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Dyadic technology oct 2018
1. Dyadic – C1 Technology
October, 2018
Reinventing biological
vaccine and drug
development &
production
2. 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.
4. DYADIC INFORMATION 4
Commercial Success in Industrial Biotech > $110 Million
Hyper productive C1 gene expression platform developed
Enzyme expression levels achieved >100 g/l with ~80% purity
Approved as safe (GRAS) by FDA for food and feed applications
C1 enzymes produced in up to 500,000 liter scale tanks
Industrial Enzymes sold to customers in 35 countries
C1 Related License Deals, Milestones & Equity in excess of $35 million
Dyadic sold its Industrial Technology business
to DuPont’s Industrial Biosciences business (“DuPont”) for
$75 million in cash
12/31/2015
5. DYADIC INFORMATION 5
Dyadic Board – Decades of Big Pharma Experience
Arindam Bose
Dr. Bose worked at Pfizer for 34 years
and held leadership roles within
bioprocess development and clinical
manufacturing and is widely
recognized as a Key Thought Leader
in the biopharmaceutical industry.
Barry Buckland
Dr. Buckland worked at Merck for 29 years
where he served in a number of senior R&D
leadership roles focusing on fermentation and
bioprocess development and the commercial
manufacturing of biologics and is widely
recognized as a Key Thought Leader in the
biopharmaceutical industry. Currently, Dr.
Buckland is the Executive Director, NIIMBL
(National Institute for Innovation in
Manufacturing Biopharmaceuticals) A public-
private consortium dedicated to advancing
biopharmaceutical manufacturing innovation.
Michael P. Tarnok
Mr. Tarnok (Dyadic chairman) spent
the majority of his career at Pfizer and
is a seasoned finance and operational
executive with extensive experience in
the pharmaceutical industry. Currently
also serves on the Board of the Global
Health Council, and Ionetix, Inc. Prior
Board service includes Keryx
Biopharmaceuticals, Inc., where he also
served as Chairman of the Board.
Vice President, Biotherapeutics
Pharmaceutical Sciences, External
Affairs and Biosimilar Strategy
Vice President, Bioprocess
R&D, Merck Research
Laboratories
Senior Vice President in
Pfizer’s US Pharmaceutical
Division
EXPERIENCELASTPOSITION
6. DYADIC INFORMATION 6
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
7. DYADIC INFORMATION 7
On-going Research Programs and Collaborations
Mitsubishi Tanabe
Pharma
To help Mitsubishi Tanabe
overcome specific gene
expression challenges of two
important therapeutic
compounds using C1
technology.
Israel Institute for Biological
Research (IIBR)
To further advance C1 for the
development and manufacture of
recombinant vaccines and
neutralizing agents comprising
targeted antigens and
monoclonal antibodies to combat
emerging disease and threats.
Other funded proof of concept
research collaborations
To explore the potential of C1
technology to produce active
moiety.
To test the feasibility of C1
technology to produce seven
different molecular biology
enzymes for pharmaceutical use.
Sanofi-Aventis
Fully funded collaboration with
Sanofi-Aventis to explore the
potential of C1 to produce
multiple classes of biologic
vaccines & drugs
8. DYADIC INFORMATION 8
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)-
9. 9
C1 Strain Development for Therapeutic Protein Production
LC strain
Low background
High proteolytic
HC strain
High Background
High proteolytic
0.1 g/L
1.0 g/L
2.0 g/L
10 g/L
15 g/L
? g/L
DNL103 - DNL115
Lower proteolytic
background
DNL120 -
Lower proteolytic
background
2016 2017 2018 2019 2020
DNL ?
Very Low proteolytic
background
(80 g/l enzyme for Bioindustrials application)
(120 g/l cellulosic enzyme for Biofuel)
New C1 strains
for biologics
Glycoengineering
DYADIC INFORMATION
10. DYADIC INFORMATION 10
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
11. 11
The Protease Expression Library
C1 proteases were identified and expressed individually in P. pastoris as secreted proteins
The proteases were identified by RNASeq, Zymogram and genome analysis.
Example: identification of proteases that affecting the stability of a specific protein: (e.g., an Antigen)
• Purified Antigen (produced in C1) was spiked into culture supernatants of the protease library and
degradation was analyzed after 3h incubation (only half of the selected proteases are presented)
• The proteases that were found to be involved in Antigen degradation were subjected to the
protease deletion strain process.
Conrol
Standard
384
393
390
393
401
410
413
416
417
426
430
436
388
398
381
N
start
Prot.A
Prot.B
100
75
50
37
25
20
100
75
50
37
25
20
Prot.C
Prot.D
Prot.G
Prot.E
Prot.F
Prot.H
Example of the identification of several
active proteases by the C1 expression library
DYADIC INFORMATION
12. DYADIC INFORMATION 12
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
13. DYADIC INFORMATION 13
Glycoengineering in C1
The goals for C1 glycoengineering
1. High levels of G0 glycans
2. Adding fucosylation: high FG0
3. Adding galactosylation: high G2 and FG2
4. Good N-glycosylation site occupancy on therapeutic proteins (Mabs)
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
14. 14
Applying alg3 deletion pathway
We have deleted the C1 Δalg3 gene and over-expressed a native or a
heterologous Mannosidase I from alg3 locus
With Δalg3 deletion only the Man3 level is about 50%
With both modifications Man3 level rises up to 80%
All fungal type high mannose and hybrid glycans are omitted
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
DYADIC INFORMATION
15. 15
Near future goals in glycoengineering
50% G0 levels by the end of 2018
80% G0 levels by end of Q2 2019
80% FG0 levels by September 2019
G0 G0FM3
DYADIC INFORMATION
16. DYADIC INFORMATION 16
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
17. DYADIC INFORMATION 17
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
18. DYADIC INFORMATION 18
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
Ligand
19. DYADIC INFORMATION 19
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
20. DYADIC INFORMATION 20
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 12.0 g/l, corresponding to 2.6 g/l/day production rate.
By further optimization of the fermentation process up to 7 days we would expect to see even higher
expression levels.
7 days = 18 g/L
2 X 7 days = 36 g/L
Certolizumab production (g/L)
21. DYADIC INFORMATION 21
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
22. 22
Protease deficient strains 9Δ-10Δ: - spiking with Fc-Fusion (*)
24-well cultivation of protease deficient strains in standard production medium at 35°C, day 4 supernatants
Fc-Fusion reference, ProA purified, 25ng/µl in spiking reactions at 35°C
Time points 0h, 1h, 3h, o/n and o/2n
Detection with Fc-antibody (red) and Fused protein antibody (green) -> combined signal is yellow
Fc-Fusion stays intact in all supernatants, especially in the one from 10Δ Prot. D strain
Different FC-Fusion molecule than in slide 28
DYADIC CONFIDENTIAL INFORMATION
23. 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
our collaborator which they were not able to do previously using other
expression systems after two years of work.
Purified samples of bispecific protein
24. 24
Blue – C1-produced bispecific
Red – CHO-produced bispecific
Bispecific produced in C1 and CHO have identical/similar activity
• C1- and CHO-produced bispecifics were assayed in an in vitro cellular activity assay
• The dose response curves are very similar
• No negative effects of the C1-derived potential impurities in cellular assays
DYADIC CONFIDENTIAL INFORMATION
25. 25
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
DYADIC INFORMATION
26. DYADIC INFORMATION 26
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
27. DYADIC INFORMATION 27
Success in expressing secreted VLP by C1
VLP is expressed into DNL121 under bgl promoter.
Productivity reaches 300mg/L
Intracellular remains around 70mg/L
Extracelular-VLP
Intracelular-VLP
28. DYADIC INFORMATION 28
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. cerevisiae
300mg/L (112,5H) 70mg/L (112,5H) 70mg/L
29. DYADIC INFORMATION 29
The Expression of Recombinant HAs by C1
Expression and performance of 5 different HA’s
Influenza strain Expression Functional HA
New Caledonia, A (H1N1) Yes Yes
Texas, A (H1N1) Yes Yes
Puerto Rico A (H1N1) Yes Yes
California, A (H1N1) Yes Yes
Florida B Yes Yes
Agglutination test• Expressed as a membrane-bound trimer
• Titers variable
30. DYADIC INFORMATION 30
Immunogenicity Study of HA/NC Produced by C1
HA/New Caledonia
The full length recombinant HA
produced in C1 did not induce any
negative clinical signs in mice.
• No weight loss.
• No negative clinical signs during
the experiment (visual
observations taken each day).
The full length of HA/New
Caledonia produced in C1
showed excellent immunogenic
properties in mice better than
the control of HA/New
Caledonia produced by
Baculovirus.
C1 can potentially produce levels of 1 g/L of
HAs and other antigens in 5 days fermentation
therefore:
• In seasonal Influenza Vaccine—total doses
distributed = 146M/year
• Each 0.5 mL dose is formulated to contain: 15
µg of HA for each strain.
• Thus, 3 X 1000L scale fermentation runs will be
able to supply the annual global HA/strain
needs against Influenza of 2,175 g.
31. DYADIC INFORMATION 31
Metabolic modeling and Proteomic Analysis for Next
Stage of Strain Engineering
Based on C1 genomic sequence, CoReCo platform for metabolic model reconstruction was
used to build a genome-scale metabolic model of C1 for further strain development work
CoReCo:
Esa Pitkänen et al. 2014 Plos Comput Biol.
Sandra Castillo et al. 2016 Biotechnol Biofuels.
32. DYADIC INFORMATION 32
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