new technologies for manufacturing recombinant products in embryonated eggs
TRANSCRIPT
Nov 13, 2012
New technologies for manufacturing recombinant
products in embryonated eggs
Eluemuno R. Blyden, Ph.D.AfriVax, Inc.
AfriVax, Inc.
• Founded 2007• USA registered • Private company• Tel: 646-596-9081• [email protected]• 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.
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
Contributing to the Global Health Mission
● MANUFACTURING VACCINES
o In response to influenza pandemics…
● DEVELOPING DIAGNOSTICS, THERAPIES & COUNTERMEASURESo 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 WORKFORCEo To sustain national, regional and international capabilities for
responding to emerging bio-threats…
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
2008 © AfriVax, Inc.
Recombinantproteins/virus
Allantoic Fluid
Virus Driven Molecular Process in the Egg
Allantoic Membrane
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-InterferonHuman CRPHuman 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: PMC1852797High 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.
rProtein Production with Sendai Virus
BMC Biotechnol. 2007; 7: 17. Published online 2007 April 5. doi: 10.1186/1472-6750-7-17 PMCID: PMC1852797High level expression of soluble glycoproteins in the allantoic fluid of embryonated chicken eggs using a Sendai virus minigenome systemTeresa 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
rProtein Production with Fowl Adenovirus
Embryonated Hen Egg
72 hr Incubation
AdCEV construct vector DNA + wt FAV1 particles
Transfect
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
2008 © AfriVax, Inc.
rHuman C-reactive protein (rHCRP)
1. w.t. virus infected2. uninfected3. rCRP virus infected affinity purified4. rCRP virus infected5. control human CRP6. MW Standards
1. w.t. virus infected2. uninfected3. rCRP virus infected affinity purified4. rCRP virus infected5. control human CRP6. MW Standards
1. rhCRP Coumassie Stain2. CRP Coumassie3. rhCRP Western Blot4. CRP Western Blot
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.
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
● 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
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!
Cost of establishing flu vaccine production with new egg technologies v. cell culture
Slide adapted from WHO publication.
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
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
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
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
