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Production of Recombinant HumanInsulin in Transgenic Safflower Seeds
Presented to:ABIC
Aug 26, 2008University College Cork, Ireland
History of Insulin
1922 1978 2008
Can we makeenough insulinfor everybodywho needs it, ata price that iscompatible withtheir means?
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
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 (
Cross-section of safflower seed
Seed oilbodies
Oilbody-Oleosin Technology Platform
NutritionalOils
Proteins
Oilbodies
Safflower
Oleosin
Floret
Seed
Partitioning recombinant proteinsusing oilbodies
Coomassie Stained Protein GelCoomassie Stained Protein Gel
rOleosinrOleosin--FPFP
OleosinsOleosins
Meeting the Emerging Exploding Demand
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
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
Insulin Program Overview andStatus
Insulin biosynthesis
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
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
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
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
Plant-derived Insulin is properly folded
V8 protease fingerprinting
HPLC Run Time (min)
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
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
Supplying World Insulin Demand
15,0003 commercial farms
Supplyfor the entire planetin 2012
acres or
1 commercial farm
1 mile
Scale-up
~ 500kg seed Pilot scale process
Insulin Process Development
Scalable, Cost-effective Solution
Fermentation vat
Field of safflower
Insulin fermentation facility
Safflower
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.
Regulatory Strategy: NorthAmerica and Europe
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
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
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)