Dyadic International, Inc. · NASDAQ:DYAI
Dyadic International — PEGS Lisbon Conference 2019
28 slides from Dyadic International's conference presentation, Nov 25, 2019
Slide 1: Novel Highly Productive Production System for Biotherapeutics: Filamentous Fungus C1
Novel Highly Productive Production System for Biotherapeutics: Filamentous Fungus Myceliophpthora thermophila C1 Markku Saloheimo VTT Technical Research Centre of Finland Ltd PEGS Lisbon Conference, November 21, 2019 1
Slide 2: VTT Cell Factories for Recombinant Protein Production
VTT Cell Factories for Recombinant Protein Production Host systems • Prokaryotic - Escherichia coli • Yeasts - Saccharomyces cerevisiae - Pichia pastoris Trichoderma reesei • Filamentous fungus - Trichoderma reesei Nicotiana benthamiana - Myceliophthora thermophila • Higher eukaryotes - Plants and p
Slide 3: Myceliophthora thermophila (Thermotelomyces heterothallica)
Myceliophthora thermophila (Thermotelomyces heterothallica) � Thermophilic fungus originally isolated from alkaline soil in Russia � Designated earlier as Chrysosporium lucknowense and currently as Myceliophthora thermophila � Isolated for its ability to produce neutral/alkaline cellulases for use i
Slide 4: Our Goal
Our Goal To further develop C1 into efficient gene expression system of biologic vaccines and drugs, to help speed up the development, lower production costs and improve the performance at flexible commercial scales. Efficient Expression Reducing Proteolytic Glycoengineering Activity � Library of pr
Slide 5: Efficient Expression: C1 Fermentation Technology
Efficient Expression C1 Fermentation Technology � Easily available defined media � No need for induction components – glucose, salts, micro � Protein is secreted to the media Fed-batch and macro elements, AA and vitamins � 20-30 % biomass Process � Fed-batch technology with glucose � pH: 5-8, Temp:
Slide 6: VTT Synthetic Expression System (SES)
Efficient Expression VTT Synthetic Expression System (SES) � Constitutive and extremely high-level expression permits use of any production conditions � Expression level is tunable by changing the number of sTF binding sites � Has been shown to function in multiple organisms: yeasts, filamentous fun
Slide 7: C1 Expression Technology
Efficient Expression C1 Expression Technology Different genetic modification methods can be Transformation applied: efficiency � Single site directed integration � 2 sites directed integration � Random integration � Episomal vectors – Transient expression system � Transformation procedure based on c
Slide 8: Reducing Proteolytic Activity
Reducing Proteolytic Activity Isolation and identification of extracellular proteases Protease activity assays Fermentation Culture supernatant Affinity Chromatography Analysis of fractions Spiking with target proteins Zymogram gels with target protein Protease A Protease B Protease C LC-ESI MS/MS F
Slide 9: C1 Protease Library in Pichia pastoris
Reducing Proteolytic Activity C1 Protease library in Pichia pastoris � More than 50 proteases were expressed individually in Pichia pastoris � Culture supernatants of each strain expressing a C1 protease were tested in spiking experiments Identification of critical proteases which are problematic fo
Slide 10: C1 Lineage of Protease Deficient Strains
Reducing Proteolytic Activity C1 Lineage of protease deficient strains DNL104 4xΔ Systematic deletion of protease genes based on: � Isolation and identification of extracellular proteases DNL110 5xΔ � C1 protease library in Pichia pastoris � Effect of different protease inhibitors on protease activi
Slide 11: Total Extracellular Protease Activity Greatly Reduced
Reducing Proteolytic Activity Total extracellular protease activity is greatly reduced in C1 protease deficient strains � Direct fluoresence-based assay with casein substrate � The activity in the 8xΔ strain is reduced over 50-fold compared to the 1xΔ strain (fermentation cultures) � The activity in
Slide 12: Protease Deletions Improving Stability and Production of Fc-fusion protein
Protease Deletions are Improving Stability and Production of a Fc-fusion protein 5xΔ 6xΔ 10xΔ 11xΔ 0h 4h o/n 0h 4h o/n 0h 4h o/n o/2n 0h 3h o/n o/2n Yellow full-length Target protein was spiked into the culture supernatant Red degradation product of the different protease deletion strains 21.11.2019
Slide 13: Protease deletion strains improve stability of target proteins
Reducing Proteolytic Activity Protease deletion strains are improving stability of target proteins - Difficult-to-express protein Vaccine antigen 9xΔ 10xΔ 0h 3.5h o/n o/2n 0h 3.5h o/n o/2n std Target protein spiked into the culture supernatant of the protease deletion strains 21.11.2019 VTT – beyond
Slide 14: Glycoengineering: Advantage of C1 over Yeast and CHO
Glycoengineering Advantage of C1 over Yeast and CHO � C1 glycan structure is more mammalian like than typically in yeasts � The native C1 glycans are mostly high mannose type (Man3-Man9) including some hybrid glycans � Less engineering steps needed for C1 � Stable genome - defined glycan structure i
Slide 15: Two main approaches to gain high Man3
Two main approaches to gain high Man3 � Man3 is the important precursor of G0 glycans ALG3 � Two approaches for high Deletion Man3: 1. Deletion of alg3 and over-expression of Mannosidase I 2. Deletion of alg3 and alg11 Flippase is the membrane protein enabling turning of the lipid-linked Asn Asn Ada
Slide 16: Alternative approach for G0: classical animal pathway
Alternative approach for G0: classical animal pathway The animal glycan modification pathway coud be built into C1 � Requires expression of Mannosidase I, GNT I, G0 Mannosidase II and GNT II to gain G0 glycans � Is more complicated than alg3 or alg3/alg11 approaches � Not pursued in C1 presently Mod
Slide 17: The effect of alg3 deletion in C1
The effect of alg3 deletion in C1 Wild type strain Alg3 deletion strain � The alg3 gene was deleted by replacing it with a marker gene. Glycans on native proteins were analyzed � The glycan pattern became much more simple • All higher MW glycans and hybrid glycans were omitted • Substantial amounts
Slide 18: Applying alg3-alg11 deletion strategy to produce G0 glycans
Applying alg3-alg11 deletion strategy to produce G0 glycans Alg3-alg11 deletion + GNT1+2 Alg3 deletion strain RT Glycoform Area Relative (Abundance) Abun (%) 10,48 M3 30,93 1,63 12,46 M3GlcNac 85,79 4,52 14,21 G0 1781,79 93,85 sum 1898,51 100,00 � The alg11 gene was deleted from an alg3 deletion str
Slide 19: The effect of flippase expression
The effect of flippase expression No flippase o/e Expression of flippase 1 Expression of flippase 2 RT Glycoform Area Relative RT Glycoform Area Relative RT Glycoform Area Relative (Abundance) Abun (%) (Abundance) Abun (%) (Abundance) Abun (%) 10,48 M3 69,34 7,05 10,48 M3 116,77 2,84 10,48 M3 259,86
Slide 20: Production of Biologics in C1
Production of Biologics in C1 21.11.2019 VTT – beyond the obvious 20
Slide 21: Fermentations for mAbY production
Fermentations for mAbY production Fermentation Vessel Initial (final) Antibody # volume (1) culture volume (1) titre (g/l) 15 10 8.0 (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 � SDS gel analysis of the mAbY antibody purified from the fermentations by protein A affinity chrom
Slide 22: mAbY Binding assay by Biacore T200
mAbY Binding assay by Biacore T200 Studying the interaction of mAbs in real time Ligand mAbY Immobilization: anti-Human-Fc & anti-Mouse-Fc CHO-produced � The binding properties of C1 produced mAbY were compared to the CHO produced mAbY in a Biacore T200 assay � The C1-produced mAbY and CHO- produced
Slide 23: Success in Bispecific mAb expression
Success in Bispecific mAb expression � Production level 1 g/L in a 6-day fermentation process � Difficult to express in several hosts r: reduced conditions � The function of C1-produced mAb was compared to the CHO- nr: non-reduced conditions produced control in a bioassay o C1-produced purified usin
Slide 24: Success in expressing high level of ZAPI antigen
Success in expressing high level of ZAPI antigen � Schmallenberg virus antigen coupled with Spytag - difficult-to-express protein in animal cells and microbial systems � First strain had a native C1 promoter in 6xΔ protease strain � Using synthetic promoter (SES) for expression, higher copy number a
Slide 25: Expression of Fc-Fusion by C1
Expression of Fc-Fusion by C1 Production of an Fc-fusion protein in C1 � The Fc-fusion protein was expressed with three synthetic promoters with different strengths � The strains were grown in fermentors at 1 litre scale • Growth and total protein production was similar between the strains • The Fc
Slide 26: Summary
Summary � Myceliophthora thermophila C1 is an industrialized protein Product Production level production host that is now developed for therapeutic protein g/L manufacture - with several large biopharma companies entering into collaborations. Mabs 22 � We have identified critical proteases to deal w
Slide 27: Acknowledgements
Acknowledgements VTT Protein Production Dyadic International Inc. Anne Huuskonen Ronen Tchelet Marika Vitikainen Gabor Keresztes Marilyn Wiebe Mark A. Emalfarb Georg Schmidt Veera Korja Anssi Rantasalo VTT Analytics Kari Koivuranta VTT Biosensors Heli Nygren Kristina Iljin Outi Koivistoinen Natalia
Slide 28: Thank You
THANK YOU rtchelet@dyadic.com markku.saloheimo@vtt.fi 21.11.2019 VTT – beyond the obvious 28