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

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)

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Strain

development

Product development

Protein

characterization

Protein

production

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Analysis Tools

PNGase F of glycosylated proteinsMALDI-TOF of N-glycans

and purified proteinsHPLC analysis of N-glycanLarge scale separation of N-glycans

(P4 column)Monosaccharides compositional

analysis of N-glycans (HPAEC-PAD)

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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

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CHO Cell

Gene for protein of interest

P

P

Human Cell

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Human Cell

Gene for protein of interest

Yeast

P

PCell Engineering

and Strain development

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Human Cell

HumanizedYeast

Gene for protein of interestP

P

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Golgicis

medialtrans

ER

Nucleus

TGN

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-glucosidases-glucosidases

To Cis Golgi To Cis Golgi

-1,2-mannosidase

-1,2-mannosidase

Human Glycosylation

Endoplasmic ReticulumFungal Glycosylation

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Golgicis

medialtrans

ER

Nucleus

TGN

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Fungal

EarlyGolgi

Medial Golgi

GolgiHuman

Medial Golgi

Late Golgi

EarlyGolgi

Late Golgi

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‘Humanized’ Yeast

Golgi

OCH1 MNN1

Human

Medial Golgi

Late Golgi

EarlyGolgi

Fungal

EarlyGolgi

Medial Golgi

Late Golgi

6-21-04 Mass (m/z)

% I

nten

sity

Man 9+Na

Man 10+Na

Man11+Na

Man 12+Na

IFN-/P.pastoris NRRL11430

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IFN-/P.pastoris , OCH1

Man 8+Na

Man 9+Na

Man 10+Na

Man 11+Na

% I

nten

sity

Mass (m/z)

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Yeast

GolgiHuman Humanized Yeast

-1,2-mannosidase

OCH1 MNN1

Medial Golgi

Late Golgi

EarlyGolgi

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Golgicis

medialtrans

ER

Nucleus

TGN

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Golgicis

medialtrans

ER

Nucleus

TGN

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Golgicis

medialtrans

ER

Nucleus

TGN

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Golgicis

medialtrans

ER

Nucleus

TGN

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Golgicis

medialtrans

ER

Nucleus

TGN

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Golgicis

medialtrans

ER

Nucleus

TGN

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Leader sequences

Schematic diagram of a typical type II membrane protein

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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

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Schematic diagram of a typical type II membrane protein

Catalytic domains (e.g. mannosidases)

FungiC.elegansDrosophilaXenopusH.sapiensMurineR.norvegicusA.thaliana

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pVM 1(2764 bp)

Ampicillin

ColE1 Ori

Lac Z

Poly linker

Not I

Asc I

2764/ 1

pVM 2(2781 bp)

Ampicillin

Lac Z

Poly linker

ColE1 Ori

Not I

Asc I

Pac I

2781/ 1

Leader sequences

Catalytic domains (human/murine/fungal/flies/worms/plants etc)

Construction of Fusion-library

Chimeric fusions

pVM 2(2781 bp)

Ampicillin

Lac Z

Poly linker

ColE1 Ori

Not I

Asc I

Pac I

2781/ 1

Composition of ER-targeted Mannosidase Library for Pichia pastoris has over

1,300 DNA constructs

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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

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IFN-/P.pastoris , OCH1

Man 8

Man 10

Man 11

Mass (m/z)

Man 9

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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)

SHxxMan8 + Na

Man8 + Na SHxy

Mass (m/z)

SHyx

Man8 + Na

Mass (m/z)

Man5 + Na SHyy

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Man5

GFI 2.0

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GolgiHuman

Medial Golgi

EarlyGolgi

UDP-GlcNAc Transporter

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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

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Man5

In vivoUDP-GlcNAc Transporter

+ GnTI

In vitroHexosaminidase

digest

Man5GlcNAc

Choi et al,. 2003

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Human

Medial Golgi

EarlyGolgi

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Man3GlcNAc2

Hamilton et al., 2003

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Medial Golgi

Late Golgi UDP-Gal Transporter

GolgiHuman

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√

GlcNAc2Man3

och1, mnn1

Man5

GlcNAcMan5

Gal2GlcNAc2Man3

NANA2Gal2GlcNAc2Man3

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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In vivo Activity of rhErythropoieitin

0

20000

40000

60000

80000

100000

120000

140000

160000

0 1 2 3 4 5 6 7 8 9

Sample

in v

ivo

In vivo Activity of rhErythropoeitinObtained from different sources

0

20000

40000

60000

80000

100000

120000

140000

160000

0 1 2 3 4 5 6 7 8 9

Sample

in v

ivo

500%

Yuen, C.T. et al., 2003

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GFl 1.0

GlycoFi’s Humanized Yeast Strains

GFl 2.0 GFl 3.0 GFl 4.0 GFl 5.0 GFl 6.0

GFl 1.1 GFl 2.1 GFl 3.1 GFl 4.1 GFl 5.1 GFl 6.1

GFl 3.2 GFl 4.2 GFl 5.2GFl 6.2

GFl 3.3 GFl 4.3 GFl 5.3 GFl 6.3

GFl 4.4 GFl 5.4 GFl 6.4

GFl 1.2GFl 2.2

GFl 2.3

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6-21-04 Fc Fusion 2.6g/l in a 3 day fermentation process

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GFl 1.0

GlycoFi’s Humanized Yeast Strains

GFl 2.0 GFl 3.0 GFl 4.0 GFl 5.0 GFl 6.0

GFl 1.1 GFl 2.1 GFl 3.1 GFl 4.1 GFl 5.1 GFl 6.1

GFl 3.2 GFl 4.2 GFl 5.2GFl 6.2

GFl 3.3 GFl 4.3 GFl 5.3 GFl 6.3

GFl 4.4 GFl 5.4 GFl 6.4

GFl 1.2GFl 2.2

GFl 2.3

6-21-04

6-21-04 Thank you!

Tillman Gerngross, PhDStefan Wildt, Ph.D.Juergen Nett, Ph.D.Piotr Bobrovicz, Ph.DBeata Bobrovicz, M.S.Stephen Hamilton, Ph.D.Robert C. Davidson, Ph.D.Andy Stadheim, Ph.D.Bianka Prinz, Ph.DHarry WischnewskiEduard RenferNikolei HodelAlissa ThompsonLeah O’Rouke

Robert Miele, Ph.D.Warren Kett, Ph.D.Theresa Mitchell, M.S.Martha ArchambeaultHeather Lynaugh

Byung-Kwon Choi, Ph.D.Dongxing Zha, Ph.D.Jim Cook, Ph.D.Angela Kull, M.S.Dunja Wildt-Perinic, M.SNatarajan Sethuraman, Ph.D.Sandra Rios, Ph.D. Thomas Potgieter, Ph.D.Erin CiacconeNathan SharkeyNam KimAdam NylenJolene Provencher

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