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)