Dyadic International, Inc. · NASDAQ:DYAI
Dyadic International — Technical Presentation (February 2020)
18 slides from Dyadic International's technical presentation, Feb 24, 2020
Slide 1: Development of Myceliophthora thermophila C1 into a next-generation therapeutic protein production system
Development of the filamentous fungus Myceliophthora thermophila C1 into a next-generation therapeutic protein production system Anne Huuskonen VTT Technical Research Centre of Finland Ltd
Slide 2: Myceliophthora thermophila (Thermothelomyces heterothallica)
Myceliophthora thermophila (Thermothelomyces 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
Slide 3: 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 4: C1 lineage of protease deficient strains
C1 lineage of protease deficient strains DNL104 4xΔ Systematic deletion of protease genes based on: � Isolation and identification of extracellular proteases � C1 protease library in Pichia pastoris DNL110 5xΔ DNL150 13xΔ � More than 50 proteases � Effect of different protease inhibitors on protease
Slide 5: Total extracellular protease activity is greatly reduced in C1 protease deficient strains
Total extracellular protease activity is greatly reduced in C1 protease deficient strains � Direct fluoresence-based assay with casein substrate � The activity of the 12xΔ strain is reduced over 50-fold compared to the 1xΔ strain (fermentation cultures) � The activity of the 13xΔ strain is reduced o
Slide 6: Protease deletions are improving stability of target proteins
Protease deletions are improving stability of target proteins Target proteins were spiked into the culture supernatants of the different protease deletion strains ΔprotH-2 ΔprotI-9 ΔprotI-10 ΔprotI-11 ΔprotJ-23 ΔprotJ-16 std Mw 0h 3h o/n o/2n 0h 3h o/n o/2n 0h 3h o/n o/2n 0h 3h o/n o/2n 0h 3h o/n o/
Slide 7: Production of Biologics in C1 - Success in expressing high level of ZAPI antigen
Production of Biologics in C1 - 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
Slide 8: Advantage of C1 over Yeast and CHO
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 is stable from cul
Slide 9: Two main approaches to gain high Man3
Two main approaches to gain high Man3 � Man3 is the important precursor of G0 glycans � Two approaches for high Man3: � Deletion of alg3 and over-expression of Mannosidase I � Deletion of alg3 and alg11 Asn Asn Man3 G0 Adapted from Stanley et al., 2008, N- glycans. In: Essentials in Glycobiology. Va
Slide 10: Two main approaches to gain high Man3
Two main approaches to gain high Man3 � Man3 is the important precursor of G0 glycans � Two approaches for high Man3: � Deletion of alg3 and over-expression of Mannosidase I ALG3 Deletion � Deletion of alg3 and alg11 Asn Asn Man3 G0 Adapted from Stanley et al., 2008, N- glycans. In: Essentials in Gl
Slide 11: Two main approaches to gain high Man3
Two main approaches to gain high Man3 � Man3 is the important precursor of G0 glycans � Two approaches for high Man3: � Deletion of alg3 and over-expression of Mannosidase I ALG3 Deletion � Deletion of alg3 and alg11 Asn Asn Man3 G0 Adapted from Stanley et al., 2008, N- glycans. In: Essentials in Gl
Slide 12: The effect of alg3 deletion in C1
The effect of alg3 deletion in C1 Wild type strain alg3 deletion strain alg3 deletion strain - native proteins – native proteins – Nivolumab � The alg3 gene was deleted by replacing it with a marker gene � Glycans on native proteins and on purified Nivolumab from shake flask cultures were analyzed �
Slide 13: Applying alg3-alg11 deletion strategy to produce G0 glycans
Applying alg3-alg11 deletion strategy to produce G0 glycans alg3 deletion strain alg3-alg11 deletion with GNT1+2 alg3 deletion strain alg3-alg11 deletion with GNT1+2 – native proteins - native proteins – Nivolumab - Nivolumab � The alg11 gene was deleted from an alg3 deletion strain. Simultaneously
Slide 14: The effect of flippase expression
The effect of flippase expression No flippase Expression of Expression of overexpression flippase 1 flippase 2 Glycan Area Relative Glycan Area Relative Glycan Area Relative (Abundance) amount (Abundance) amount (Abundance) amount M3 69.34 7.05% M3 116.77 2.84% M3 259.86 3.00% G0 914.85 92.95% M3-Gl
Slide 15: Towards G2 glycans on target proteins
Towards G2 glycans on target proteins alg3-alg11 deletion with GNT1+2 G0 strain with GalT - native proteins - native proteins Glycan Relative amount M3 3.16% M3-GlcNAc 0.42% G0 5.81% G1 6.97% G1 5.53% G2 76.38% � The human GalT1 gene was expressed in a strain with G0 glycan machinery � Glycans on na
Slide 16: Summary
Summary � Myceliophthora thermophila C1 is an industrialized protein Product Production level production host that is now developed for therapeutic g/L protein manufacture – with several large biopharma companies entering into collaborations. Mabs 24.5 � We have identified critical proteases to deal
Slide 17: Acknowledgements
Acknowledgements VTT Protein Production Dyadic International Inc. Anne Huuskonen Ronen Tchelet Marika Vitikainen Gabor Keresztes Georg Schmidt Mark A. Emalfarb Marilyn Wiebe Veera Korja Anssi Rantasalo VTT Analytics VTT Biosensors Andriy Kovalchuk Heli Nygren Kristina Iljin Kari Koivuranta Natalia M
Slide 18: THANK YOU!
THANK YOU! rtchelet@dyadic.com anne.huuskonen@vtt.fi
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