Even after all production technology has been approved, there is no established regulatory pathway to licensure and approval, as there is for egg-based platforms.
Production of TIV vs LAIV Using Cell Culture Systems Only TIVs are currently licenced for production in cell culture systems. Comparisons made to LAIV production will thus be theoretically based on current literature as well as legislative and managerial factors. The regulatory pathway to approval and licensure is more complex for LAIV than for TIV due to the uncertainties associated with an, as yet, unlicensed platform . On top of this, the intellectual property issues surrounding cell culture are more restricting for LAIV production as it requires patented reverse genetics technology to produce cold adapted master strains as well as a new cell line or an already patented one . Nevertheless, once the initial barriers of LAIV production are overcome, a plant using this platform would be able to produce much larger quantities of vaccine than any other platform . It will also require a smaller facility and less infrastructure. LAIV production has the advantage of a fast response time combined with large production capacity. Despite this, start-up manufacturers would be better off opting for TIV production in cells due to its more certified use.
Figure 1. Pandemic influenza vaccine production time table shown for different platforms [32,15,24,20].
The Best way to Move Forward Using Current Technology
If a pandemic were to occur today, the TIV cell culture platform would be the first to deliver pandemic vaccines and it will be capable of supplying a larger population than the other platforms. Theoretically the LAIV cell culture production platform would deliver its vaccines at approximately the same time at TIV cell culture but will be able to deliver vaccine doses to an even larger population. The second best platform for responding to a pandemic is the LAIV embryonated egg platform. It is able to produce more vaccine doses in a limited period of time compared to the TIV embryonated egg platform. The prolonged production lead-time of the embryonated egg platforms place them last in terms of their pandemic response potential.
The TIV embryonated egg platform is the least equipped to respond to a pandemic, yet it is the most widely used platform. This is mainly due to prior establishment of the platform and the absence of intellectual property barriers, which is a major obstacle to starting up LAIV embryonated egg and TIV cell culture plants [14,21]. International bodies such as the WHO are negotiating the transfer of intellectual property to plants in developing countries . Due to such negotiations the Serum Institute of India has launched an H1N1 pandemic vaccine in 2010 and
the Government Pharmaceutical
Organization of Thailand may release a pandemic vaccine into the Thai market at the end of 2011 . International legislation and regulations may also play a role in loosening the restrictions of intellectual property; “The Trade-Related Aspects of Intellectual Property Rights Agreement” authorizes countries to make full use of intellectual property and patents in the event of a public health emergency, such as a pandemic . Reducing the stringency of regulation and approval requirements by governmental administrative agencies, such the FDA, will also facilitate the use of newer vaccine production technology. This is a challenging task as the safety of the approved products has to be maintained along with a strict ‘first, do no harm’ policy.
In terms of meeting egg supply demands production plants and governments can enter long term contracts with egg supply companies to ensure year round supply of eggs. The US government has entered a US$8 million/year contract with Sanofi Pasteur in this regard . These contracts are however expensive and may not be feasible for developing countries or individual production plants.
Starting up manufacturers in industrial countries which can afford the technology should shift towards production in cell culture. This will allow for easily scalable, high capacity production of pandemic strain vaccine in a timely manner. The larger quantity will allow for an excess which will reduce cost per dose and allow for better distribution to developing countries. Furthermore, cell culture manufacturers would be able to produce vaccines against highly virulent avian influenza viruses, whereas egg manufacturers would not, this would have relevant implications in a pandemic caused by a novel avian influenza virus . In contrast, developing countries need to invest in LAIV egg based production plants, with intellectual property transfer, as this platform is the cheapest and fastest to erect with high production capacity.
The embryonated egg and cell culture platforms are capable of producing TIVs and LAIVs, each combination of platform and production style has its own set of advantages and disadvantages.
Today, almost all influenza vaccine production plants make use of the TIV embryonated egg platform, using this platform on its own to meet pandemic influenza vaccine needs is clearly impractical. This does not mean that the platform should be removed or replaced, rather it should be used in conjunction with other platforms to improve overall production capacity and delivery.
Pandemic preparation decisions regarding which platform to invest in are complex and require consideration of a wide variety of factors. Availability of embryonated eggs, capital investment, research and development required, maintenance cost, production lead-time and infrastructure are some of the many factors that have to be assessed in order to make a truly informed decision.
The future of influenza pandemic vaccine production depends not only on the manufacturing pros and cons of the different platforms but also on legislation, regulatory approval pathways and international cooperation. Responding to a global pandemic will require international cooperation between the many different production platforms so that the advantages of all may be used to provide affordable pandemic influenza vaccine, in a timely sustainable manner to support the needs of a global population.
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