OR WAIT null SECS
Recombinant vector technologies can improve the yield and lower the cost of egg-based influenza vaccine production.
The H5N1 avian influenza A and 2009 influenza A (H1N1) pandemics have highlighted the critical importance of embryonated hen eggs as a manufacturing platform for vaccines. We discuss the implications of recombinant viral vectors that can express proteins in embryonated chicken eggs and their potential impact on manufacturing with this long-established platform. New recombinant protein expression AdCEV vectors offer a pragmatic approach to increasing the efficiency, utility, and safety of manufacturing in eggs. Their use is complementary to the single-use downstream processing systems and risk-based quality assurance that are leading to dramatic reductions in the cost of multi-use manufacturing facilities for recombinant biopharmaceuticals. This system also offers a safe and economical manufacturing process that addresses the urgent needs of influenza pandemic preparedness, especially for low and middle income countries (LMICs).
Fertilized hen eggs have been used in vaccine manufacturing for over 50 years. They are the original single-use bioreactors—inexpensive, easily scaleable, and environmentally friendly.More human vaccines are manufactured in embryonated eggs than in any other biological substrate, with more than 250 million doses of inactivated seasonal influenza vaccine distributed to 100 countries annually. Vaccines for yellow fever and many veterinary vaccines are also routinely made in eggs.1
Dimitri Vervitsiotis, Getty Images
Nevertheless, large-scale flu vaccine production in eggs poses many challenges such that significant investments have been directed toward developing cell culture-based alternatives. These new platforms, which include recombinant mammalian cell culture, plant-based vaccines, and E. coli and other microbial-based production systems, have made significant progress, but the best of these efforts are still several steps away from meeting the needs of large-scale, annual influenza prophylaxis.2,3
Given this situation, eggs are likely to remain the most important production platform for flu vaccine production for the foreseeable future. Yet, the narrow economic margins of annual flu vaccine development, licensure, and mass production are a serious challenge to pandemic flu vaccine preparedness, which must be based on the same production capacity used for seasonal flu vaccine production. Yields of inactivated flu vaccine can range from 1–3 doses per egg, depending on the strain, although the seasonality of the industry in temperate regions can leave production capacity idle for half the year.1
The World Health Organization strategy for pandemic vaccine supply calls for the establishment of an economically sustainable base of annual flu vaccine production around the world that can provide surge capacity in the event of a pandemic like the current 2009 influenza A (H1N1) pandemic.4 The need for increased flu immunization is especially important in low and middle income countries (LMICs) where flu prophylaxis is minimal or absent. Efforts are being aimed at stretching the existing egg-based vaccine supply through use of adjuvants and improved recovery of immunogens from eggs.
Here, we examine the potential impact of new recombinant vector technologies on influenza vaccine production and the wider implications of such technology for global health. Egg-based manufacturing as presented here is comprised of three interacting processes: a molecular process that takes place in the egg, a manufacturing process consisting of upstream and downstream activities that manipulate unit eggs, and an operational process that encompasses the spatial, temporal, and economic organization of egg-based vaccine manufacturing. The potential impact of the model AdCEV/egg platform on each of these organizational levels is discussed.
Several human, mammalian, and avian viruses can infect cells of the chorionallantoic membrane of chicken eggs (a monolayer of cells surrounding the fluid-filled allantoic cavity in the egg). Infection results in the accumulation of live virus particles in the allantoic fluid that can be collected after a suitable incubation period. The standard method of egg-based vaccine production consists of pre-incubation of the eggs, inoculation with a live virus (e.g., influenza, yellow fever), incubation, harvesting of allantoic fluids, downstream processing, and filling and finishing. For the classic inactived influenza vaccine, purification, inactivation, and stabilization of this harvested material yields vaccine product.
Fowl Adenovirus Type 1 (FAV1) is an adenovirus that can infect embryonated eggs. Viral vectors constructed by manipulating the FAV1 genome yield a novel class of vectors (AdCEV vectors, AfriVax, Inc., Seattle, WA) that can be used to produce recombinant proteins in eggs.4 FAV1 vector-driven expression in eggs has been demonstrated for recombinant human C-reactive protein, rabies glycoprotein (Figure 1), HEV glycoprotein, and a handful of other viral and human proteins.5 A similar approach using a Sendai virus-derived mini-genome system also has been used to produce recombinant viral glycoproteins in chicken eggs at yields 3–5 times higher than a vaccinia cell culture system.6 These and other results show that biologically functional human and avian recombinant proteins can be made in eggs.
Figure 1. AdCEV vector driven protein expression in eggs. 1) Coumassie-stained SDS-PAGE MW standards (Bio-Rad). 2) Coumassie-stained SDS-PAGE 10x concentrated allantoic fluid from SPF eggs infected with AdCEV-rabies G vector; 3) Western blot of Material in column 2, reacted with anti-rabies(Vnukovo-32) mouse monoclonal antibody (Capricorn Products, ME, USA).
Avian Adenovirus-Based Vectors Can Improve Yields From Eggs
For vaccines, the main impacts of using AdCEV vectors derive from the potential for higher yields of antigen per egg. In wild-type FAV1 infections of eggs, up to milligram amounts of viral proteins can accumulate in the allantoic fluid. This is much higher than the quantities of immunogen produced in influenza virus infected eggs (about 50 μg/egg). Recombinant flu antigens made with FAVI-based vectors should have native immunogenic characteristics, because eggs are a natural host for influenza.
Influenza immunogen production was the world's first scaffold approach to industrial protein production.1 Annually, the epitopes on the scaffold are modified by viral evolution, and vaccine selections for manufacture are recommended by regulatory agencies to match viral strains in circulation. The ability to express immunogens from vectors that can be manipulated in E. coli and only introduced into eggs at the production batch stage offers great flexibility for molecular engineering scaffolds for flu and other diseases. For example, innovative molecular chimera constructs that include TLR-stimulating components3 or that produce self-assembling virus-like particles (VLPs) have been shown to result in efficacious flu vaccines containing 10-fold less immunogen.3
The expression of such advanced immunogen constructs in eggs, with the yield improvements provided by AdCEV vectors, could result in a rapid and readily implementable solution to the global shortage of manufacturing capacity. Using expression vectors also decouples product yield from strain-to-strain variations that affect traditional flu vaccine manufacturing in eggs.
Egg-based recombinant platforms create a new category of production system that combines the advantages of recombinant cell culture with inactivated vaccine manufacturing approaches while eliminating many of their drawbacks. A recent comparison of bulk costs per liter for different flu vaccine production systems shows that they can be divided into high, medium, and low-cost categories (Table 1).7
Table 1. Cost categories for influenza vaccines manufactured with different production technologies.7
The cost of flu vaccines per course derives from a complex equation of molecular, manufacturing, and operational factors that affect the number of courses per liter. These include: antigen yield per liter, required antigen dosage per strain, number of strains per dose, and number of doses required per course.7 Egg-derived recombinant flu antigens could shift the cost of inactived influenza vaccine manufacturing into a lower cost category similar to, or better than, that of live attenuated flu vaccines. Efforts are underway to (i) evaluate production yields achievable for pandemic and epidemic strain vaccines made with AdCEV; (ii) reduce the quantity of antigen per dose and number of doses required; and (iii) evaluate the potential of recombinant chimeric molecular adjuvant strategies.
Eggs account for approximately 50% of bulk vaccine cost in inactived influenza vaccine manufacturing.7 Recombinant vectors offer a way of lowering vaccine cost by reducing the number of eggs required for a given yield. For example, a 10-fold increase in yield could result in a 40% reduction in vaccine cost. In addition, by producing more vaccine per unit operating time, inactived influenza vaccine production can be accelerated. Even a 3- to 5-fold reduction in the number of eggs required would dramatically improve manufacturing logistics and have the follow-on effect of enhancing quality control.
Simplifying production can improve quality. In recent years, the complexity of large-volume vaccine manufacturing has led to quality problems and FDA regulatory actions among even the most experienced manufacturers.8 The AdCEV/egg system facilitates production by allowing a common approach to facility design, wherein multiple vaccines can be made with process differences mostly confined to downstream steps.
The higher expression yields achieved through molecular-level improvements would enable a reduction in facility size and complexity, dramatically reducing the costs of design, construction, licensure and operation.
Higher yields result in smaller processing volumes (<1,000 L), which work well with new single-use manufacturing technologies. For example, process streams could be easily conveyed between processing stages in single-use containers, eliminating the need for complex transfer lines and piping systems. Such a facility might typically use a modest 8,000 ft2 of ISO 7 and 8 classified space, with a similar amount of space for utilities, enabling manufacturers to enhance the quality of vaccines and better comply with GMP requirements.
A scaleable, integrated manufacturing concept called the Poultry Pharm is being developed as a deployment model for AdCEV/egg-based manufacturing. A turn-key Poultry Pharm consists of a fertilized egg production facility (or supply source) operationally linked to an incubation and bioprocessing suite where recombinant proteins can be manufactured using AdCEV and other egg-based vector technologies. Lean design and construction makes it possible for Poultry Pharms to be quickly and inexpensively deployed with a reduced timeline for design, construction, and commissioning—in months rather than years (Figure 2). We achieved a dramatic reduction in projected facility cost estimates from $45 to 85 million to <$15 million at full validation (Table 2) using modern modular manufacturing approaches.
Figure 2. Rapid 12-month timeline for the design and construction of a modular GMP vaccine facility.
Furthermore, the Poultry Pharm franchise concept enables a bottom-up approach to technology transfer for vaccine production, building on existing agro-industrial and human capacity in the developing world, while providing support to ensure international standards of quality control for locally produced products. Future developments of such manufacturing platforms will require (i) the development of disease surveillance that can integrate with product development cycles, (ii) the evolution of design specifications for manufacturing facilities using the AdCEV/egg platform and single-use equipment, and (iii) review of modern manufacturing facility design and operation approaches with regulatory agencies such as the FDA, EMEA, and WHO to gain support for licensure.
Table 2. Cost centers and actual budget for the design and construction of an 8,000 ft2 GMP vaccine facility.
Upgrading the embryonated egg platform with the technologies and systems described here could help to update the flu vaccine manufacturing process. The shorter lead time needed for producing an AdCEV production vector would offer near real-time development of vaccines for emerging pathogen strains. Vector technologies can reduce the time from strain isolation to full-scale flu vaccine production from the current norm of 28 weeks to 20 weeks (Figure 3).
Figure 3. Comparison of estimated timelines for influenza vaccine development and manufacture using recombinant vectors versus live influenza virus.2
AdCEV vectors are being optimized for use within the framework of small- to medium-scale facilities that can manufacture a mix of products targeting state, national, or regional biopharmaceutical markets for added sustainability. Such a manufacturing system adds a long sought-after component to the global influenza surveillance and manufacturing system by offering the potential for rapid, regional level responses to the threat of rapidly evolving strains of pathogenic influenza.
Funding Surge Capacity from Other Market Opportunities
Financing an increase in global flu vaccine manufacturing capacity requires infrastructure to sustain preparedness, and to rapidly surge production. The cost of implementing upgrades to vaccine production as outlined by the WHO is estimated at $3–10 billion and depends heavily on private sector investment and the commitment of existing manufacturers.4 Manufacturers have responded to the current swine flu pandemic generously, committing nearly 157 million of the estimated 200 million dose target set by the WHO for immunization of target populations in poor countries.9 Using the AdCEV/egg platform, the growing demand for recombinant products in diagnostics and research reagent industries can be used as an economic foundation for sustaining pandemic preparedness. Five non-vaccine biologic market segments could sustain the growth of a global vaccine manufacturing network: human biologics, human diagnostics, agricultural, specialties, and non-medical diagnostics. They offer products with market values ranging from $5 to 20,000 per mg of recombinant protein, which is an amount reasonably produced in a single chicken egg using AdCEV technology.
Recombinant vector technology offers a powerful way of extending long-standing experience with egg-based manufacturing to the fight against pandemic and neglected diseases. AdCEV vectors for use in eggs are being developed along with facilities that can be reconfigured to produce a sustainable mix of high value biologics and low cost vaccines in any economic context.
The recombinant adenovirus/egg technologies create a new category of production platform with unique economic, technical, and safety characteristics. The power to engineer the molecular processes of antigen production result in increases in yield, fidelity of product, and expansion of options for downstream processing. Changes in these parameters have far-reaching effects at the manufacturing process level including higher and more predictable yields from fewer eggs. This will potentially effect quality control issues that have traditionally dogged the egg-based vaccine industry.
A global system that can identify a pathogen, qualify targets in its lifecycle, then rapidly design and manufacture specific interventions to neutralize it is one of the most exciting promises of the post-genomic era. Ironically, such a system has existed for influenza for many decades, successfully operating on an annual cycle. Although it is ultimately desirable to move away from manufacturing in embryonated eggs, much can still be learned from this system. By offering significant improvements in biosafety and disruptive economics of scale, AdCEV/egg based systems could empower a sustainable, distributed manufacturing model more accessible to low and middle income countries and more resistant to biosecurity threats.
Eluemuno R. Blyden, PhD, is the founder and CEO of AfriVax, Inc., Seattle, WA, email@example.comPeter K. Watler, PhD, is a principal consultant and chief technology officer at Hyde Engineering + Consulting, Inc., South San Francisco, CA.
1. Matthews JT.Egg-Based Production of Influenza Vaccine: 30 Years of Commercial Experience. The Bridge. 2006;36(3): 17–24.
2. Hayden FG, Howard WA, Palkonyay L, Kieny MP. Report of the 5th meeting on the evaluation of pandemic influenza prototype vaccines in clinical trials: World Health Organization, Geneva, Switzerland, 12–13 February 2009. Vaccine. 2009 Jun 24;27(31):4079–89.
3. Fedson DS.New technologies for meeting the global demand for pandemic influenza vaccines. Biologicals. 2008 Nov;36(6):346–9.
4. Kieny MP, Costa A, Hombach J, Carrasco P, Pervikov Y, Salisbury D, et al. A global pandemic influenza vaccine action plan. Vaccine. 2006;24:6367–70.
5. Grabko VI, Blyden ER. Recombinant eggs and gene cloning and expression vectors based on avian adenoviruses. 2001. PCT Publication: WO0119968.
6. Corral T, Ver LS, Mottet G, Cano O, Garc쟭Barreno B, Calder LJ, et al. High level expression of soluble glycoproteins in the allantoic fluid of embryonated chicken eggs using a Sendai virus minigenome system. BMC Biotechnol. 2007;7:17.
7. Oliver Wyman. Influenza Vaccine Strategies for Broad Global Access. Seattle, Wa: Program for Appropriate Technology in Health (PATH); 2007.
8. FDA Warning Letter. Merck & Company, Inc., April 28, 2008.