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Production of Recombinant HumanInsulin in Transgenic Safflower Seeds
Presented to:ABIC
Aug 26, 2008University College Cork, Ireland
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History of Insulin
1922 1978 2008
Can we makeenough insulinfor everybodywho needs it, ata price that iscompatible withtheir means?
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Why plants?
Advantages of plant systems:• Higher capacity production and lower
production costs for bulk protein
• Economical Manufacture of large volumeproducts
Seed-based expression systems:• Seeds have evolved as natural storage
organs with high capacity for protein• Low hydrolytic environment provides for
stable storage• Enables production to be decoupled
from processing
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Testing and production vehicles
Safflower• Safflower: Carthamus tinctorius, Family: Asteraceae• Origin: Central Asia• Semi-arid regions of North America, Australia and
AsiaAdvantages for PMP production:• Low production acreages (
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Cross-section of safflower seed
Seed oilbodies
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Oilbody-Oleosin Technology Platform
NutritionalOils
Proteins
Oilbodies
Safflower
Oleosin
Floret
Seed
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Partitioning recombinant proteinsusing oilbodies
Coomassie Stained Protein GelCoomassie Stained Protein Gel
rOleosinrOleosin--FPFP
OleosinsOleosins
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Meeting the Emerging Exploding Demand
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Not Enough Insulin To Go Around
Diabetes Incidence: 2006
India
41.3 MChina
25.1 MCAN/US
22.5 MWEU
18.0 M
Insulin Consumption Insulin Consumption and Demand
600 kginsulin
Rest of World30% Insulin Consumption
Western World70% Insulin Consumption
Source: WHO, International Diabetes Federation
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It’s Going To Get Worse
WEU
23.4 M
China
42.3 M
India
79.4 M
Diabetes Incidence: 2030
CAN/US
33.9 M
600 kginsulin
Rest of World65% Insulin Demand
Western World35% Insulin Demand
Source: WHO, International Diabetes Federation
Insulin Demand Insulin Demand
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Insulin Program Overview andStatus
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Insulin biosynthesis
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Expression construct
insulin expression: under β-phaseolinpromoter/terminator
selectable marker: pat gene underubiquitin promoter/terminator for PPTresistance
post-extraction maturation ofinsulin: functional insulin (5.7 kDa)generated by in vitro processing offusion protein
transformation: into AgrobacteriumEHA101 then into the plant using theflower dipping method (Arabidopsis)or infection of explants (safflower)
Construct
Mature Insulin
Fusion Protein
Pha P Pha T PAT Ubi T RBLB Ins FP Ubi P
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Insulin expression inArabidopsis
ER Apoplast Oilbodies
Gel Blot Gel Blot Gel Blot
Coomassie blue stained gels and western blots (anti-insulin mab) of total seed protein
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Insulin expression in safflower
250
150
100
75
kDa
50
37
25
20
Arab.Insulin
WT 1 2 3 4 5 1 2 3 4 5
Safflower Insulin Line A Safflower Insulin Line B
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Insulin - Chemical Equivalence
Safflower-derived insulin:Molecular mass5807 Da
Plant-derived insulin:
• Is chemically equivalent tocommercially-available humaninsulin
• Folds identically to commercially-available human insulin
Commercialpharmaceutical-grade insulin:Molecular mass5807 Da
Electrospray Mass Spectrometry
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Plant-derived Insulin is properly folded
V8 protease fingerprinting
HPLC Run Time (min)
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Insulin – In Vitro Functionality
-20
0
20
40
60
80
100
120
0.01 0.1 1 10 100 1000
Insulin (SemBioSys)
%M
ax
imu
mB
ou
nd
Insulin (ng/ml)
error bars =/- SEM
IC50 (ng/ml)
2.28
2.08
2.10
USP Insulin
Pharmaceutical RLD Insulin
Receptor Binding Assay
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Saline
USP Insulin (pharma grade standard)
Insulin (Humulin® R-Eli Lilly)
Insulin (SemBioSys)
12
Rabbit Safflower Insulin Tolerance Test
Minutes Post Injection
20
40
0 100 200
60
80
100
%B
loo
dG
luco
se
lev
el
Pharmacokinetics andPharmacodynamics wereindistinguishable from thereference drug
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Supplying World Insulin Demand
15,0003 commercial farms
Supplyfor the entire planetin 2012
acres or
1 commercial farm
1 mile
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Scale-up
~ 500kg seed Pilot scale process
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Insulin Process Development
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Scalable, Cost-effective Solution
Fermentation vat
Field of safflower
Insulin fermentation facility
Safflower
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Insulin Process Advantages
Harrison, R.G., Todd, P., Rudge, S.R. and Petrides, D.P. Bioseparations science and engineering. New York: Oxford University Press, 2003, pp. 349-362.
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Regulatory Strategy: NorthAmerica and Europe
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Regulatory framework
• FDA and EMEA have both publishedguidelines on the manufacture of biologics inplants
• Plant-based systems will be expected to meetthe standards of quality established forconventional production platforms (GMPstarts in the field)
• Insulin Product Regulatory- EMEA published draft guidance on Biosimilar
Insulin- FDA: insulin approved by CDER under FD&C Act.
Therefore eligible for 505(b)(2) application
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Reversesplaque build-up
Regulatory Path
There is a
clear
Conclusions from the FDA meeting:
• Insulin can follow the abbreviated505(b)(2) rule
• First human trial will be a Phase II forpharmacokinetics & pharmacodynamics
• 50 subjects, 1 month study
• Second human trial will be aPhase III for longer-term safety
• 500 subjects, 6 months, 2 armssafflower insulin and Humulin®
• 500 subjects, 6 months, 1 armsafflower insulin only
• No special regulations related to plant
safflower-derived insulin
Met with the FDA for a pre INDconsultation in October 2006Met with EMEA in May 2008
processregulatory
for
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Program Status
• Completed manufacture of sub-chronic toxicologybatch
• Repeat dose (28-day) toxicology studies in rats andmonkeys completed in-life phase Apr 20/08. Alltoxicology studies were “clean”
• IND submission achieved in July 2008• Meeting and Briefing Book submission for EMEA
SAWP took place May 2008• Manufacture of Phase 1 trial batch completion end
Aug• Initiation of Phase 1 PK/PD clinical trial planned for
Q3-Q4• Phase III clinical trial planned for late 2009-2010 (12
month study)