The Production of Therapeutic Proteins in Plants: Apo AI and Insulin

The Production of Therapeutic Proteins in Plants:Apo AI and InsulinLiz MurrayTeam Leader,Bioanalytical

SemBioSys Genetics, Inc.SemBioSys Genetics, Inc. is a Canadian biotechnologycompany developing a broad pipeline of protein-basedpharmaceuticals and non-pharmaceutical productsusing a safflower plant production system.

SemBioSys OverviewLocation: Calgary, Alberta, CanadaSymbol: SBS.TOEmployees: 35Patents: 170 issued93 pending

SemBioSys’ NicheSemBioSys focuses on finding existing protein based drugsof therapeutic interest or other products such as specialty oilsfor which there is an unmet need.SemBioSys addresses demand/protein availability issueswith a lower cost production system. SemBioSys is aplatform technology company.

SemBioSys’ TechnologyProof-of-ConceptArabidopsis thalianaCommercial Production SpeciesSafflower (Carthamus tinctorius)

Transformation of SafflowerGerminationSeedselectionInfection ofCotyledons byAgrobacteriumAcclimationto SoilEarly stagegrowth – noselectionRootingShoot Elongation& Selection

Field Grown GMO SafflowerPlanting sites: Alberta, United States and Chile -federal regulatory approval is required beforeplanting each season.Isolation distances are required – 1600 m from othersafflower crops in Canada, 2 miles in the US.Seed is harvested and cleaned before use.

The Oilbody Technology PlatformSafflowerSeedProteinsFloretOilbodies:Seed structures with aphospholipid membraneand protein coat thatstore energy forgermination in the form ofoil, protecting the oil fromoxidization anddeteriorationOleosinOleosome

Purification of OilbodiesThe Process: flotation-centrifugation to separate theoilbody fraction

Plant Impurity Assays – HCP and OleosinRabbit anti-host cell protein antibodies were raised to thesoluble and oilbody fractions of safflower seed grindpreparations.Antibodies were supplemented with other polyclonals thatrecognized oilbody proteins for the ELISA.Limit of Detection =

Plant Impurity AssaysPhytate (IP 6 ) is the plant’s phosphate storage moleculeand requires phytase to release the phosphate.Phytate is an anti-nutrient – will bind important mineralsin the body such as calcium, zinc and iron.Phytate is assayed by digestion with phytase thenquantitation of phosphate using a commercial phosphateassay reagent (Bio-Mol Green) in a microplate format(620 nm).Limit of Detection =

Plant Impurity AssaysCarbohydrate – simple and complex, some unique to plants.Detected using an HPLC method involving TFA hydrolysisof the protein to release monosaccharides.Limit of Detection =

Product Impurity Assays – Fusion PartnerFusion partner - usually a single chain antibody (scFv) thatinteracts with oleosin on the oilbody surface.Solubility issues are usually a problem with this once it iscleaved.Product is cleaved from the fusion partner using enzymes.Limit of Detection =

Other Impurity AssaysHeavy metals – present in soil, detected using ICP-MS(inductively coupled plasma mass spectrometry).Pesticides – including herbicides, detected using standardagricultural methods using CFIA and EPA guidelines.

Viral Load/Viral ClearanceRegulators recognize that plant associated viruses are of norisk to humans (EMEA CHMP/BWP/48316/2006). Accidentalviral contamination through agricultural practices is aconcern and must be addressed (i.e. animals, fertilizers, etc).Seed viral load - testing of surface contamination for virusesViral clearance of process – testing same as for any othermanufacturing process.

Leading Product CandidatesProduct Indication Stage of DevelopmentPharmaceuticalBiosimilar Insulin(SBS-1000)Apo AI MilanoDiabetesAcute CoronarySyndrome(atherosclerosis)Phase I/II• SBS-1000 demonstrated to bebioequivalent to commerciallyavailable insulinPreclinical• Positive preliminaryregression/remodeling data• Pre-IND meeting held Q3 2009

InsulinPositioned to Meet the Growing Demand• Diabetes is a worldwide crisis• Increasing emphasis on earlier use in Type 2 diabetes• Escalating demand in the Developing World will further outstrip supply

Biosimilar Insulin (SBS-1000)• Very well characterized biologic with establishedmonograph assays and reference standards• FTO – very little restricting intellectual property• Unmet need – increasing incidence of diabetesworld wide with limited supply• Reduced manufacturing costs – allows accessibilityfor poorer countries

Economics of Plant-Produced InsulinSafflowerEnabling technology to meetinsulin demand1.2 %~1.0insulin expression oftotal seed proteinacre produces 1 kg of insulinFloretSeed15,000acres to supply 2012projected insulin demandworld-wide$80Mest. capital cost for 1,000 kgof plant-produced insulin70%Capital costreductioncompared tofermentation

Insulin – Expression in PlantsPlant Transformation Vector for InsulinstaRBphaPPRSscFvD9pVS1 reppSBS443812797 bpER-HInphaTbomoriSpecRLBubiTpatubiPDerived from an Agrobacterium plasmid using selection based on herbicide tolerance.Expression is targeted to the seed.1 st generation construct was not an oleosin fusion system.

Insulin – Equivalence DataV8 Protease FingerprintingHPLC Run Time (min)USP Insulin (reference standard)SemBioSys InsulinV8 protease digest isdiagnostic for correctdisulfide binding in insulin

Insulin – Equivalence DataInsulin Receptor Binding Analysis of SBS-1000 DSIC50 Prediction Curves - Insulin300250USPy = 96415x -1.9211uU/ml insulin20015010050SBS-1000y = 532058x -2.3245R 2 = 0.98R 2 = 0.96Humuliny = 201360x -2.1042R 2 = 0.98NIBSCy = 217904x -2.1133R 2 = 0.9800 10 20 30 40 50 60 70 80 90 100USP Humulin SBS-1000 NIBSC% no insulin controlIC 50 value comparison (µU/ml)USPSBSInsulinNIBSCHRRatio toUSPRatio toNIBSCRatio toHR52.51 59.80 55.95 53.58 1.14 1.07 1.12

Insulin – Equivalence DataIGF-1 Receptor Binding Analysis of SBS-1000 DSIC50 Prediction Curves - IGF-1nM IGF10010IGF-1y = 27104x -2.3766R 2 = 0.98NIBSCy = 381217910928x -4.8196R 2 = 0.95USPy = 238270451661x -4.7054R 2 = 0.98SBS-1000y = 527721407540x -4.8927R 2 = 0.9610000100010010nM insulin10 10 20 30 40 50 60 70 80 90 1001IGF-1 USP NIBSC SBS-1000% no ligand controlIC 50 value comparison (µU/ml)IGF-1SBSInsulinStandardRatio toIGFRatio to InsulinStandard2.48 2570 2471 1036 1.04 (NIBSC)2414 1.06 (USP)

Insulin – Equivalence DataRabbit Safflower Insulin Tolerance Test% Initial Blood Glucose10080604020June 2008SalineUSP Insulin (reference standard)Insulin (Humulin ® R - Eli Lilly)Insulin (SemBioSys)0 100 200Minutes Post Injection

Insulin Characterization: Clinical Bioequivalence250.0200.0Comparison of Blood Insulin Levels betweenSBS-1000 and Reference InsulinsHumulin® RHumulin® SSBS-1000150.0pmole/L*min100.050.00.00 1 2 3 4 5 6 7 8 9Hours25

Apo AINext Generation Therapy in AtherosclerosisStatins (LDL therapy)Halts plaque build-upApo AI (HDL therapy)Reverses plaque build-up

Apo AI Milano (SBS-2000)• FTO – SBS has the patents for expression of Apo AIin plants• Volume/Dosing – likely 50-100 mg/kg which meansseveral grams are required for each dose in eachperson• Manufacturing – bacteria free system avoids theinteraction of Apo AI with bacterial endotoxin•Unmet need – no drug on the market is able toreverse plaque buildup in blood vessels

Chemical Characterization of Purified Apo AI MilanoMS & N-terminal SequencingApo AI StandardMassExpected 28078.6 DaObserved 28076.0 DaN-terminal SequenceExpected DEPPQObserved DEPPQMassSafflower Apo AI MilanoMassExpected 27781.3 DaObserved 27780.0 DaN-terminal SequenceExpected PPQSPObserved PPQSPMass

Functional Characterization of Purified Apo AI MilanoDMPC Clearance Assay1.2001.0000.800Abs @ 325 nm0.6000.4000.2000.0000 1 2 3 4 5 6 7 8 9 10 11 12 14 16 18 20 22 24 26 28 30Time (min)DMPC (non red) ApoAI (non red) SBS Milano (non red)

Cholesterol Efflux in vitro6.0In vitro Cholesterol Efflux5.0% efflux4.03.02.01.00.0no stimulation vehicle only Apo AI alone SBS Apo AI Milano Plasma HDLalone (native Apo AI)SBS Apo AI MilanoHDLTest article

Cholesterol Mobilization in vivoSBS Apo AI Milano HDLVehicle only4.0Total cholesterol (mmol/l)3.53.02.52.01.51.00.50.00 5 30Minutes

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Non-Pharma ProductsProducts and Commercial PartnersGLA (omega-6 oil) - ArcadiaChymosin (protein) - INDEARDHA (omega-3 oil) - MartekHydresiaTM (oilbodies) - Botaneco