Clearing up the facts on egg-based vaccine production

By Ken Christiansen, president of RAME-HART
Vaccines serve as a critical line of defense in disease prevention and control. As global communities interact more, there is a pronounced importance on rapidly delivering greater quantities of vaccine that reach more people as quickly as possible. Health organizations are increasingly concerned about the potential of an influenza virus pandemic amidst a growing global population. This fear has been   exacerbated by the 2009 H1N1 (swine flu) epidemic and more recently, the H7N9 (avian flu) outbreak in China[1].  The latter is a stark example of how this threat affects livestock and humans alike. Developers of both human and veterinary vaccines must address this challenge and deliver superior protection in shorter periods of time.
As new technologies are explored, health organizations stress the severity of an impending influenza epidemic. In 2005, the United Nations System Coordinator for Avian and Human Influenza outlined a global initiative designed to prevent the potentially devastating impact of an influenza virus, which could cause anywhere from five to 150 million deaths[2]. Alternatives such as cell-culture-based growth systems, recombinant protein expression systems and DNA-based vaccines face several limitations (e.g., tumorigenicity risks, ongoing clinical development, simply being cost-prohibitive) that would currently not make them viable options in the event of a pandemic[3].
Chicken-egg-based influenza vaccines, in contrast, remain the most effective manufacturing method available. As a part of its H7N9 preparedness strategy, the CDC is currently distributing viral samples grown in eggs to develop a vaccine. The H7N9 avian flu virus samples have been replicating well in eggs according to CDC reports[4]. The process is highly regulated, stable and predictable. Nonetheless, despite continual advances in the technology, misconceptions remain about the validity and effectiveness of egg-based vaccine production. In the following article, we discuss and evaluate the ‘myths’ around this proven vaccine development process.
Myth #1: “Egg-based Influenza Vaccine Production is Slow and Outdated”
Egg-based vaccine production is on occasion characterized as outdated and old-fashioned. Originally developed in the 1950’s, the technology has been used to produce seasonal influenza vaccines for over 30 years. While long-standing, the process has evolved to address various challenges, including yield, automation, capacity, quality assurance and production speed. Improvements to egg supply variability have also been made, minimizing and often eliminating the periods of time that eggs were previously unavailable[5] for use.
One area where this mistaken perception arises is in the face of pandemic preparedness. Inadequate vaccine supply is an industry-wide challenge but egg-based production comes under duress from the belief that livestock management is an erratic, un-evolved process. Vaccine manufacturer Sanofi Pasteur, however, recently developed new technologies to improve egg supply and increase availability in advance of vaccine production dates.  With the advent of restructured flock management, embryonated eggs, previously unavailable for certain periods of time, can now support vaccine production year round4.
Newer manufacturing methods such as cell-based production are predicated on improving areas like speed-to-market, risk of contamination and vaccine potency. But cell-based vaccines undergo many of the same critical processes as the egg-based method such as vaccine isolation, extraction and purification. Furthermore, in cell-based technologies, the use of animal cells in media is disadvantageous because of concerns over bio-burden potential and batch variability, two factors which compromise viral yield.  Another shared concern is strain variability. Because of the influenza virus’ propensity to change composition, trivalent vaccines which consist of different influenza strains must be formulated annually. While cell-based production is designed for faster response, it is still subject to the physical constraints of strain availability4.
As a pioneer of inoculating and harvesting machines, RAME-HART has continuously developed advancements in egg-based vaccine production technologies. Thirty years ago, egg-based production was completely manual and very labor intensive, however over time RAME-HART has automated multiple steps in the manufacturing process including harvesting and inoculation, reducing the frequency of human error, bio-burden and the risk of contamination. Egg-based influenza vaccine production has gone from a manual operation to an almost completely automated process where eggs are loaded, inspected, inoculated, de-capped, harvested and unloaded without virtually any human interference.  
Advances to the egg-based process like recombinant vector technologies are being explored to develop faster response times to an impending influenza pandemic. This method manipulates an adenovirus capable of infecting embryonated eggs to produce recombinant proteins. These egg recombinant technologies are designed to increase harvesting yields, reduce the cost of production and abridge the time of full-scale influenza vaccine production from 28 weeks (the standard timetable) to 20 weeks[6].
Overall, egg-based flu vaccine manufacturing has continually evolved, stabilizing egg supply variability, increasing vaccine yield, reducing human error, minimizing the incidence of bio-burden and raising production capacity.
Myth #2: “Egg-based Influenza Vaccine Manufacturing is a Messy, Error-filled Manual Process”
As we have already seen, egg-based vaccine production has evolved beyond a completely manual process. Increased automation and advances in technology have now cut down on limitations caused by human involvement – physical manipulation, potential of increased bio-burden, damage to the egg supply and subjective evaluation of egg suitability. Nonetheless, egg-based production is often characterized as a primitive process marked by broken egg shells and spilled yolk.
In the past, manual operators trained extensively to recognize egg impurities and rejections before and after inoculation, but repetitive daily inspection innately led to increased error rates and longer inspection times. Another critical step inhibited by operator subjectivity was de-capping and egg-shell removal, an operation which repeatedly introduces bacterial contaminants into the egg harvest. Improper execution of these steps (e.g., incomplete de-capping, broken egg shells) can lead to loss of egg supply and deliver lower doses overall. However, new technologies in de-capping, candling and inspection systems (harvesting and inoculation) have increased automation while minimizing subjective operator assessments. As a result, these technologies enable egg-based vaccine production to deliver high yields and ramp up production times. Operator handling has also been removed from the manufacturing equation and automation of the loading, transfer and delivery processes has reduced the incidence of broken eggs, spilled yolk and contamination.
For example, a recent project between RAME-HART and vaccine manufacturer MedImmune minimized human involvement from the vaccine manufacturing process, reducing the need for subjective operator decision-making from 100 percent to 10 percent[7]. The project resulted in a fully automated manufacturing line for egg-based vaccine production. Automating each operation from egg tray handling to candling, reduced the facility’s seasonal headcount by 25 percent.  Instances of manual handling fell from 5,850 events per batch to four events per batch and the facility increased production from 2,500 eggs per hour to 10,000 eggs per hour.
As egg-based vaccine technologies continue to advance, there will be fewer manual processes, less human errors and better harvest yields and production times.
Myth #3: “There is a Greater Incidence of Contamination/Bio-burden by Growing Vaccines in Eggs”
Vaccine production carries distinct inherent elements of risk and safety. Development and production involves interaction with live viruses, pathogens and bacteria. In its final form, a vaccine must be proven safe for wide human application[8]. The egg-based method of vaccine production has had in place, for over 30 years, rigorous regulatory and compliance standards set forth by health organizations, but it continues to be mischaracterized as a “messy” process filled with greater incidences of bio-burden and contamination. Concerns over diminished vaccine yields stem from the health of livestock, the presence of trace-egg protein and bacterial contaminants (e.g., salmonella and campylobacter).
Advances to egg-based processes have become much more rigorous in recent years. Flocks associated with vaccine production are housed in air-filtered environments, kept under bio-secure regulations and examined using validated biological assays. Specific pathogen-free (SPF) eggs are sanitized and candled to remove infertile eggs and dead embryos, minimizing rejected candidates prior to incubation and manufacturing operations[9].
This challenge – directly managing the risk of virus and pathogen interaction while producing and ensuring a safe product – is not exclusive to the egg-based method. Cell-culture based vaccine technologies also encounter these obstacles. With cell-based vaccines, the cell substrates used to grow viruses possess tumorigenic and oncogenic potential and it is this risk that has created regulatory limitations for wider cell-based vaccine application6.
Egg manufacturing innovations have introduced greater efficiencies to the downstream process. RAME-HART’s equipment has significantly reduced bio-burden and product contamination by automating key processes (e.g., inspection, inoculation, harvesting and cleaning), integrating these processes into an interconnected manufacturing line and minimizing operator involvement and assessment. These applications helped vaccine producer MedImmune increase harvest yield approximately 15 percent per egg harvested7. Furthermore, by integrating several automated processes into a single production line, operator interactions that previously lead to contamination (e.g., de-capping, inoculation and harvesting) were virtually eliminated.
Behind long-standing regulatory safeguards, egg-based influenza vaccine production continues to progress. By integrating bio-secure processes to minimize the risk of internal contaminants and precise, automated manufacturing processes that minimize human interaction, egg-based technologies are delivering higher vaccine yields.

In the end, preserving global health in the face of seasonal or pandemic influenza outbreaks is not about the pursuit of one technology. Currently, egg-based influenza vaccine production is the strongest tool health organizations and nations can employ. Companies like RAME-HART are developing smarter technologies to improve the process and give vaccine manufacturers a head start in rapidly delivering higher quality vaccines that keep the global population safe. In the event of a seasonal outbreak, or worse, a global pandemic, the egg-based platform is the preferred method of influenza vaccine production – in terms of scale, yield and safety – to provide communities adequate vaccine supply.  Whether it’s cell-based, egg-based or the development of a universal flu vaccine, the biomedical community must evolve these processes, not discard them.

[1] Bardiya, N., and J. H. Bae. "Influenza Vaccines: Recent Advances in Production Technologies." Applied Microbiology and Biotechnology 67.3 (2005): 299-305. Print.
[2] Nabarro, David. "PRESS CONFERENCE BY UN SYSTEM SENIOR COORDINATOR FOR AVIAN, HUMANINFLUENZA." UN News Center. UN, 29 Sept. 2005. Web. 02 May 2013.
[3] Chua, Joel V., and Wilbur H. Chen. "Bench-to-bedside Review: Vaccine Protection Strategies during Pandemic Flu Outbreaks." Critical Care 14.2 (2010): 218. Print.
[4] Center for Infectious Disease Research and Policy. H7N9 News. CDC: Labs Take Lead Role in H7N9. CIDRAP. University of Minnesota, 26 Apr. 2013. Web. 3 May 2013.
[5] Matthews, James T. Egg-Based Production of Influenza Vaccine: 30 Years of Commercial Experience. Rep. 3rd ed. Vol. 36. Washington, DC: National Academy of Engineering, 2006. Print. The Bridge.
[6] Blyden, Eluemuno R., PhD, and Peter K. Walter, PhD. "New Approaches to Improved Vaccine Manufacturing in Embryonated Eggs." BioPharm International Magazine. Advanstar Communications, 2 Jan. 2010. Web. 28 Apr. 2013.
[7] MedImmune. UK Automation Upgrade Project: A Paradigm Shift in the Egg-based Vaccine Industry. N.p.: MedImmune, 2012. Print.
[8] Ray, Suma, PhD. "Challenges and Trends in Vaccine Manufacturing." BioPharm International Magazine 2 Oct. 2011: 3-11. BioPharm International Magazine. Advanstar Communications, 2 Oct. 2011. Web. 29 Apr. 2013.
[9] Charles River Laboratories International, Inc. Technical Guidelines - SPF Eggs. North Franklin, CT.: Charles River Laboratories International, 2012. Print.


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