GlobalBioprocessingandBioanaly2csCongress2018Prague,May24-25,2018

Reinven&ngbiologicalvaccineanddrugdevelopment&produc&onDYADIC®

Dyadic–C1TechnologyMay24,2018

Safe Harbor Regarding Forward-Looking Statements

DYADIC INFORMATION 2

Certain statements contained in this presentation are forward-looking statements within the

meaning of the federal securities laws. These forward-looking statements involve risks,

uncertainties and other factors that could cause Dyadic’s actual results, performance or

achievements to be materially different from any future results, performance or achievements

expressed or implied by such forward-looking statements. Any forward-looking statements

speak only as of the date of this presentation and, except as required by law, Dyadic expressly

disclaims any intent or obligation to update or revise any forward-looking statements to reflect

actual results, any changes in expectations or any change in events. Factors that could cause

results to differ materially are discussed in Dyadic’s publicly available filings, including

information set forth under the caption “Risk Factors” in our December 31, 2016 Annual Report

filed with OTC Markets on March 24, 2017. New risks and uncertainties arise from time to time,

and it is impossible for us to predict these events or how they may affect us.

C1 – A Powerful Scientific Anomaly

3

Unique Morphology (Propagules)

High Purity of target secreted

protein

Shorter Development &

Production Cycle

§  Translates into better growth conditions •  Higher yields of

secreted protein •  Lower viscosity •  MTP •  At scales ranging

from laboratory shake flasks to 20,000l tanks and above

• 

§  Greater retention of target secreted protein through downstream processing

§  Requires only low cost synthetic media

§  No Viruses which eliminates 2 purification steps typical in CHO •  No Low pH viral

inactivation •  No Virus

nanofiltration

§  C1 has received GRAS (Generally Recognized as Safe) designation from FDA and is considered fit for human consumption

§  Develop g/l/d C1 cell lines in 15 weeks

§  From seed flask to fermenter •  Savings of

nearly 10 -14 days vs CHO

§  Fermentation Cycle time 4-7 days •  1/2 to 1/3rd the

time of CHO

C1 has received US FDA GRAS recognition

DYADIC INFORMATION

Dyadic Overview

DYADIC INFORMATION 4

1979 FOUNDED

20+ YEARS EXPERIENCE IN PHARMA / FUNGAL GENE EXPRESSION PLATFORMS

HQ:Jupiter,FL

BD&L:LondonR&DManagement:Budapest

R&D:Spain

R&D:Finland

C1 Strain Development for Therapeutic Protein Production

LCstrainLowbackgroundHighproteoly&c

HCstrainHighBackgroundHighproteoly&c

0.1g/L

1.0g/L

2.0g/L

10g/L

15g/L

?g/L

DNL103-DNL115LowerbackgroundLowerproteoly&c

DNL120-LowbackgroundLowproteoly&c

2016 2017 2018 2019 2020

DNL?LowbackgroundLowproteoly&c

7daysfermenta&on

(80g/lenzymeforBioindustrialsapplica&on)

(120g/lcellulosicenzymeforBiofuel)

FromBioIndustrialapplica2onstoBiologics

5

NewC1strainsforbiologics

Glycoengineering

DYADIC INFORMATION

Flow Diagram of C1 Expression Technology

6

Currenthighestproduc&vityofmAbs–2.4g/l/d

DYADIC INFORMATION

Construc&ng Strainconstruc&ng

MTPscreeningandanalysis

1Lscalefermenta&on

Purifica&onandanalysis

Genesynthesis

•  ThesynthesisoftheGOIisbeingdonebyoutsourcing

2+weeks 2weeks 2weeks 3weeks 1week

•  CloningisdoneinYeastorE.coli.

•  Prepara&onoflinearfragments

•  Protoplasttransforma&on

•  1-4DNAfragmentscanco-transformed

•  Sitespecificintegra&onor

•  Singleormul&-copyrandomintegra&on

•  Coloniesappearsaber4-7days.

•  Star&ngre-isola&on

•  Removalofselec&onmarkerforre-transforma&on2+weeks

•  96or24wellplatescanbeused

•  Sourceofinoculatecanbeeitherafrozencellstockormyceliafromaplate.

•  Shakerincuba&on

•  Inoculumofvegeta&vecellsorspores

•  4-7daysprocess•  Fedbatch

technology•  Definedmedia

withoutYeastExtract

•  Glucosefeeding.•  NoInduc&onis

needed

•  Proteinissecretedtothemedia

•  Biomasssedimenta&on

•  ProteinApurifica&onformAbsor

•  Standardpurifica&onmethodologybyfiltra&onandchromatography

•  Noneedforvirusclearance

C1 Expression Technology

DYADIC CONFIDENTIAL INFORMATION 7

Construc&ng

LibraryofpromotersincludingSynthe&cPromoters(VTTpropriety)Libraryofsignalsequences(secre&on)LibraryofterminatorsSelec&vemarkers•  Severalauxotrophicmarkers•  U&liza&onmarkers•  Dominantselec&on/resistancemarkers

C1 Expression Technology

8

Differenttransforma&onmethodscanbeapplied:o  Singlesitedirectedintegra&ono  2sitesdirectedintegra&on(inprogress)o  Randomintegra&ono  Episomalvectors

Transforma&onefficiency

1integra*onsite 2integra*onsite

Randomintegra*on

Episomes

Ø  Transforma&onprocedurebasedonchemical(PEG)methodwithprotoplastsorelectropora&on

Ø  Frequenciesfor1μgDNA:•  20transformantsforsitespecificintegra&on•  Upto100transformantsforrandomintegra&on•  13,000transformantsfortelomericvectortransforma&on

DYADIC INFORMATION

C1 Expression Technology

9

Fermenta&on

SeedTank

200L–500m3

FERMENTER

HarvestTank

Seedflask

Inoculum

Defined medium!

NH4OH for pH control!

Glucose feeding!

Processing & Recovery!Packaging! Assembly! Filling! Formulation!

centrifugation,!Protein A, !

Purification!

Fed-Batch technology!

48 hrs! 24 hrs! 120-168 hrs!

o  Easilyavailabledefinedmediacomponents–glucose,salts,microandmacroelements,AA.

o  Fed-batchtechnologywithglucosefeedingo  Lowviscositycultureduetomorphologychanges(propagule)o  Noneedforinduc&ono  Proteinissecretedtothemediao  30-40%biomasso  pH:5-8,Temp:25-42°C.o  1Lto500,000Lfermenta&onscale

FromMTPtoLargescalemAbsproduc2vity

24wellsMTP–1mg/4ml1Lfermentor–1.5/g/l/d30Lfermentor–1.71g/l/d

DYADIC INFORMATION

Development of Different Biologics by C1

10

Applica&ons

mAbs

Fc-Fusions

mAbmimics

Bi-specific

Human&Animalvaccines

(*)

(*)

(**)

(*)

(*)

(*)SuccessfulexpressionbyC1system(**)C1expressioninprogress(***)Futureplan

DYADIC INFORMATION

Therapeu&cEnzymes&Proteins

(***)

In-house Products Development for Biosimilars Market

DYADIC INFORMATION 11

Valida&onat30Lfermenta&on-soon Laststageofconstruc&on Construc&onongoing

(*)GlobalData2017

(*)Candidate Structure Applications Market Volume (*)

($M) Status

(30L Fermentation)

VLP Animal Vaccines N/A

mAb (IgG1)

Oncology

1,402

7,000

6,900

mAb (IgG4)

Inflammatory diseases

4,700 2.1 g/L (112h)

PEGYlated-Fab 1,500 7.5 g/L (112h)

Bi-specifc scFV

Oncology

~250

Fab 4,250

Candidate Structure Applications

Status

Sequence

definition

In-vitro Synthesi

s Cloning Transfor-

mation Screening at

flask

Base case productivity

at 30L

mAB (IgG1)

Oncology

Candidate Structure Applications

Status

Sequence

definition

In-vitro Synthesi

s Cloning Transfor-

mation Screening at

flask

Base case productivity

at 30L

mAB (IgG1)

Oncology

High Yield & Purity of C1 Expressed mAb’s

DYADIC INFORMATION 12

We have expressed 100% of the mAbs tested in our 3rd party research collaborations

Initial unoptimized level of mAbs usually reach 2-5 g/L in 4-7 days fermentation

Highest level of unoptimized expressed mAb is 1.54 g/l/d, (Fc-fusion – 1.35 g/l/d)

The mAbs are integrated specifically to a “Hot spot” in the genome

After the integration the selective marker is being eliminated

The mAbs are secreted to the media and are being properly

folded

A) SDS-PAGE B) Western Blot

Contr

ols

C1+ m

Ab4

LC

HC

LC

HC

mA

b4

Mar

ker

C1 P

S

C1+ m

Ab4

mA

b4

(1) (2)

(*) Samples were taken from the 24-well plate culture.

1

2

3 4

5

6

13"

Success in MAbX Expressions by C1 Fermenta2onscarriedoutformAbXproduc2onwithvesselvolumes,culturevolumes,andan2body2tres.

SDSgelanalysisofthemAbXan2bodypurifiedfromthefermenta2onsbyproteinAaffinitychromatography:A.Fermenta2onMT107ina10litrevessel,B.Fermenta2onsMT111-113ina1litrevessel.InputdepictsthesampleloadedtotheproteinAcolumn,fr3-fr7aretheelu2onfrac2onsobtainedfromthechromatography.SamplesofCHO-producedmAbXareshownascontrols.

A. B.07 11 12 13

mA

bX

mA

bX

Product Fermentation #

Vessel volume (l)

Initial (final) culture

volume (l)

Feed volume (l)

Feed rate (g glc l-1 h-1)

Antibody titre (g/l)

mAb2 MT107 10 8 (10.5) 7.2 2.6 8.0

MT111 1 0.8 (~1.1) 0.78 2.5 6.3

MT112 1 0.8 (~1.1) 0.77 2.5 6.5

MT113 1 0.8 (~1.1) 0.78 2.5 7.9

Product Fermentation #

Vessel volume (l)

Initial (final) culture

volume (l)

Feed volume (l)

Feed rate (g glc l-1 h-1)

Antibody titre (g/l)

mAb2 MT107 10 8 (10.5) 7.2 2.6 8.0

MT111 1 0.8 (~1.1) 0.78 2.5 6.3

MT112 1 0.8 (~1.1) 0.77 2.5 6.5

MT113 1 0.8 (~1.1) 0.78 2.5 7.9

DYADIC INFORMATION

14!

Success in MAbY Expressions by C1

Fermenta2onscarriedoutformAbYproduc2onwithvesselvolumes,culturevolumes,andan2body2tres.

SDSgelanalysisofthemAbYan2bodypurifiedfromthefermenta2onsbyproteinAaffinitychromatography.Thefermenta2onnumbersandvolumesareshownabovethepanels.‘Start’depictsthesampleloadedtotheproteinAcolumn,fr4-fr6aretheelu2onfrac2onsobtainedfromthechromatography.AsampleofCHO-producedmAbYisshownasacontrol.

15 16 17 18

mA

bY

FermentationNo.

Vesselvolume(l)

Initial(final)culture

volume(l)

Feedvolume(l)

Feedrate(gglucosel-1h-1)

Antibodytitre(g/l)

15 10 8(10) 8.2 3.0 9.3316 1 0.8(~1.1) 0.74 2.7 7.117 1 0.8(~1.1) 0.72 2.9 7.3518 1 0.8(~1.1) 0.72 2.8 7.6

FermentationNo.

Vesselvolume(l)

Initial(final)culture

volume(l)

Feedvolume(l)

Feedrate(gglucosel-1h-1)

Antibodytitre(g/l)

15 10 8(10) 8.2 3.0 9.3316 1 0.8(~1.1) 0.74 2.7 7.117 1 0.8(~1.1) 0.72 2.9 7.3518 1 0.8(~1.1) 0.72 2.8 7.6

DYADIC INFORMATION

C1 Expression Technology

15

mAbYproduc2on2ter(g/L SpecificmAbYproduc2on(g/gtotalprotein)

3

5

6 8

23

4

64

89

0

2

4

6

8

10

12

0 24 48 72 96 120

mAb

(g/L)

Hours

+50%

0%

10%

20%

30%

40%

50%

60%

0 24 48 72 96 120 Hours

X2.3-fold

MediumplusfeedingimprovementleadtoamAbY2terof9g/Lat90h,andincreaseinspecificproduc2vity

Ini$alprocesswithYE 6 1.46 0.24 0.69

MediumimprovementWithoutYE 8 1,71 0,36 0,23

Medium+feedingimp.WithoutYE 9 2.4 0.54 0.23

g/L g/L/d g/g €/g

Produc$vity Mediumcostcontribu$on

Summary

2.4g/l/d

DYADIC INFORMATION

MAbX Binding Assay by Biacore T200

Studyingtheinterac2onofmAbinreal2me

Ø  MAbXforwhichtheligandwascommerciallyavailablewasproducedinCHO(controlMab)andC1(C1-produedmAb)

Ø  Thebindingproper&esofapharma’smAbstotheligandwerecomparedinaBiacoreT200assay

Ø  ThecontrolmAbXandC1-producedMAbXshowedvirtuallyindis2nguishablebindingkine2cs.

Ø  SimilarresultswereobtainedwithmAbY

16

ka (1/Ms): 1,033E+5 ± 80 kd (1/s): 3,539E-4 ± 6,2E-7 KD (M): 3,424E-9

ka (1/Ms): 1,056E+5 ± 63 kd (1/s): 4,821E-4 ± 7,3E-7 KD (M): 4,565E-9

ka (1/Ms): 1,069E+5 ± 88 kd (1/s): 3,651E-4 ± 8,6E-7 KD (M): 3,417E-9

ka (1/Ms): 1,085E+5 ± 230 kd (1/s): 5,067E-4 ± 8,4E-7 KD (M): 4,669E-9

Single cycle

Single cycle

Multi-cycle

Multi-cycle

Capture:An%-mAb

Analysed:mAb

Immobiliza6on:α-HumanFc&α-MouseFc

CHO-produced

C1-produced

mAbX

DYADIC INFORMATION

FC

Ø  SuccessfulexpressionofFc-Fusionprotein

Ø  C1 expressing Fc-Fusion was cul&vated in 1 litre fermentors at 38oC and theproductwasanalysedbyWesternBlorng

Ø  TheproteinApurifica&onyieldfromday6was8.1g/l,correspondingto1.35g/l/dayproduc&onrate.

Ø  Thefermenta&onwasnotfullyop&mized

17!

Success In Fc-Fusion Expressions by C1

1 2 3 4 6 FC standards Fermentation (days)

DYADIC INFORMATION

18 DYADIC INFORMATION

C1 Lineage of Proteases Deletion Strains

DNL1207xΔ(B)prot.

DNL1217xΔ(B)prot.DNL1197ΔTF

DNL1115xΔ(B)prot.

DNL1044xΔprot.

DNL1156xΔprot.

DNL1258xΔ(A)

DNL1258xΔ(D)prot.

DNL1258xΔ(B)prot. DNL1258xΔ(C)prot.

DNL1258xΔ(E)prot. DNL1258xΔ(F)prot.

DNL118(A)7xΔ

DNL1105xΔ(A)prot.

Ø  Theviabilityoftheproteasedele&onstrainswerenotnega&velyaffected

Ø  Growthrateofproteasedele&onstrainsincreased–2.0hdoubling&me

%0

19

CaseinassayatdifferentpHs

Improvementoftheprotease-dele2onstrains(4ΔProteases,5Δproteases,5Δproteasesand7Δproteases)wasmeasuredbyCaseinassay(*)

0

10

20

30

40

50

60

70

80

4Δ 5Δ 6Δ 7Δ

pH5 pH6,7 pH8

Normalized to 1mg/ml total protein

DYADIC INFORMATION

(*)Theproteoly2canalysisof8Δproteasesstrainsisongoing

Proteolytic Activity of Proteases Deletion Strains

Glycoengineering in C1

20

Glycoengineering of C1 strain will provide the formation of various glycan structures to evaluate immunogenicity

C1 typical Glycan structure

Ø  Unlikemost fungi and yeasts, C1 does not have ‘high’mannose(branched30-50mannosespecies),butratherhas‘oligo’mannoseandhybrid-typestructure.

Ø  Thena&veC1glycanpatern is rela&velycomplexwithhigh mannose type (Man3-Man9) and hybrid type(Man3HexNac-Man8HexNac)glycanforms

Ø  Sofar,O-glycosyla&onwasnotiden&fiedintherapeu&cproteinsexpressedinC1butminorleveliss&llpossible

C1 future Glycostructures

Ø GlycoengineeringworkisbeingappliedtoC1strain to create a strain that producesproteinswithdefinedhumanglycoforms

Ø  2 approaches are being applied: i) ’Classical’mammalianpathway,ii)Alg3pathway.

Ø  13stepswillbeappliedfor1.5–2yearswork

Ø  The first steps of Glycoengineering C1 cellshasbegunandweresuccessful

Ø Nonega&veeffectsoncellviabilityhavebeenobservedwithanyofthemodifica&onsdone

G0 G0F G2 G2F Man3 Man6 Man9 Man8 Man7 Man5

High mannose Core 5 – 25%

DYADIC INFORMATION

Glycoengineering C1 Strains

21

ImprovingtheglycoformstructureinC1Glycoengineeredstrains:

Ø  Proteodynamics(France)analysedglycansfromna&veproteinsamplesofglycoengineeredC1strains(indicated)bypermethyla&on+MALDI-TOFanalysiso  Nofungalhighmannosestructurespresento  Upto80%ofMan3structure,theimportantprecursorforhumanglycoforms

Ø  Nonega&veeffectsoncellviabilityhavebeenobservedwithanyofthemodifica&onsdone

ComparisonofNglycanprofilesBydifferentC1engineeredstrains

Step1Dele)on

Step2Expressionofheterologousenzyme

Step2’ExpressionofIndigenousenzyme

DYADIC INFORMATION

Compara2vegenome-scalereconstruc2onofgaplessmetabolicnetworksforpresentandancestralspecies.

22 DYADIC INFORMATION

CoReCoplauormformetabolicmodelreconstruc&onwasusedtobuildagenome-scalemetabolicmodelofC1star&ngfromitsproteinsequencesThefirstdraboftheC1metabolicmodelcontains5compartments(predictedusingadeeplearningmodel),4719reac2ons,3661metabolitesand992genes

CoReCo:EsaPitkänenetal.2014PlosComputBiol.SandraCas&lloetal.2016BiotechnolBiofuels.

Metabolic modeling and Proteomic Analysis for Next Stage of Strain Engineering

C1Technology

Reinven&ngbiologicalvaccineanddrugdevelopment&produc&onDYADIC®

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