6-21-04 Engineering the Secretory Pathway of the Yeast P. pastoris to Produce Mammalian...
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Transcript of 6-21-04 Engineering the Secretory Pathway of the Yeast P. pastoris to Produce Mammalian...
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)
6-21-04
Strain
development
Product development
Protein
characterization
Protein
production
6-21-04
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)
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
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CHO Cell
Gene for protein of interest
P
P
Human Cell
6-21-04
Human Cell
Gene for protein of interest
Yeast
P
PCell Engineering
and Strain development
6-21-04
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
6-21-04
Fungal
EarlyGolgi
Medial Golgi
GolgiHuman
Medial Golgi
Late Golgi
EarlyGolgi
Late Golgi
6-21-04
‘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
6-21-04
IFN-/P.pastoris , OCH1
Man 8+Na
Man 9+Na
Man 10+Na
Man 11+Na
% I
nten
sity
Mass (m/z)
6-21-04
Yeast
GolgiHuman Humanized Yeast
-1,2-mannosidase
OCH1 MNN1
Medial Golgi
Late Golgi
EarlyGolgi
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Golgicis
medialtrans
ER
Nucleus
TGN
6-21-04
Golgicis
medialtrans
ER
Nucleus
TGN
6-21-04
Golgicis
medialtrans
ER
Nucleus
TGN
6-21-04
Golgicis
medialtrans
ER
Nucleus
TGN
6-21-04
Golgicis
medialtrans
ER
Nucleus
TGN
6-21-04
Golgicis
medialtrans
ER
Nucleus
TGN
6-21-04
Leader sequences
Schematic diagram of a typical type II membrane protein
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
6-21-04
Schematic diagram of a typical type II membrane protein
Catalytic domains (e.g. mannosidases)
FungiC.elegansDrosophilaXenopusH.sapiensMurineR.norvegicusA.thaliana
6-21-04
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
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
6-21-04
IFN-/P.pastoris , OCH1
Man 8
Man 10
Man 11
Mass (m/z)
Man 9
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)
SHxxMan8 + Na
Man8 + Na SHxy
Mass (m/z)
SHyx
Man8 + Na
Mass (m/z)
Man5 + Na SHyy
6-21-04
Man5
GFI 2.0
6-21-04
6-21-04
GolgiHuman
Medial Golgi
EarlyGolgi
UDP-GlcNAc Transporter
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
6-21-04
Man5
In vivoUDP-GlcNAc Transporter
+ GnTI
In vitroHexosaminidase
digest
Man5GlcNAc
Choi et al,. 2003
6-21-04
Human
Medial Golgi
EarlyGolgi
6-21-04
Man3GlcNAc2
Hamilton et al., 2003
6-21-04
Medial Golgi
Late Golgi UDP-Gal Transporter
GolgiHuman
6-21-04
√
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√
6-21-04
6-21-04
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
6-21-04
6-21-04
6-21-04
6-21-04
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 Fc Fusion 2.6g/l in a 3 day fermentation process
6-21-04
6-21-04
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
Support:NIHNIST