New non-GMO vector technologies for expressing recombinant proteins in hen eggs offer a novel development and production platform for biologics and vaccines. By upgrading egg-based manufacturing, these technologies bring a wealth of experience and resources to the 21st Century fight against emerging diseases. The AdCEV(TM) vector technology developed by AfriVax and the Sendai Virus vector technologies can potentially improve manufacturing in eggs by increasing yields, expanding the repertoire of possible products, and increasing biosafety. With AdCEV(TM) technology, eggs can be used as miniature bioreactors for rapidly manufacturing a wide range of high-value biopharmaceuticals. The speed with which recombinant products can be developed, scaled and manufactured with these technologies offers a unique competitive advantage compared to other industrial technologies like cell culture and plants.

1. Nov 13, 2012
New technologies for
manufacturing recombinant
products in embryonated eggs
Eluemuno R. Blyden, Ph.D.
AfriVax, Inc.

2. AfriVax, Inc.
• Founded 2007
• USA registered
• Private company
• Tel: 646-596-9081
• info@afrivax.com
• http://www.afrivax.com
Industry leading projects for:
• Rational development of
recombinant products targeting
human and animal diseases.
• Translational Research CRO
services for scale-able production
platforms including eggs.
• Integration of technologies and
services to support the product
development value chain.
• Global network of partners serving
the One Health field.

3. Summary: Why an Egg-based Platform?
• Recombinant proteins can be expressed in eggs
• Combines advantages of recombinant technology
with proven methods of egg-based manufacturing
• Broadens applications in biopharmaceutical
production-- subunit vaccines, VLPs, biogenerics,
diagnostics, rMAbs, etc. could be made in eggs
• Environmentally friendly single-use/disposable
bioreactor technology!
• Suits rapid, high-throughput product development
and distributed manufacturing for a sustainable
global health solution

4. Contributing to the Global Health Mission
● MANUFACTURING VACCINES
o In response to influenza pandemics…
● DEVELOPING DIAGNOSTICS, THERAPIES &
COUNTERMEASURES
o To protect against chemical, biological, radiological, and nuclear
threats…
● ACCELERATING PRODUCT AVAILABILITY
o From proof-of-concept through licensure with rapid automated
development and scale-up in eggs
● TRAINING TOMORROW’S WORKFORCE
o To sustain national, regional and international capabilities for
responding to emerging bio-threats…

5. Vertical Integration is the key challenge for
next-generation egg-based manufacturing
●Unit process level technologies that create or
improve manufacturing processes in the egg
●Machine level technologies that create or improve
manufacturing processes with eggs
●Systems level technologies for innovation in the
egg-based manufacturing enterprise

6. 2008 © AfriVax, Inc.
Recombinant
proteins/virus
Allantoic
Fluid
Virus Driven Molecular Process in the Egg
Allantoic
Membrane

7. Recombinant vector technologies
System Genetics Molecules Demonstrated Yield rProtein
estimate/egg
Sendai Virus SS RNA RSV F glycoprotein ~ug range
Fowl Adenovirus DS RNA Rabies Glycoprotein G
Human Beta-Interferon
Human CRP
Human Lactoferrin
50ug-- 0.8mg/egg
Influenza A SS RNA Virus particles 50-100ug/egg
Various other SS RNA Virus particles ~ug range
PCT Publication: WO0119968 (2001)
Recombinant Eggs and Gene Cloning and Expression Vectors Based on Avian Adenoviruses.
V.I. Grabko, E.R. Blyden
Protein Expr Purif. 2009 May;65(1):100-7. Epub 2009 Jan 11.
Production of recombinant human lactoferrin in the allantoic fluid of embryonated chicken eggs and its characteristics.
Tutykhina IL, Bezborodova OA, Shmarov MM, Logunov DY, Neugodova GL, Nemtsova ER, Naroditsky BS, Yakubovskaya RI, Gintsburg AL
BMC Biotechnol. 2007; 7: 17. Published online 2007 April 5. doi: 10.1186/1472-6750-7-17 PMCID: PMC1852797
High level expression of soluble glycoproteins in the allantoic fluid of embryonated chicken eggs using a Sendai virus minigenome
system Teresa Corral,1 Lorena S Ver,1 Geneviève Mottet,2 Olga Cano,1 Blanca García-Barreno,1 Lesley J Calder,3 John J Skehel,3
Laurent Roux,2 and José A Melero1.
J Virol. 1999 Nov;73(11):9679-82.
Rescue of influenza A virus from recombinant DNA. Fodor E, Devenish L, Engelhardt OG, Palese P, Brownlee GG, García-Sastre A.

8. rProtein Production with Sendai Virus
BMC Biotechnol. 2007; 7: 17. Published online 2007 April 5. doi: 10.1186/1472-6750-7-17 PMCID: PMC1852797
High level expression of soluble glycoproteins in the allantoic fluid of embryonated chicken eggs using a Sendai virus
minigenome system
Teresa Corral,1 Lorena S Ver,1 Geneviève Mottet,2 Olga Cano,1 Blanca García-Barreno,1 Lesley J Calder,3 John J Skehel,3 Laurent
Roux,2 and José A Melero1

9. rProtein Production with Fowl Adenovirus
Embryonated Hen Egg
72 hr Incubation
AdCEV construct vector DNA + wt FAV1 particles
Transfect

10. Recombinant Rabies Glycoprotein G
1. Sample 7ul of Rabies G -AdCEV transfected allantoic fluid.
2. Pre-stained MW markers.
3. Sample x10 nanofiltered and concentrated Rabies G -AdCEV
transfected allantoic fluid.
• Soluble form
• Weakly immunogenic in
hamsters and mice (tested
at CDC and Rocky
Mountain Laboratories,
USA)
• Commercially viable
diagnostic reagent
• Commercially
viable research reagent

11. 2008 © AfriVax, Inc.
rHuman C-reactive protein (rHCRP)
1. w.t. virus infected
2. uninfected
3. rCRP virus infected affinity purified
4. rCRP virus infected
5. control human CRP
6. MW Standards
1. w.t. virus infected
2. uninfected
3. rCRP virus infected affinity purified
4. rCRP virus infected
5. control human CRP
6. MW Standards
1. rhCRP Coumassie Stain
2. CRP Coumassie
3. rhCRP Western Blot
4. CRP Western Blot

12. Advantages of AdCEV™/Egg Platform
• DNA mini-chromosome or replacement vector strategies
• Eggs have ideal phylogenetic relationship for human
recombinant products
• FAV1 virus is considered harmless to humans
• FAV1 derived vectors could produce up to ~3mg/egg
(300mg/litre) of recombinant protein with optimization
• Scale up of production is easy-- just process more eggs.
• Amenable to automation
• Purification of antigens from allantoic fluid is well
established.
• Eggs are already widely deployed biological substrate
especially in emerging developing world.

13. Business Model: End-to-end service-- from
discovery to fully licensed product
● CRO for discovery, design and engineering of proteins with bioinformatics and HTS
● Clone and express proteins in eggs using our technologies
● Offer expertise in translational research for commercializing recombinant vaccine,
diagnostic, biosimilar or new therapeutic products using egg platform
● CMO for pilot lots to support clinical trials
● Complete small-medium scale facility design, construction and validation

14. ● Combine bioinformatics, robotics
and automation for rapid
gene/protein engineering.
● Identify optimum product candidates
by combinatorial screening.
● Scale-up candidates in the same
system-- just use more eggs!
An egg-based HTS system for rapid design
and engineering

16. Next-generation egg-based manufacturing of
vaccines, diagnostics & biosimilars
Bringing it all together in small and medium scale modern modular manufacturing
facilities for regional applications in human and veterinary health...
AdCEV
inside!

17. Cost of establishing flu vaccine production
with new egg technologies v. cell culture
Slide adapted from WHO publication.

18. AfriVax egg-based business offers
• CRO projects to discover, design and engineer
biosimilars, subunit vaccines, VLPs or rMAbs
• CRO projects to express recombinant proteins in
eggs and develop purification procedures
• CMO projects to produce bulk protein for vaccine,
diagnostic or therapeutic applications
• Commercialization projects for complete
manufacturing facilities and franchiseable systems

19. Wider Opportunity offered by the
technologies
• Sustainable platform for pandemic preparedness
and rapid response to biothreats
• Private equity opportunity to commercialize
biologic products for local markets
• Collaboration with vaccine manufacturers to
create alternative revenue streams or improve
ROI from existing egg-based production assets
• Distributed franchise manufacturing of global
health products

20. References
● Blyden, E.R.; Christiansen, K., "Poultry Pharming: Next-Generation
Technologies for Egg-Based Biopharmaceutical Manufacturing," Global
Humanitarian Technology Conference (GHTC), 2011 IEEE , vol., no.,
pp.51,55, Oct. 30 2011-Nov. 1 2011 doi: 10.1109/GHTC.2011.17
● Eluemuno R. Blyden, PhD [1], Peter K. Watler, PhD [2] “New Approaches to
Improved Vaccine Manufacturing in Embryonated Eggs” BioPharm
International Supplements Jan 02, 2010
● AdCEV™ Egg Technology

21. Summary: Why an Egg-based Platform?
• Recombinant proteins can be expressed in eggs
• Combines advantages of recombinant technology
with proven methods of egg-based manufacturing
• Broadens applications in biopharmaceutical
production-- subunit vaccines, VLPs, biogenerics,
diagnostics, rMAbs, etc. could be made in eggs
• Environmentally friendly single-use/disposable
bioreactor technology!
• Suits rapid, high-throughput product development
and distributed manufacturing for a sustainable
global health solution

22. Thank You!
http://www.afrivax.com