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“C1”, Speeding Up Development & Lowering The Cost of Biologics

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“C1”, Speeding Up Development & Lowering The Cost of Biologics

Industry & Society Challenges

DYADIC INFORMATION 3

1 Estimated Industry Average CHO Yield for a 12-14 day fermentation run, results vary by company.

0.1 g/l

4 g/lCHO Productivity Appears To Have Plateaued1

1985 1995 2005 2015

Current Industry Solution: Build more expensive

Industry Problems

Therapeutic protein production is expensive

Involves using enormous quantities of expensive

growth medium

Requires costly manufacturing facilities

Few advances in the protein production process during the past decade, particularly in the area of

CHO cell improvement

Current productivity is not adequate to meet future

commercial manufacturing demand

DYADIC INFORMATION

• ADD PRIOR SAMSUNG SLIDE

CHO Technology is Highly Capital Intensive and Costly

Samsung Biologics plant in Incheon, South Korea, Cost $740 million

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Dyadic is Developing What the Industry Refers to As a “CHO stopper”

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CHO stopper? Biogen looks to alternative cell lines for future of bioproduction.

The Chinese hamster ovary (CHO) cell line is not the future for biomanufacturingsays Biogen, MIT & Gates Foundation

BioPharma Reporter Bioprocessing survey report, 11/03/2017

“Nearly half the respondents of our second state of the global biomanufacturing survey believe we are too reliant on Chinese Hamster Ovary (CHO) expression systems.”

Dyadic’s Goal To further develop C1 into a safe and efficient gene expression system to help speed up the development, lower production costs and improve the performance of biologic vaccines and drugs at flexible commercial scales.

, you may use the headline, summary and link below:

04-Apr-2018 By Dan Stanton

Dyadic confident Big Biopharma will move away from CHO cell reliance

A string of deals shows biopharma is beginning to embrace its low-cost and high-yielding …– fungus-based C1 Gene Expression Platform.deals shows biopharma is beginning to embrace its – yet commercially unproven – fungus-based

C1: Speeding up the development of biologics while lowering costs

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BioPharma- Reporter.com

Ø Collaborations With Two Top Ten Pharma, Mitsubishi Tanabe Pharma, & Various Other Pharma and Biotech Companies.

C1 – A Powerful Scientific Anomaly

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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

• Micro Titer• Fermenters

§ 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

§ At scales ranging from laboratory shake flasks to 20,000l tanks and above

§ 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 receivedUS FDA GRAS recognition

Flow Diagram of C1 Expression Technology

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Constructing Strain constructing

MTP screening and analysis

1L scale fermentation

Purification and analysis

Gene synthesis

• The synthesis

of the GOI is

being done by

outsourcing

2+ weeks 2 weeks 2 weeks 3 weeks 1 week

• Cloning is done

in Yeast or E.

coli.• Preparation of

linear

fragments

• Protoplast

transformation

• 1-4 DNA

fragments can

co-transformed

• Site specific

integration

or

• Single or multi-

copy random

integration

• Colonies

appears after 4-

7 days.

• Starting re-

isolation

• Removal of

selection

marker for re-

transformation

2+ weeks

• 96 or 24 well

plates can be

used

• Source of

inoculate can

be either a

frozen cell

stock or

mycelia from a

plate.

• Shaker

incubation

• Inoculum of

vegetative cells

or spores

• 4-7 days

process

• Fed batch

technology

• Defined media

without Yeast

Extract

• Glucose

feeding.

• No Induction is

needed

• Protein is

secreted to the

media

• Biomass

sedimentation

• Protein A

purification for

mAbs

or

• Standard

purification

methodology by

filtration and

chromatography

• No need for virus

clearance

Current highest productivity of mAbs – 1.71 g/l/d

C1 Expression Technology

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Constructing

Library of promoters including Synthetic PromotersLibrary of signal sequences (secretion)Library of terminatorsSelective markers• Several auxotrophic markers• Utilization markers • Dominant selection / resistance markers

C1 Expression Technology

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o 96- or 24- well plates or shake flasks can be usedo Proteins can be analysed by:

• HPLC, UPLC, MS/MS• Western blots• SDS-PAGE• etc.

MTP screening and analysis

Ø  Fermentation broth fractions are run on PAGE gel"

Ø  POI solution is covered the gel"

Ø  Identified white bands are being extracted and the proteases are being identified by MS analysis"

A" CB"

F1" F2" F3" F4" F5"

Ø  Genome analysis and Blast"Ø  I d e n t i fi c a t i o n o f C 1

proteases"Ø  Constructing single protease

deletion libraries"

Creating POI-specific proteases deletion strain"

Creating set of single protease deletion strains"

A. Specific approach! B. General approach!

(-)-P

rot1"

(-)-P

rot1

2"

A) SDS-PAGE B) Western Blot

Cont

rols

C1+

mA

b4

LC

HC

LC

HC

mA

b4

Mar

ker

C1 P

S

C1+

mA

b4

mA

b4

(1) (2)

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

A) SDS-PAGE B) Western Blot

Cont

rols

C1+

mA

b4

LC

HC

LC

HC

mA

b4

Mar

ker

C1 P

S

C1+

mA

b4

mA

b4

(1) (2)

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

C1 – A Powerful Scientific Anomaly / Very Low Viscosity

11DYADIC CONFIDENTIAL INFORMATION

C1fermenta+on

C1 Expression Technology

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Fermentation

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 Easily available defined media components – glucose, salts, micro and macro elements, AA.

o Fed-batch technology with glucose feedingo Low viscosity culture due to morphology changes (propagule) o No need for inductiono Protein is secreted to the mediao pH: 5-8, Temp: 25 - 42°C.o 1L to 500,000L fermentation scale

From MTP to Large scalemAbs productivity

24 wells MTP – 1mg/4ml1L fermentor – 1.5/g/l/d30L fermentor – 1.71 g/l/d

FC

Ø Successful expression of Fc-Fusion protein

Ø C1 expressing Fc-Fusion was cultivated in 1 litre fermentors at 38oC and theproduct was analysed by Western Blotting

Ø The protein A purification yield from day 6 was 8.1 g/l, corresponding to 1.35g/l/day production rate.

Ø The fermentation was not fully optimized

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Success in Fc-Fusion expressions by C1

DYADIC CONFIDENTIAL INFORMATION

1 2 3 4 6 FC standards Fermentation (days)

Ø Successful expression of mAbs

Ø The expression was done in the new developed C1 strain with low proteinsbackground

Ø This protease deficient strain expresses stable mAbs.

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Success in mAb expressions by C1

LC

HC m

Ab1

Mar

ker

SDS-PAGE Western Blot

Con

trol

s

C1

C1+

mA

b1

C1+

mA

b1

LC

HC

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Success in mAbX expressions by C1Fermentations carried out for mAbX production with vessel volumes, culture volumes, and antibody titres.

SDS gel analysis of the mAbX antibody purified from the fermentations by protein A affinity chromatography: A. Fermentation MT107 in a 10 litre vessel, B. Fermentations MT111-113 in a 1 litre vessel.

Input depicts the sample loaded to the protein A column, fr3-fr7 are the elution fractions obtained from the chromatography. Samples of CHO-produced mAbX are shown as controls.

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

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Success in mAbY expressions by C1Fermentations carried out for mAbY production with vessel volumes, culture volumes, and antibody titres.

SDS gel analysis of the mAbY antibody purified from the fermentations by protein A affinity chromatography. The fermentation numbers and volumes are shown above the panels. ‘Start’ depicts the sample loaded to the protein A column, fr4-fr6 are the elution fractions obtained from the chromatography. A sample of CHO-produced mAbY is shown as a control.

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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

MAbY expression level in 30L fermentation

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0 1 2 3 4 5 6 7 8 9

0 24 48 72 96 120

mAb

(g/L

)

Hours

mAb production (g/L)

Defined media w/o YE

Defined media with YE

Produc(vity Cost*

gmAb/L gmAb/L/day Eur/gmAb

DefinemediaW/OYE 8 1,71 0,23

DefinemediawithYE 6 1,46 0,69

Definedmediaw/oYE

DefinedmediawithYE

(*) Cost contribution of the media

Ø Current best yield with purified samples: 1.71 g/l/d (w/o yeast extract)Ø An optimized Defined Medium (w/o yeast extract) has been formulated using a balanced

mixture of vitamins, salts, minerals and AA.

C1 Benefits: Lower Production Costs, Both CAPEX and OPEX

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Stainless Steel Multiuse2 x12,000 liter

Single Use Bioreactor2,000 liter

CHO C1Annual Protein Demand in g 800,000 800,000 800,000

Tank size in Liters 12,000 2,000 2,000 Productivity g/l 4 10 15 % Yield 65% 75% 75%Batches per year 20 40 40

Tank Output in g 624,000 600,000 900,000 Tanks Needed 2.0 2.0 1.0 % Capacity Utilized 64% 67% 89%

C1 can lower CAPEX:Smaller facility footprint and related costs

C1 can lower OPEX:• Low cost media• High Yield / Produce at smaller scale

MAbY binding assay by Biacore T200

Studying the interaction of mAb in real time

Ø MAbY for which the ligand was commercially available was produced in CHO (control Mab) and C1 (C1-produed mAb)

Ø The binding properties of a pharmas mAbs to the ligand were compared in a Biacore T200 assay

Ø The control mAbY and C1-produced MAbY showed virtually indistinguishable binding kinetics.

Ø Similar results were obtained with other mAb

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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

MAbY

C1 Strain Lineage for Biologics

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DNL110 5xΔ

DNL111 6xΔ

DNL120 7xΔ DNL 119 7xΔ DNL118 6xΔ + Δ TF

DNL 125 8xΔ

DNL104 4xΔ()

Ø The viability of the protease deletion strains were not negatively affected

Ø Growth rate of protease deletion strains increased – 2.0h doubling time

DNL115 6xΔ

Proteolytic Activity of Proteases Deletion Strains

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Casein assay at different pHs

Improvement of the protease-deletion strains (4ΔProteases 5Δproteases and 6Δproteases) was measured by Casein assay

0

10

20

30

40

50

60

70

80

4Δ 5Δ 6Δ 7Δ

pH5 pH6,7 pH8

Normalized to 1mg/ml total protein

Glycoengineering of C1

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

C1 typical Glycan structure

Ø Unlike most fungi and yeasts, C1 does not have ‘high’mannose (branched 30-50 mannose species), but rather has‘oligo’ mannose and hybrid-type structure.

Ø The native C1 glycan pattern is relatively complex with highmannose type (Man3-Man9) and hybrid type (Man3HexNac-Man8HexNac) glycan forms

Ø Unlike yeast and other fungi, no O-glycosylation structurewas detected in proteins expressed from C1.

C1 future Glycostructures

Ø Glycoengineering work is being applied to C1strain to create a strain that produces proteinswith defined human glycoforms

Ø 2 approaches are being applied: i) ’Classical’mammalian pathway, ii) Alg3 pathway.

Ø 13 steps will be applied for 1.5 – 2 years work

Ø The first steps of Glycoengineering C1 cellshas begun and were successful

G0 G0F G2 G2FMan3 Man6 Man9 Man8 Man7 Man5

High mannose Core 5 – 25%

Glycoengineering C1 Strains

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Improving the glycoform structure in C1 Glycoengineered strains:

Ø Proteodynamics (France) analysed glycans from native protein samples of glycoengineered C1 strains (indicated) by permethylation + MALDI-TOF analysiso No fungal high mannose structures presento Up to 80% of Man3 structure, the important precursor for human glycoforms

Ø No negative effects on cell viability have been observed with any of the modifications done

ComparisonofNglycanprofilesBydifferentC1engineeredstrains

Step1Dele)on

Step2Expressionofheterologousenzyme

Step2’ExpressionofIndigenousenzyme

C1 Strain Development for Therapeutic Protein Production

LC strainLow backgroundHigh proteolytic

HC strainHigh BackgroundHigh proteolytic

0.1 g/L

1.0 g/L

2.0 g/L

10 g/L

15 g/L

? g/L

DNL103 - DNL115Lower backgroundLower proteolytic

DNL120 -Low backgroundLow proteolytic

2016 2017 2018 2019 2020

DNL ?Low backgroundLow proteolytic

7 daysfermentation

(80 g/l of target enzyme for Bio industrial application)

(120 g/l cellulosic enzyme for Biofuel)

C1 From BioIndustrial applications to Biologics

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New C1 strains for biologics

Glycoengineering

Dyadic Board – Decades of Big Pharma Experience

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Arindam Bose, Ph.D.

EXPERIENCE

Dr. Bose worked at Pfizer for 34 yearsand held leadership roles withinbioprocess development and clinicalmanufacturing and is widelyrecognized as a Key Thought Leaderin the biopharmaceutical industry.

Barry Buckland, Ph.D.

EXPERIENCE

Dr. Buckland worked at Merck for 29 yearswhere he served in a number of senior R&Dleadership roles focusing on fermentation andbioprocess development and thecommercial manufacturing of biologics and iswidely recognized as a Key Thought Leader inthe biopharmaceutical industry. Currently, Dr.Buckland is the Executive Director, NIIMBL(National Institute for Innovation inManufacturing Biopharmaceuticals) A public-private consortium dedicated to advancingbiopharmaceutical manufacturinginnovation.

Michael P. TarnokChairman

EXPERIENCE

Mr. Tarnok spent the majority of hiscareer at Pfizer and is a seasonedfinance and operational executivewith extensive experience in thepharmaceutical industry. Currentlyalso serves on the Board of the GlobalHealth Council, and Ionetix, Inc. PriorBoard service includes KeryxBiopharmaceuticals, Inc., where healso served as Chairman of the Board.

LAST POSITIONVice President, BiotherapeuticsPharmaceutical Sciences, External Affairs and Biosimilar Strategy

LAST POSITIONVice President, Bioprocess R&D, Merck Research Laboratories

LAST POSITIONSenior Vice President in Pfizer’s US Pharmaceutical Division

Summary

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Shorter development & production cycles

Higherprotein yields

Lower CapEx/OpEx

Higher purity & greater protein recovered

Low Cost Media / No Viral Inactivation

No negative clinical signs in mice studies

R&D Collaborations Licensing Arrangements

Other Commercial Opportunities

Dyadic is looking for partners in the biopharmaceutical space to exploit the

potential of C1. Contact mjones@dyadic.com

Reinventing biological vaccine and drug development & productionDYADIC®

THANK YOU