producing biologics with c1...c1 – the most productive fungal expression system for biologics 4 a...
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(OTCQX: DYAI)
Producing Biologics with C1
BIO Conference 2017June 18, 2017
Safe Harbor Regarding Forward-Looking Statements
2
Certain statements contained in this presentation are forward-looking statements within themeaning of the federal securities laws. These forward-looking statements involve risks,uncertainties and other factors that could cause Dyadic’s actual results, performance orachievements to be materially different from any future results, performance or achievementsexpressed or implied by such forward-looking statements. Any forward-looking statements speakonly as of the date of this presentation and, except as required by law, Dyadic expresslydisclaims any intent or obligation to update or revise any forward-looking statements to reflectactual results, any changes in expectations or any change in events. Factors that could causeresults to differ materially are discussed in Dyadic’s publicly available filings, includinginformation set forth under the caption “Risk Factors” in our December 31, 2016 Annual Reportfiled with OTC Markets on March 15, 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.
Dyadic Overview
Revolutionary protein expression technology “C1”: based on Myceliopthora thermophila fungus
Technology covered by over 20 patent families
Listed on the stock exchange (OTCQX: DYAI), liquidity of > 50m USD (1)
Experienced management & board– 20+ Years of Experience with Fungal Production Systems– 20+ Years in Pharmaceuticals
20+ Years of Commercial Enzyme Production with C1 technology
Hyper productive strain developed: >100 g/l with ~80% purity
Approved as safe (GRAS) by FDA for food and feed applications
Produced in up to 500,000l scale tanks
Biopharmaceuticals
Strategic focus since 2016 Powerful molecular toolbox enables
expression of complex proteins Successful Proof of Concept studies
mAbsVaccinesNon-
GlycosylatedProteins
Dyadic has demonstrated the power of C1 for the expression of biologics and is now looking to establish partnerships with biopharmaceutical companies
(1) As of March 31 , 2017, including ~ $7.4 million of restricted cash held in escrow until July 2017 from the DuPont transaction.
3DYADIC INFORMATION
C1 – The Most Productive Fungal Expression System for Biologics
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A safe and reliable protein production platform, C1-derived vaccine showed no adverse clinical effects in mice5 Safety &
Reliability
Produces batches of proteins that are significantly purer than traditional production methods 12 Purity
Cuts preparation and production time in half compared to CHO3 Time Saving
Achieves much higher yields than traditional production systems in CHO, E. coli, S. cerevisiae, P. pastoris1 Yield
Grows under broader temperature & pH ranges and is easily scalable compared to CHO4 Robustness
C1 can minimize CapEx investments, production costs of biologics and overcome the limitations of traditional production systems
DYADIC INFORMATION
C1’s Unique Morphology Enables Non-Viscous Fungal Production
The low viscosity allows C1 to be used in established microbial production facilities,
requiring no additional CapEx investment
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Visc
osity
(cP
)
Protein Yield
500
400
300
200
100
100
80
60
40
20
Pro
tein
(g/l)
Viscosity
Standard Fungal Line C1
Low Viscosity, High Yield
Filamentous fungi face challenges for their use in production due to high viscosity
C1 exhibits a unique morphology resulting in low viscosity
DYADIC INFORMATION
C1 Highly Robust Production Host
40ºC37ºC-32ºC25ºC
pH range
71 14
5 8C1 CHO
Rob
ustn
ess
1 After 2 Purification Steps, 2 No purification steps 3 Optimal range 32 - 37ºC. Source: Sellick, C. et al (2009) Optimizing CHO Cell Culture Conditions. Genetic Engineering and Biotechnology News Tutorial.
CH
O3
45ºC-
25ºC
C1
Operates under a wider temperature than CHO
Operates under a wider pH range than CHO
At scales ranging from laboratory shake flasksto 20,000l tanks and above
Using defined medium.
Purit
y
C1 White Strain 2.0 2
C1 1st
Generation2E. Coli 1 C1 delivers Higher levels
of the target protein (100 g/L)
Significantly higher purity(80% purity
CHO 2
WT
LC
HC
6DYADIC INFORMATION
C1 Fermentation Process
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Seed Tank
200L – 500m3
FERMENTER
Harvest Tank
Seed flask
Inoculum
Defined medium
NH4OH for pH control
Glucose feeding
Processing & Recovery
Packaging Assembly Filling Formulation Purification
Fed-Batch technology
Fed-batch technology, simple defined media and scalable process with commercial success
DYADIC INFORMATION
Reproduction rate of cell 2x higher than for CHO Protein production rate at least 1.5 fold Higher purity of protein achieved may decrease recovery time
C1 Enables Shorter Production Cycles in Comparison to CHO
1
1
2
2
3
3
0 1 2 3 4 5
CHO
C1
Duration of Steps in Production
*Note: Protein Recovery may be faster due to higher purity of C1 production
Week 1 Week 2 Week 3 Week 4
Batch Cycle time is reduced by >50% in comparison to CHO, freeing up capacity
Production time reduced by >14 days
1: Biomass Expansion 2: Protein Production 3: Protein Recovery*
8DYADIC INFORMATION
Protein Production by C1Fermentation profile of total protein production by HC strain Vs. single proteins production by LC strain
9DYADIC INFORMATION
C1 Production strain = HC strainC1 White strain = LC strain
LC strain expressing Indigenous enzyme by licensee partner
LC strain expressing heterologous enzyme for licensee partner
(1)
(2)
(1)
(2)
0
5
10
15
20
25
30
35
40
Per.C6 CHO C1 ComplexBiologics Yield
3
10
30
9
15
< 60
0
10
20
30
40
50
60
S. cerevisiae P. pastoris C1Industrial/Simple
Protein Yield
C1 Has the Potential to Produce Significantly More Protein
Simple/Non-Glycosylated Proteins Complex Proteins, e.g. mAbs
Yiel
d in
g/l
>3 times higher yield 2 to 10 times
higher yield
Sources: 1 Boehringer Ingelheim, BioXcellence production: www.bioxcellence.com. & Shane Cox Gad (2007) Handbook of Pharmaceutical Biotechnology. Wiley Interscience, New Jersey. 2 Non-GMP conditions, non purified. 3 Susan Gotensparre (2007) Crucell. InPharmaTechnologist.com. 4 Non-GMP conditions, not purified, expected based on small scale production experience
High cell density attainable More protein produced Protein is secreted Both for small and large scale production Codon optimization established For heterologous proteins of both bacterial and mammalian origin
High productivity of C1 proven
10
1
2
34
2 3
20
KeyHighest yield claimedRealistic estimate
1
40
15
20
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C1 for Biologics
Production of heavy & light chain successful MS/MS data reveals correct structure Binding to target confirmed via ELISA
Heavy chain
Light chain
Case Study 1: Humira Production of Fab successful The structure was confirmed by MS analysis Specificity of binding confirmed via ELISA
72h 96h
Case Study 2: Lucentis
C1 has produced biologically-active monoclonal antibodies Protease deficient strains with no mAb degradation successfully generated
Successful Initial Engineering of C1 for mAb Production
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C1 Expresses mAbs: Generic Humira and Lucentis
Prod
uctiv
ity
0 20 40 60 80 100 120 140 160
Time (h)
Ranibizumab level
DYADIC INFORMATION
Proteases Deletion for Stabilizing Humira
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0 4 8 24Single deletion
0 4 8 24Triple deletion
Hrs:
ΔproA ΔproA, ΔproB, ΔproC
First stage - Three proteases deletions enabled the development of a strain in which In vitro stability of heavy chain against C1. fermentation culture filtrates could be obtained
0 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 buffer
Humira spiking
HC
LC
DYADIC INFORMATION
Successful expression of new mAbs for third party
The expression was done in the new developed C1 strain with lowproteins background
This protease deficient strain expresses stable mAbs.
Fermentation process optimization is underway to increase theproductivity further
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C1 Expresses mAb: More Success in mAb Expressions by C1
SDS-PAGE Western Blot
Con
trol
s
C1+
mAb
4
LC
HC
LC
HCm
Ab4
Mar
ker
C1
PS
C1+
mAb
4
DYADIC INFORMATION
Further Improvement of C1 Production Platform for BiologicsDyadic has experience with each of the molecular tools necessary to optimize the strain for high productivity and functionality for the targeted protein class
Genetic manipulation
Computational biology
Man9 G0 G2F
Genome sequence
Extensive genetic tools
Changing the cellular
regulatory circuit
+
Libraries of efficient strong
promoters
+/-
Libraries of TF and signal
peptides and / or carrier proteins
+/-
Libraries of protease
deletion strains
+/-
Glycoengineering to form mammalian-like
glycan structures in progress
Juststarted
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Gene 1Pr Carrier
DYADIC INFORMATION
C1 Strains DevelopmentSteps in developing better C1 strain for therapeutic protein production
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HCproduction strain
LC 0production strain
LC 1production strain
LC 103production strain
LC 104production strain
Basic therapeutic protein Productivity:
Proteolytic Activity:
0 g/L 20 g/L
High Low
Proteolytic Activity:
0 g/L 20 g/L
High Low
Proteolytic Activity:
0 g/L 20 g/L
High Low
Proteolytic Activity:
0g/L 20 g/L
High Low
Proteolytic Activity:
0 g/L 20 g/L
High Low
Basic therapeutic protein Productivity:
Basic therapeutic protein Productivity:
Basic therapeutic protein Productivity:
Basic therapeutic protein Productivity:
DYADIC INFORMATION
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C1 for Vaccines
C1 for Vaccine Production – Lower Costs & Higher Efficacy
Easy scale up, lower production costs due to higher yield (in comparison to CHO/yeast/ E. coli)
C1 produced antigen generated an equal, or better, immune response in mice than the industry standard antigen
International collaboration ongoing in vaccine development
Key Advantages
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The C1 technology platform:
A leap in technology that shows the potentialto change the way in which both Human andAnimal Health Biopharmaceutical companiesbring their biologic vaccines and drugs tomarket faster, in greater volumes, at lowercost, and with newer beneficial properties.
DYADIC INFORMATION
ZAPI, is a research and development program sponsored by the EU withthe goal of developing a platform suitable for the rapid development andproduction of vaccines and protocols to fast-track registration ofdeveloped products to combat epidemic Zoonotic diseases that have thepotential to effect the human population.
ZAPI Project goal
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ZAPI Project
Western blot
Δ Δ
72 h 96 h
WTWT Three different antigens each one for different virus were expressed by C1 and secreted to the medium
A specific protease that was responsible for cleaving one antigen was identified and knocked out
C1 antigen passed the first animal test – with more animal tests expected to be performed
An optimized fermentation process is now being developed
DYADIC INFORMATION
C1 Advantages as Production Host for Biologics
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High productivity
Low viscidity
High purity protein secretion
Defined media based on Glc
Fed batch technology no need for perfusion
5-7 days fermentation
C1 culture
Advance genetic tool box
Site specific integration Vs. random integration
DYADIC INFORMATION
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Human vaccine
Animal health
mAbs
FC-Fusions
Mimic mAbs
Hormones
(*)
(***)
(*)
(**)
(**)
(**)(*) Successful expression by C1 system(**) C1 expression in progress(***) Future plan
C1 culture
C1 – Potential Products
DYADIC INFORMATION
Summary - Key Advantages of C1
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Dyadic is looking for partners in the biopharmaceutical space to exploit the potential of C1.
For further inquiry, please [email protected]
Further benefits: Unique properties that can be
engineered for the desired product profile
A toolbox for strain engineering to optimize production of different biologics (vaccines, simple proteins, antibodies)
Short production
cycles
2
High purity of produced
protein Robust and reliable
manufacturing
3
4
First product shown to be safe in animal studies
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Highprotein yields
1
Thank You
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