dyadic – c1 technology c1-production in single use or … · 2018-10-01 · dyadic expressly...

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 EmalfarbBPI, 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 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 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


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)-


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


MTP Screening for Drug Discovery

Plasmid construction

Strain construction

MTP screening and analysis

Gene synthesis

The synthesis of the GOI is being done by outsourcing

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


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 outsourcing

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


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


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


C1 Fermentation Technology

Fed-batch Process

From MTP to Large scale mAbs productivity

24 wells MTP – 1mg/4ml1L fermentor – 1.7/g/l/d30L 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


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

day

s5

days

Passage 2 flask

Passage 1 plate


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 3Passage 4

Passage 5

Passage 6

18-2

8 d

9-12

day

s14

day

s


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


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


Media and process Development

Medium plus feeding improvement lead to a mAbYtiter 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


Saving Plant CapEx with C1 production (*)C1 - 6 X 2000 L fermenters

Broth harvestCentrifugation/filtr

ation

Protein-A affinity chromatography

Additional chromatography

Additional Chromatography

Low feeding rateFermentation time – 7 daysProductivity – 14.0 g/Lstarting volume – 950 LFinal volume – 1620 LBiomass – 30%Total protein per 6 runs – 95.3 KGX 2 weeks – 190.5 KG

High feeding rateFermentation time – 7 daysProductivity – 16.7 g/LWorking volume – 1600 LFinal volume – 2720 LBiomass – 30%Total protein per 6 runs – 159.9 KGX 2 weeks – 319.8 KG

Continuous purification

process

Batch purification

process

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


Saving Plant CapEx with C1 production (*)C1 - 6 X 2000 L fermenters

Broth harvestCentrifugation/filtr

ation

Protein-A affinity chromatography

Additional chromatography

Additional Chromatography

Low feeding rateFermentation time – 7 daysProductivity – 14.0 g/Lstarting volume – 950 LFinal volume – 1620 LBiomass – 30%Total protein per 6 runs – 95.3 KGX 2 weeks – 190.5 KG

High feeding rateFermentation time – 7 daysProductivity – 16.7 g/LWorking volume – 1600 LFinal volume – 2720 LBiomass – 30%Total protein per 6 runs – 159.9 KGX 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 KG6 X 2000L CHO – 48 KG


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)


C1 Can Operate Successfully in Single Use Bioreactor

Single use Bioreactor

Equipment: 50L XDR-50MO Single Use GE bioreactor

Product protein: Certolizumab


Potential Benefit of Using SUB with C1 for Commercial Manufacturing

Stainless Steel Multiuse2 x12,000 liter

Single Use Bioreactor1X 2,000 liter

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 yieldsC1 can lower OPEX Smaller facility footprint and related

costs Low cost media

IgG1 mAb


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


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

our collaborator which they were not able to do previously using other

expression systems after two years of work.

Purified samples of bispecific protein


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) were identified


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

VP2protein

VP2 gene

(+34) 983 54 85 63

[email protected]

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


Success in expressing secreted VLP by C1VLP is expressed into DNL121 under bgl promoter. Productivity reaches 300mg/L

Intracellular remains around 70mg/L

Extracelular-VLP

Intracelular-VLP


Visualization of VLPs Produced by C1Intracellular 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. cerevisiaereached a level of 70 mg/L

VLP produced by C1 Control

Extracellular fraction

Intracellular fraction

S. cerevisiae

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


Summary

Shorter development & production cycles

Higher protein yieldsLower 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 [email protected]

Thank You

August 2018


production

Backup

August 2018


production

5


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

Typical Yeast Glycan Structure

Man30-50

Dyadic C1 Glycan Structure

Man3-9

Targeted Mammalian Glycoform structuress

G0 G0F G2 G2F


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-typestructure.

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 inC1 but minor level is still possible

C1 future Glycostructures

Glycoengineering work is being applied to C1 strain to create astrain that produces proteins with defined human glycoforms

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

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

The first steps of Glycoengineering C1 cells have been donesuccessfully.

G0 G0F G2 G2F


Glycoengineering C1 StrainsImproving 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

observed with any of the modifications done


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: TNFαneutralization by binding and inhibition of cell signaling for proliferation

Secondary: activation of 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

of an Infliximab (anti-TNFα) biosimilar (2013)


GRAS certificate for C1


Saving Plant CapEx with C1 production

CHO - 6 X 12,000 L fermentors

Broth harvestCentrifugation/f

iltration

Protein-A affinity

chromatograpy

Additional polishing


C1 - 6 X 2000 L fermentors

Broth harvestCentrifugation/f

iltration

Protein-A affinity

chromatograpy



dyadic – c1 technology c1-production in single use or … · 2018-10-01 · dyadic expressly...

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