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6-21-04 Engineering the Secretory Pathway of the Yeast P. pastoris to Produce Mammalian Glycoproteins June 21st, 2004 Huijuan Li, Ph.D. GlycoFi Inc, Lebanon,

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Presentation on theme: "6-21-04 Engineering the Secretory Pathway of the Yeast P. pastoris to Produce Mammalian Glycoproteins June 21st, 2004 Huijuan Li, Ph.D. GlycoFi Inc, Lebanon,"— Presentation transcript:

1 6-21-04 Engineering the Secretory Pathway of the Yeast P. pastoris to Produce Mammalian Glycoproteins June 21st, 2004 Huijuan Li, Ph.D. GlycoFi Inc, Lebanon, NH

2 6-21-04 Corporate Overview Founded in 2000 45 employees – 36 scientists/9 G&A $17.6M in venture capital raised to-date -- Polaris Ventures, SVLS, Boston Millennia Partners Locations: -- Lebanon, NH (HQ) -- Cambridge, MA Revenues: -- 2002: $233K -- 2003: $1.45M -- 2004: $2.75M (booked to date) -- 2005: $1.00M (booked to date)

3 6-21-04 Strain development Product development Protein characterization Protein production

4 6-21-04 Analysis Tools  PNGase F of glycosylated proteins  MALDI-TOF of N-glycans and purified proteins  HPLC analysis of N-glycan  Large scale separation of N-glycans (P4 column)  Monosaccharides compositional analysis of N-glycans (HPAEC-PAD)

5 6-21-04 Inadequate Manufacturing technology Current manufacturing technologies invariably rely on the use of plasma derived proteins and growth factors at some part of the manufacturing process (Safety?) Current manufacturing technology delivers a heterogenous mixture of ‘glycoforms’ Two major trends are driving the need for more efficient, and safer therapeutic protein production technology. A growing pipeline of protein based therapeutics: Genomics, and Proteomics approaches are fueling the discovery of new protein based therapies Novel technologies for the generation of humanized antibodies are expected to drastically increase the demand for manufacturing capacity The increased understanding of structure/function relationships of glycoproteins is leading to the development of new therapeutic entities

6 6-21-04 CHO Cell Gene for protein of interest P P Human Cell

7 6-21-04 Human Cell Gene for protein of interest Yeast P P Cell Engineering and Strain development

8 6-21-04 Human Cell Humanized Yeast Gene for protein of interest P P

9 6-21-04 Golgi cis medial trans ER Nucleus TGN

10 6-21-04  -glucosidases To Cis Golgi  -1,2-mannosidase Human Glycosylation Endoplasmic Reticulum Fungal Glycosylation

11 6-21-04 Golgi cis medial trans ER Nucleus TGN

12 6-21-04 Fungal Early Golgi Medial Golgi Golgi Human Medial Golgi Late Golgi Early Golgi Late Golgi

13 6-21-04 ‘Humanized’ Yeast Golgi OCH1 MNN1 Human Medial Golgi Late Golgi Early Golgi Fungal Early Golgi Medial Golgi Late Golgi

14 6-21-04 Mass (m/z) % Intensity Man 9+Na Man 10+Na Man11+Na Man 12+Na IFN-  /P.pastoris NRRL11430

15 6-21-04 IFN-  /P.pastoris,  OCH1 Man 8+Na Man 9+Na Man 10+Na Man 11+Na % Intensity Mass (m/z)

16 6-21-04 Yeast Golgi Human Humanized Yeast  -1,2-mannosidase OCH1 MNN1 Medial Golgi Late Golgi Early Golgi

17 6-21-04 Golgi cis medial trans ER Nucleus TGN

18 6-21-04 Golgi cis medial trans ER Nucleus TGN

19 6-21-04 Golgi cis medial trans ER Nucleus TGN

20 6-21-04 Golgi cis medial trans ER Nucleus TGN

21 6-21-04 Golgi cis medial trans ER Nucleus TGN

22 6-21-04 Golgi cis medial trans ER Nucleus TGN

23 6-21-04 Leader sequences Schematic diagram of a typical type II membrane protein

24 6-21-04 Library of 66 secretory targeting sequences for P.pastoris ER Early Golgi Medial Golgi Late Golgi All leaders are sequence verified and ‘ligation-ready’ in 96-well plates

25 6-21-04 Schematic diagram of a typical type II membrane protein Catalytic domains (e.g. mannosidases) Fungi C.elegans Drosophila Xenopus H.sapiens Murine R.norvegicus A.thaliana

26 6-21-04 Leader sequences Catalytic domains (human/murine/fungal/flies/worms/plants etc) Construction of Fusion-library Chimeric fusions Composition of ER-targeted Mannosidase Library for Pichia pastoris has over 1,300 DNA constructs

27 6-21-04 Grow colonies in 96 well format Induce secretion of IFN-  Centrifuge and collect supernatant Purify IFN-  by DEAE and C-18 chromatograpyhy / Ni affinity Transform IFN-  expressing strain of P.pastoris,  OCH1 with library and pick colonies Screening for transformants that perform desired N-glycosylation MALDI -TOF Cleave N-glycan with PNGase, purify and analyze

28 6-21-04 IFN-  /P.pastoris,  OCH1 Man 8 Man 10 Man 11 Mass (m/z) Man 9

29 6-21-04 N-glycans of IFN-  expressed in a  OCH1 mutant of P.pastoris which has been transformed with a mannosidas/leader fusion library (4 constructs out of >1200) SHxx Man8 + Na SHxy Mass (m/z) SHyx Man8 + Na Mass (m/z) Man5 + Na SHyy

30 6-21-04 Man5 GFI 2.0

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32 Golgi Human Medial Golgi Early Golgi UDP-GlcNAc Transporter

33 6-21-04 Milestone III N-Acetylglucosamine transferase I (GnTI) library GnTI cDNAs from: H.sapiens C. elegans Xenopu Drosophila melongaster Nicotiana tabacum 13 catalytic domains and deletions have been fused to the leader library Size of library: >700 GnTI/leader fusions

34 6-21-04 Man5 In vivo UDP-GlcNAc Transporter + GnTI In vitro Hexosaminidase digest Man5GlcNAc Choi et al,. 2003

35 6-21-04 Human Medial Golgi Early Golgi

36 6-21-04 Man 3 GlcNAc 2 Hamilton et al., 2003

37 6-21-04 Medial Golgi Late Golgi UDP-Gal Transporter Golgi Human

38 6-21-04 √ GlcNAc 2 Man 3  och1,  mnn1 Man 5 GlcNAcMan 5 Gal 2 GlcNAc 2 Man 3 NANA 2 Gal 2 GlcNAc 2 Man 3 GFI 3.0, Choi et al 2003 GFI 4.0, Hamilton et al 2003 GFI 2.0 √ √ √ GFI 5.0, Davidson et al 2004 √

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40 In vivo Activity of rhErythropoeitin Obtained from different sources 0 20000 40000 60000 80000 100000 120000 140000 160000 0123456789 Sample in vivo 500% Yuen, C.T. et al., 2003

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44 GFl 1.0 GlycoFi’s Humanized Yeast Strains GFl 2.0GFl 3.0GFl 4.0GFl 5.0GFl 6.0 GFl 1.1GFl 2.1GFl 3.1GFl 4.1GFl 5.1GFl 6.1 GFl 3.2GFl 4.2GFl 5.2 GFl 6.2 GFl 3.3 GFl 4.3 GFl 5.3GFl 6.3 GFl 4.4GFl 5.4GFl 6.4 GFl 1.2 GFl 2.2 GFl 2.3

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46 Fc Fusion 2.6g/l in a 3 day fermentation process

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48 GFl 1.0 GlycoFi’s Humanized Yeast Strains GFl 2.0GFl 3.0GFl 4.0GFl 5.0GFl 6.0 GFl 1.1GFl 2.1GFl 3.1GFl 4.1GFl 5.1GFl 6.1 GFl 3.2GFl 4.2GFl 5.2 GFl 6.2 GFl 3.3 GFl 4.3 GFl 5.3GFl 6.3 GFl 4.4GFl 5.4GFl 6.4 GFl 1.2 GFl 2.2 GFl 2.3

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50 Thank you! Tillman Gerngross, PhD Stefan Wildt, Ph.D. Juergen Nett, Ph.D. Piotr Bobrovicz, Ph.D Beata Bobrovicz, M.S. Stephen Hamilton, Ph.D. Robert C. Davidson, Ph.D. Andy Stadheim, Ph.D. Bianka Prinz, Ph.D Harry Wischnewski Eduard Renfer Nikolei Hodel Alissa Thompson Leah O’Rouke Robert Miele, Ph.D. Warren Kett, Ph.D. Theresa Mitchell, M.S. Martha Archambeault Heather Lynaugh Byung-Kwon Choi, Ph.D. Dongxing Zha, Ph.D. Jim Cook, Ph.D. Angela Kull, M.S. Dunja Wildt-Perinic, M.S Natarajan Sethuraman, Ph.D. Sandra Rios, Ph.D. Thomas Potgieter, Ph.D. Erin Ciaccone Nathan Sharkey Nam Kim Adam Nylen Jolene Provencher Support: NIH NIST


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