Dyadic C1 Technology C1-Production In Single Use or Reinventing - - PowerPoint PPT Presentation

Dyadic – C1 Technology C1-Production In Single Use or Stainless Steel Bioreactors

September 7th, 2018

Reinventing biological vaccine and drug development & production

Ronen Tchelet & Mark Emalfarb BPI, Boston.

DYADIC INFORMATION 2

Safe Harbor Regarding Forward-Looking Statements

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, 2017 Annual Report filed with OTC Markets on March 27, 2018 and our March 31, 2018 Quarterly Report filed with the OTC Markets on May 10, 2018. 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.

DYADIC INFORMATION 3

Dyadic Overview

• Revolutionary protein expression technology “C1”: based on Myceliophthora thermophila fungus
• Technology covered by over 20 patent families
• Listed on the stock exchange (OTCQX: DYAI), cash and liquid investments ~ 45.8m USD(1)
• Experienced management & board
• 20+ Years of Experience with Fungal Production Systems
• 20+ Years in Pharmaceuticals

Demonstrated the power of C1 for the production of biologics now seeking partnerships with biopharmaceutical companies

(1) As of June 30, 2018

20+ Years of Commercial Enzyme Production

• Platform optimized 2009 – 2015
• Hyper productive strain developed with

purity: >100 g/l with ~80% purity

• Produced in up to 500,000L tanks
• GRAS FDA certified

Biopharmaceuticals

• Strategic focus since 2016
• Powerful molecular toolbox enables expression of complex proteins
• Application proven successful:
• Vaccines
• Non-Glycosylated Proteins
• mAbs

DYADIC INFORMATION 4

Dyadic Overview

HQ: Jupiter, FL BD&L: London R&D Management: Budapest R&D: Valladolid R&D: Helsinki

• 1979 FOUNDED
• 20+ YEARS EXPERIENCE IN

PHARMA / FUNGAL GENE EXPRESSION PLATFORMS

DYADIC INFORMATION 5

How Dyadic Leverages C1 Advantages for Biologics

Efficient vast screening system for drug discovery

Growing on 24 or 96 MTP

Fast development timeline for Biologics Simple fermentation process in stainless steal bioreactors Success in Single use reactors Low cost of USP & DSP

• High productivity -
• Advanced genetic tools (Efficient transformation) -
• Efficient secretory system -
• Low viscosity -
• Wide range of fermentation conditions -
• Fast growing -
• Grow on simple defined media -
• Can tolerant high glucose concentration –
• Easy scaling up (was scaled up to 100m3)-

DYADIC INFORMATION 6

C1 Expression Technology

Transformation efficiency Different transformation methods can be applied:

• Single site directed integration
• 2 sites directed integration (in progress)
• Random integration
• Episomal vectors
• Transformation procedure based on chemical

(PEG) method with protoplasts or electroporation

• Frequencies for 1μg DNA:
• 20 transformants for site specific

integration

• Up to 100 transformants for random

integration

• ~13,000 transformants for telomeric

vector transformation

DYADIC INFORMATION 7

MTP Screening for Drug Discovery

Plasmid construction Strain construction MTP screening and analysis Gene synthesis

• The synthesis of the

GOI is being done by

• utsourcing

1+ weeks 2 weeks 2 weeks

• Cloning is done in

Yeast or E. coli.

• Preparation of linear

fragments

• Telomeric vector
• Protoplast

transformation

• Colonies appears after 4-

7 days.

• Starting re-isolation

procedure

• 96 or 24 well plates

can be used

• Source of inoculum

can be either a frozen cell stock or mycelia from a plate.

• Shaker incubation 4

days

• Use of telomeric

vector for drug discovery.

• 24 wells plates:

1mg/4ml - stable product

DYADIC INFORMATION 8

Drug Development

Plasmid construction Strain construction MTP screening and analysis 1L scale fermentation Purification and analysis Gene synthesis

• The synthesis of the

GOI is being done by

• utsourcing

1+ weeks 2 weeks 2 weeks 3 weeks 1 week

• Cloning is done in

Yeast or E. coli.

• Preparation of linear

fragments

• Vectors for site

directed integration

• Protoplast

transformation

• Colonies appears after 4-

7 days.

• Starting re-isolation

procedure

• Removal of selection

marker for re- transformation 2+ weeks

• 96 or 24 well plates

can be used

• Source of inoculum

can be either a frozen cell stock or mycelia from a plate.

• Shaker incubation 4

days

• Inoculum of

vegetative cells

• 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

1L fermentor: 1–10 g stable product for further development Removal of selection marker for re- transformation 2+ weeks

DYADIC INFORMATION 9

Production of Stable Proteins

• The viability of the protease

deletion strains was not negatively affected

• Growth rate of protease deletion

strains increased at one of the steps – 2.0h generation time Under construction C1 Lineage of Proteases Deletion Strains

DYADIC INFORMATION 10

Reducing the Proteolytic Activity

1) Protease deletion strains 2) Wide range of Temperature 3) Wide range of pHs

The 10-12 X protease deletion strains, under production at optimized temp. and pH will be used to produce stable Biologics

DYADIC INFORMATION 11

C1 Fermentation Technology

Fed-batch Process

From MTP to Large scale mAbs productivity 24 wells MTP – 1mg/4ml 1L fermentor – 1.7/g/l/d 30L fermentor – 2.4 g/l/d

• Easily available defined media components – glucose, salts, micro and macro elements, AA, vitamins.
• Fed-batch technology with glucose feeding
• Low viscosity culture due to morphology changes (propagule)
• No need for induction
• Protein is secreted to the media
• 30-40% biomass
• pH: 5-8, Temp: 25 - 42°C.
• 1L to 500,000L fermentation scale

DYADIC INFORMATION 12

Generic Process Flow Chart for C1 (12 – 14 days)

Inoculum expansion - bioreactors Seed train: inoculum expansion in flak Production bioreactor N-3 ~1.6L scale N-2 ~40L scale N-1 ~1000L scale N ~12,000L scale Pre-inoculum: Mycelium activation 1 – 1,5 days 0,75 -2,5 days 5 days Passage 2 flask Passage 1 plate

DYADIC INFORMATION 13

Generic Process Flow Chart for CHO (41 – 54 days)

Inoculum expansion - bioreactors Seed train: inoculum expansion Production bioreactor N-3 ~80L scale N-2 ~400L scale N-1 ~2,000L scale N ~12,000L scale Passage 1 Passage 2 Passage 3 Passage 4 Passage 5 Passage 6 18-28 d 9-12 days 14 days

DYADIC INFORMATION 14

MAbY Expressions by C1

Fermentations 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:

• A. Fermentation MT15 in a 10 litre vessel,
• B. Fermentations MT16-18 in a 1 litre vessel.

Input depicts the sample loaded to the protein A column, fr4-fr6 are the elution fractions obtained from the chromatography. Samples of CHO-produced mAbY are shown as controls.

Ferm entation # Vessel volume (1) Initial (final) culture volume (1) Antibody titre (g/l) 15 10 8 (10.5) 8.0 16 1 0.8 (1.1) 6.3 17 1 0.8 (1.1) 6.5 18 1 0.8 (1.1) 7.9

DYADIC INFORMATION 15

MAbY Binding Assay by Biacore T200

Studying the interaction of mAbs in real time

• MAbY for which the ligand was commercially

available was produced in CHO (control Mab) and C1 (C1-produed mAb)

• The binding properties of a pharma’s mAbs to the

ligand were compared in a Biacore T200 assay

• The control mAbY and C1-produced MAbY

showed virtually indistinguishable binding kinetics.

• Similar results were obtained with other mAb

mAbY

DYADIC INFORMATION 16

Media and process Development

Medium plus feeding improvement lead to a mAbY titer of 9 g/L at 90 h, and increase in specific productivity + 50%

mAbY production titer (g/L)

X 2.3

Specific mAbY production (g/g total protein)

€/g

DYADIC INFORMATION 17

Saving Plant CapEx with C1 production (*)

C1 - 6 X 2000 L fermenters

Broth harvest Centrifugation/filtr ation Protein-A affinity chromatography Additional chromatography Additional Chromatography

Low feeding rate Fermentation time – 7 days Productivity – 14.0 g/L starting volume – 950 L Final volume – 1620 L Biomass – 30% Total protein per 6 runs – 95.3 KG X 2 weeks – 190.5 KG High feeding rate Fermentation time – 7 days Productivity – 16.7 g/L Working volume – 1600 L Final volume – 2720 L Biomass – 30% Total protein per 6 runs – 159.9 KG X 2 weeks – 319.8 KG

Continuous purification process Batch purification process

(*) based on 2.0 g/L/day and 30% biomass

DYADIC INFORMATION 18

Saving Plant CapEx with C1 production (*)

C1 - 6 X 2000 L fermenters

Broth harvest Centrifugation/filtr ation Protein-A affinity chromatography Additional chromatography Additional Chromatography

Low feeding rate Fermentation time – 7 days Productivity – 14.0 g/L starting volume – 950 L Final volume – 1620 L Biomass – 30% Total protein per 6 runs – 95.3 KG X 2 weeks – 190.5 KG High feeding rate Fermentation time – 7 days Productivity – 16.7 g/L Working volume – 1600 L Final volume – 2720 L Biomass – 30% Total protein per 6 runs – 159.9 KG X 2 weeks – 319.8 KG

Continuous purification process Batch purification process

(*) based on 2.0 g/L/day and 30% biomass

6 X 12,000L CHO – 288 KG 6 X 2000L CHO – 48 KG

DYADIC INFORMATION 19

Success in Expressing Certolizumab (Fab) by C1

• Successful expression of Certolizumab
• ELISA kit was used to measure and conform Certolizumab expression level (triplicates of samples were

quantified)

• The calculated expression level was 9.6 g/l, corresponding to 2.0 g/l/day production rate.
• By using promoter and strain and by further optimized fermentation process we would expect to see similar, if

not higher, expression levels as we see with our best mAb (2.4 g/l/day). Certolizumab production (g/L)

DYADIC INFORMATION 20

C1 Can Operate Successfully in Single Use Bioreactor

Single use Bioreactor Equipment: 50L XDR-50MO Single Use GE bioreactor Product protein: Certolizumab

DYADIC INFORMATION 21

Potential Benefit of Using SUB with C1 for Commercial Manufacturing

Sta inle ss Ste e l Multiuse 2 x12,000 lite r Sing le Use Biore a c tor 1X 2,000 lite r Single Use Bioreactor

• C1 can be operated in 2000L SUB for

commercial applications

• No commercial 12,000L USB is

available C1 can lower CAPEX:

• Produce at smaller scale while

dramatically increasing protein yields C1 can lower OPEX

• Smaller facility footprint and related

costs

• Low cost media

IgG1 mAb

DYADIC INFORMATION 22

Success In Fc-Fusion Expressions by C1

• Successful expression of Fc-Fusion protein
• C1 expressing Fc-Fusion was cultivated in 1 litre fermentors at 38oC and the product was analysed by Western

Blotting

• The protein A purification yield from day 6 was 8.1 g/l, corresponding to 1.35 g/l/day production rate.
• The fermentation was not fully optimized

DYADIC INFORMATION 23

Success In Bispecific Expression

In a few months work we have been able to express a bispecific antibody using C1 and provide sufficient quantities of this antibody to

• ur collaborator which they were not able to do previously using other

expression systems after two years of work.

Purified samples of bispecific protein

DYADIC INFORMATION 24

Success in Expressing High Level of ZAPI Antigen

The New strain using SES promoter system significantly increased the production and stability of the target antigen when 723 mg/L was reached in 94 hrs.

• SES construct was transformed in two 8x protease deletion strains transformants were cultivated in 24-well

MTP with the addition of protease inhibitors.

• SES clones with several fold increase in production (compared to bgl

gl) were identified

DYADIC INFORMATION 25

Ch-VLP Platform Technology Basis

VP2 protein is a structural protein of the Infectious Bursitis virus (IBDV; Gumboro) what naturally auto assemble forming Virus Like Particles

Translation Assembling process x60

VLPs VP2 protein VP2 gene

(+34) 983 54 85 63 info@bdibiotech.com

C/ Louis Proust, 13 47151 Boecillo (Valladolid) - Spain

DYADIC INFORMATION 26

Success in expressing secreted VLP by C1

VLP is expressed into DNL121 under bgl gl promoter.

• Productivity reaches 300mg/L
• Intracellular remains around 70mg/L

Extracelular-VLP Intracelular-VLP

DYADIC INFORMATION 27

Visualization of VLPs Produced by C1

Intracellular and extracellular fractions of SP-VLP have been visualized by Transmission Electronic Microscopy (TEM)

• Extracellular VLPs produced by C1 are

perfectly conformed. The structure is homogeneous in size and aspect.

• The production level of the extracellular VLP

produced by C1 was 300 mg/L.

• The production level of the intracellular

remained VLP produced by C1 was 70 mg/L

• In comparison, extracellular fraction couldn’t

be produced by S. cerevisiae.

• Intracellular VLP produced by S. cerevisiae

reached a level of 70 mg/L

VLP produced by C1 Control

Extracellular fraction Intracellular fraction

• S. c

cer erev evisiae e

300mg/L (112,5H) 70mg/L (112,5H) 70mg/L

DYADIC INFORMATION 28

Summary

Shorter development & production cycles Higher protein 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

Thank You

August 2018

Reinventing biological vaccine and drug development & production

Backup

August 2018

Reinventing biological vaccine and drug development & production

5

DYADIC INFORMATION 31

C1 Glycoengineering In Progress

Advantage of C1 over Yeast and CHO

• Dyadic’s C1’s glycan structure is more mammalian like than typical yeast
• The native C1 glycan pattern is relatively complex with high mannose type (Man3-Man9)
• O-glycosylation was not identified in therapeutic proteins expressed in C1
• Less engineering steps needed for C1
• Stable genome - defined glycan structure is stable from culture to culture and batch to batch
• The first steps of Glycoengineering C1 cells has begun and were successful
• No negative effects on cell viability have been observed with any of the modifications done

T ypic a l Ye a st Glyc a n Struc ture

Ma n30-50

Dya dic C1 Glyc a n Struc ture

Ma n3-9

T a rg e te d Ma mma lia n Glyc oform struc ture ss

G0 G0F G2 G2F

DYADIC INFORMATION 32

Glycoengineering in C1

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

C1 typical Glycan structure

• 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.

• The native C1 glycan pattern is relatively complex with high mannose type

(Man3-Man9) and hybrid type (Man3HexNac-Man8HexNac) glycan forms

• So far, O-glycosylation was not identified in therapeutic proteins expressed in

C1 but minor level is still possible

C1 future Glycostructures

• Glycoengineering work is being applied to C1 strain to create a

strain that produces proteins with defined human glycoforms

• 2

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

• About 13 steps will be applied for 1.5 – 2 years work
• The first steps of Glycoengineering C1 cells have been done

successfully.

G0 G0F G2 G2F

DYADIC INFORMATION 33

Glycoengineering C1 Strains

Improving the glycoform structure in C1 Glycoengineered strains:

• Proteodynamics (France) analyzed glycans from

native protein samples of glycoengineered C1 strains (indicated) by permethylation + MALDI-TOF analysis

• No fungal high mannose structures present
• Up to 80% of Man3 structure, the important

precursor for human glycoforms

• No negative effects on cell viability have been
• bserved with any of the modifications done

DYADIC INFORMATION 34

Roadmap for Biosimilar Development (Cetrolizumab)

Cell line develop- ment CMC develop- ment Biological activity compari- son Structural compari- son Non- clinical compari- son Clinical compari- son

CMC development and batches production (GMP for clinical assays) Optimization of process & product quality attributes profile, cell line characterization & stability, formulation & product stability testing …

Generation of producing strain USP&DSP development Analytics to compare the structural & physicochemical characteristics of product vs. commercial reference In vitro assays to compare product mechanisms of action.

• Primary: TNFα

neutralization by binding and inhibition of cell signaling for proliferation

• Secondary: activation
• f immune

responses as CDC, ADCC….

• In vitro PD studies to

compare neutralization. activity, CDC, ADCC and apoptotic effects and cross-reaction with human tissues

• In vivo PK studies to

detect products in animal serum & to measure anti-products Ab concentration

• In vivo toxicity & toxic

kinetics assays

• 1 year Phase I trial to

determine PK equivalence (mainly safety) in AS patients (250)

• 2 year Phase I pilot

study for RA patients (19)

• 1 year Phase III trial to

mainly determine therapeutic equivalence in RA patients (606)

* Based on EMA assessment report for approval

• f an Infliximab (anti-TNFα) biosimilar (2013)

DYADIC INFORMATION 35

GRAS certificate for C1

DYADIC INFORMATION 36

Saving Plant CapEx with C1 production

CHO - 6 X 12,000 L fermentors

Broth harvest Centrifugation/f iltration Protein-A affinity chromatograpy Additional polishing Additional polishing

C1 - 6 X 2000 L fermentors

Broth harvest Centrifugation/f iltration Protein-A affinity chromatograpy Additional polishing Additional polishing