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

Dyadic – C1 Technology C1-Production In Single Use or Reinventing biological vaccine and drug Stainless Steel Bioreactors development & production Ronen Tchelet & Mark Emalfarb BPI, Boston. September 7 th , 2018

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 2

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 20+ Years of Commercial Enzyme Production Biopharmaceuticals  Platform optimized 2009 – 2015  Strategic focus since 2016  Hyper productive strain developed with  Powerful molecular toolbox enables expression of complex proteins purity: >100 g/l with ~80% purity  Application proven successful:  Produced in up to 500,000L tanks • Vaccines  GRAS FDA certified • Non-Glycosylated Proteins • mAbs Demonstrated the power of C1 for the production of biologics now seeking partnerships with biopharmaceutical companies (1) As of June 30, 2018 DYADI C INFORMATION 3

Dyadic Overview R&D: Helsinki BD&L: London R&D Management: Budapest R&D: Valladolid HQ: Jupiter, FL  1979 FOUNDED  20+ YEARS EXPERIENCE IN PHARMA / FUNGAL GENE EXPRESSION PLATFORMS DYADIC INFORMATION 4

How Dyadic Leverages C1 Advantages for Biologics - 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 100m 3 )- - Simple Efficient vast Fast development fermentation Success in Single Low cost of USP screening system timeline for process in use reactors & DSP for drug discovery Biologics stainless steal bioreactors Growing on 24 or 96 MTP DYADIC INFORMATION 5

C1 Expression Technology Different transformation methods can be applied: Transformation  Single site directed integration efficiency  2 sites directed integration (in progress)  Random integration  Episomal vectors  Transformation procedure based on chemical (PEG) method with protoplasts or electroporation  Frequencies for 1 μ g DNA: • 20 transformants for site specific integration • Up to 100 transformants for random integration • ~13,000 transformants for telomeric vector transformation DYADIC INFORMATION 6

MTP Screening for Drug Discovery 1+ weeks 2 weeks 2 weeks Plasmid Strain MTP screening Gene synthesis construction construction and analysis • Use of telomeric vector for drug discovery.  The synthesis of the  Cloning is done in  Protoplast  96 or 24 well plates GOI is being done by Yeast or E. coli . transformation can be used • 24 wells plates: outsourcing  Preparation of linear  Colonies appears after 4-  Source of inoculum fragments 7 days. can be either a frozen 1mg/4ml - stable  Telomeric vector  Starting re-isolation cell stock or mycelia procedure from a plate. product  Shaker incubation 4 days DYADIC INFORMATION 7

Drug Development 1+ weeks 2 weeks 2 weeks 3 weeks 1 week Plasmid Strain MTP screening 1L scale Purification and Gene synthesis construction construction and analysis fermentation analysis  The synthesis of the  Cloning is done in  Protoplast  96 or 24 well plates  Inoculum of  Protein is secreted to the GOI is being done by Yeast or E. coli . transformation can be used vegetative cells media outsourcing  Preparation of linear  Colonies appears after 4-  Source of inoculum  4-7 days process  Biomass sedimentation fragments 7 days. can be either a frozen  Fed batch technology  Protein A purification for  Vectors for site  Starting re-isolation cell stock or mycelia  Defined media mAbs directed integration procedure from a plate. without Yeast Extract  or  Removal of selection  Shaker incubation 4  Glucose feeding.  Standard purification marker for re- days  No Induction is methodology by filtration transformation 2+ weeks needed and chromatography  No need for virus clearance Removal of selection marker for re- 1L fermentor: 1–10 g stable transformation 2+ weeks product for further development DYADIC INFORMATION 8

Production of Stable Proteins C1 Lineage of Proteases Deletion Strains Under construction  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 DYADIC INFORMATION 9

Reducing the Proteolytic Activity The 10-12 X protease deletion strains, under production at optimized temp. and pH will be used to produce stable Biologics 1) Protease deletion strains 2) Wide range of Temperature 3) Wide range of pHs DYADIC INFORMATION 10

C1 Fermentation Technology  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 Fed-batch  Protein is secreted to the media Process  30-40% biomass  pH: 5-8, Temp: 25 - 42°C.  1L to 500,000L fermentation scale 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 DYADIC INFORMATION 11

Generic Process Flow Chart for C1 (12 – 14 days) Passage 1 Pre-inoculum: 1 – 1,5 days plate Mycelium activation Passage 2 Seed train: inoculum flask expansion in flak 0,75 -2,5 days N-3 ~1.6L scale N-2 ~40L scale Inoculum expansion - bioreactors N-1 ~1000L scale 5 days Production bioreactor N ~12,000L scale DYADIC INFORMATION 12

Generic Process Flow Chart for CHO (41 – 54 days) Passage 1 18-28 d Passage 2 Passage 3 Seed train: inoculum Passage 4 expansion Passage 5 Passage 6 9-12 days N-3 ~80L scale N-2 ~400L scale Inoculum expansion - bioreactors N-1 ~2,000L scale 14 days Production bioreactor N ~12,000L scale DYADIC INFORMATION 13

MAbY Expressions by C1 Fermentations carried out for mAbY Ferm entation Vessel volume Initial (final) Antibody # titre (g/l) (1) culture volume (1) production with vessel volumes, culture 8.0 15 10 8 (10.5) volumes, and antibody titres. 1 6.3 16 0.8 (1.1) 6.5 17 1 0.8 (1.1) 7.9 18 1 0.8 (1.1) 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. DYADIC INFORMATION 14

MAbY Binding Assay by Biacore T200 Studying the interaction of mAbs in real time mAbY 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  DYADIC INFORMATION 15

Media and process Development mAbY production titer (g/L) Specific mAbY production (g/g total protein) X 2.3 + 50% Medium plus feeding €/g improvement lead to a mAbY titer of 9 g/L at 90 h, and increase in specific productivity DYADIC INFORMATION 16