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1
“C1”, Speeding Up Development & Lowering The Cost of Biologics
2
“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
4
Dyadic is Developing What the Industry Refers to As a “CHO stopper”
5
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
6
BioPharma- Reporter.com
Ø Collaborations With Two Top Ten Pharma, Mitsubishi Tanabe Pharma, & Various Other Pharma and Biotech Companies.
C1 – A Powerful Scientific Anomaly
7
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
8
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
9
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
10
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
12
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
13
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.
14
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
15
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
16
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.
15 16 17 18
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
17
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
18
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
19
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
20
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
21
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
22
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
23
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
24
New C1 strains for biologics
Glycoengineering
Dyadic Board – Decades of Big Pharma Experience
25
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
26
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 [email protected]
Reinventing biological vaccine and drug development & productionDYADIC®
THANK YOU