Download - BIO Philadelphia yeast expression 2005
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Novel Solutions to Yeast Recombinant Protein Expression
Dr Stephen Berezenko
Bio 2005 Philadelphia
June 21 2005
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Issues with yeast expression
“S. cerevisiae glycosylation isn’t the same as higher eukaryotes”
– True– O-linked glycosylation
• Can be effectively controlled by pmt mutations and downstream processing
– N-linked glycosylation• Think smart - make the non-glycosylated protein• In majority of examples still active
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Misconceptions
• “Stable yeast episomal plasmids not available”– Whole 2µm plasmids are very stable in selective
media– Superior alternative to integration
• Curing and retransformation• “S. cerevisiae has a limited secretion capacity”
– Significant inter-strain variation– Strain engineering is not only possible, but highly
desirable• Control proteolysis• Increase expression
– Chemical mutagenesis & selection– Endogenous gene over-expression
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Enhanced Productivity
Protein Secreted Intracellular
Albumin 3 g/L WC *
Transferrin (N413Q, N611Q) 1 g/L WC *
scFv 3.6 g/L SN †
scFv-albumin 5.5 g/L SN †
Albumin-GSlinker-scFv 5.1 g/L SN †
Haemoglobin 2% CDW #
PAI-2 20% TSP ‡
Thymidine Phosphorylase 10% TSP ‡α1-antitrypsin 40% TSP ‡
* WC: Whole culture
† SN: Supernatant# CDW: Cell Dry Weight‡ TSP: Total Soluble Protein
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Expression System Performance
Delta Saccharomyces cerevisiae expression
(g.L-1) Titre
(g.L-1)P. pastoris 0.011P. pastoris 0.049S. cerevisiae ~0.0015S. cerevisiae ~0.0015S. cerevisiae 0.009S. cerevisiae 1.3
Transferrin(N413Q, N611Q)Albumin 4.0-4.5 P. pastoris ~2.8scFv-albumin fusion 5..5 P. pastoris ~0.010
~0.050
hGH 1.3
3.3 P. pastoris
Protein Competitive yeast systems
Yeast
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Recombinant Human Albumin
• Large secreted protein
– 67kDa– 585 amino acids
• Highly folded– 35 cysteines– 17 disulphide bonds– 1 free cysteine
Structure of rHA with five molecules of myristate bound.
Curry et al. (1998) Nature Structural Biology 5, 827-835
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Yeast – Positive Attributes
• GRAS status– S. cerevisiae– K. lactis
• Wide range of strains• Extensive industrial history
– 16 S. cerevisiae therapeutic products marketed
– 7 P. pastoris therapeutic products under development
Gerngross, T. (2004) Nature Biotechnology 22, 1409-1414
8m3 working volume fermentation vessel
Nottingham, U.K.
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Scale-up and Technology Transfer
• Scale up– R&D – 10L Fed-batch process– Commercial – 12m3 (total volume)
– 8m3 (working volume)– cGMP/FDA
• Technology Transfer– Successfully completed to Japanese
Pharmaceutical company
– HGSI and albumin-based fusions
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Albumin Fusion Technology
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Albumin Fusions Proteins
• Albumin joined to another protein through a peptide bond–Sequence encoding a given therapeutic protein is
ligated to the sequence encoding human albumin–High yield expression of the fusion protein (multiple
g/L) in optimised yeast strains
• Albumin has characteristics (charge distribution and size of ~70kDa) that prevent clearance via the kidney:19 day half-life
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What type of fusions can you make?
• The DNA sequence for the protein of choice can be joined to the:
– C-terminus HSA
– N-terminus HSA
– In the middle
– Combinations
• So junction site of the fusion protein can be defined at the molecular level
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Albumin Fusions
• Expressed up to 8 variants of 18 different proteins (n>50)
• hGH• IFNa-2b• IL11• IL10• IL1 receptor antagonist• Cyanovirin• gp41 peptides• 5-Helix
• scFv• Endostatin• Angiostatin• Apolipoprotein A1• Prosaptide• Kunitz domains• CNTF• vWF A1 domain
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Fusion Expression Levels (g/L)
Fusion N C
IL1-RA 6.1 3.3
IL11 - 0.6
Endostatin 1.0 2.5
HIV peptides 2.3 2.6
CNTF - 2.5
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Expressed proteins - intracellular
• α1-antitrypsin + variants• PAI-2• PAI-1• Haemoglobin (α2β2 functional tetramer)• Platelet-derived endothelial cell growth factor
(thymidine phosphorylase)• Lipoprotein associated coagulation inhibitor• Nitric oxide synthase (NOS)
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Expressed proteins - secreted
• Albumin–Albumin
fragments/mutants• Albumin-based fusions, e.g.• Fibronectin & fragments• Insulin• Fab’& scFv• Apolipoprotein A1• Pro-urokinase & ATF
• PAI-2• A. niger glucose
oxidase• Growth hormone• Interferon α-2b• Transferrin &
Lactoferrin
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Mitotically Stable Vector Systems
• Whole 2µ plasmids– pJDB219 (Yeast/E. coli shuttle vector)– pSAC35 – Disintegration vector
• pDB2244 - Disintegration vector + rHA
pDB2244, cirO
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Productivity - Host strain variation
Standards
10 – 150 mg/L
S150
-2B
cir+
JRY1
88 c
ir+
MT3
02/2
8B c
ir+
MC1
6 ci
r+
BJ19
91 c
ir+
•rHA productivity in shake flask culture–10mL YEP, 2%(w/v) glucose, 4 days, 30oC–YEp13 based vector, cir+ – rocket immunoelectrophoresis
Standards
10 – 150 mg/L
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Mitotically Unstable Vector Systems
• YEp – Yeast Episomal plasmids– YEp24, YEp13, pJDB207 (Yeast/E. coli
shuttle vectors)– Highly unstable – in cir+ yeast strains
YEp13, cir+
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Productivity - Host strain variation• rHA productivity in shake flask culture
– 10mL YEP, 2%(w/v) glucose, 4 days, 30oC
– Whole 2µm plasmid, (Disintegration vector) in cir0 yeast strains
Standards
10 – 200 mg/L
JRY1
88 c
ir0
S150
-2B
cir0
CB11
63
cir0
MT3
02/2
8B c
ir0
MC1
6 ci
r0
LL20
cir0
AH22
cir0 Standards
10 – 200 mg/L
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Host Strain Improvement Programme
• Plate assay for increased albumin expression– in vivo– Semi-quantitative
Mutants -Increased rHA expression
Parental strain
Control -Non-rHA producing
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Selection Cycle
Chemically mutate
Plate screen
Shake FlaskFermentation
Cure and Retransform
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Productivity – Shake Flask Screen
• rHA productivity in shake flask culture– 10mL YEP, 2%(w/v) glucose, 4 days, 30oC– Duplicate analysis
Standards
20 – 150 mg/L
Mutant Strains
Pare
ntal
St
rain
* ***
* Potential Up-mutants
Standards
20 – 150 mg/L
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High Cell Density Fermentation System
• Synthetic chemical defined– Simple, commercial grade materials– No animal or human derived products
• Fed-batch process• 5L batch• 5L feed• 300C ± 10C• pH5.5 ± 0.1• 1500rpm max
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Expression time course
Analysis of culture supernatant
1 2 3 4 5 61ug
1ug
LaneFeed Time
(hr)Feed Vol
(L)Biomass(g CDW/L)
1 6.5 0.1 8.9
2 14.0 0.3 14.9
3 30.5 1.1 46.8
4 38.3 1.9 67.5
5 54.5 4.8 101.8
6 55.5 5.0 101.3
12% Bis-Tris SDS Novex gel
MES Buffered
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0
1
2
3
4
DB1
DS65
DS212
DS569
DS1101 D88
DXY1
D540
D638
D674
rHA
pro
duct
ivity
g/L
yap3- hsp150- pmt1-
rHA producing yeast strains obtained byaspecific mutagenesis
1,2,7,8-diepoxyoctane (DEO)N-methyl-N'-nitro-N-nitrosoguanidine (NTG)4-nitroquinoline N-oxide (NQO)
Strains obtained by acombination of specific &aspecific mutagenesis
DEO
NTG
NQO
NTG
NTG
Yeast Strain Family
*
* Productivity of monomeric albumin assessedby densitometry / SDS PAGE
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Downstream Process Improvement through Expression Strain Modifications
YAP3
yap3
rHA monomer
45kDa fragment
-Phe-Gln-Asn-Ala-Leu-Leu-Val-Arg-Tyr-Thr-Lys-Lys-Val-Pro
•45kDa N-terminal fragment
•Observed in Pichia sp,
Kluyveromyces sp and Hansenula sp
•Carboxy terminus heterogeneous
•Terminating between Phe403 and Val409;
most common Leu407 and Val409
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Downstream Process Improvement through Expression Strain Modifications
• N-linked glycosylation – None
• O-linked glycosylation– Undetectable by ES-MS– Approx. 0.7% of rHA bound to
ConA– Average of 3-5 moles/mole– Dolichyl-phosphate-D-mannose:
protein-O-D-mannosyltransferase (PMT1 – 6)
• ConA binding material reduced approx. five-fold in a pmt1mutant yeast strain
α1-3
S/T
MNN1
PMT1-PMT6MNT1/KRE2
α1-2
α1-3
α1-2
ER Lumen
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Downstream Process Improvement through Expression Strain Modifications
• Hsp150p (Pir2p)– Host cell wall protein– Large
• ~150kDa • extensively O-linked
glycosylated• 47kDa deglycosylated
– Removed by gel permeation chromatography
– Antigenic in yeast sensitive subjects
Enrichment by ConAchromatography
HSP150+ HSP150-
0.2m
g
2mg
10m
g
0.2m
g
2mg
10m
g
Western blot with anti-Hsp150p
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Translational read-through
L G L stop A L D F F A R G 34aa S K stopTTA GGC TTA TAA GCT TTG GAC TTC TTC GCC AGA GGT...........TCT AAA TAA ..
C-Terminus Albumin ADH1 Terminator
L G L stop stop A stopTTA GGC TTA TAA TAA GCT TAA TCC ..........
C-Terminus Albumin ADH1 Terminator
Anti-Adh1p immunoaffinity purificationrHA-Adh1p rHA
Load
Fl T
hru
Elua
te
Load
F Th
ru
Elua
te
• Estimated translational read-through– 0.002% (w/w) rHA-Adh1p fusion
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ESMS (MaxEntTM) comparison of RecombuminTM
rHA and Pichia-derived rHA
66000 66250 66500 66750 67000 67250mass0
100
%
RecombuminTM 20%Pichia-derived rHA
∆ = 124Da⇒ Cys34 blocked
?
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Summary
• Whole 2µ episomal plasmid systems have high mitotic stability
• Inter-strain variation• Strain improvement is obtainable
– Increased productivity– Control of post-translational modifications– Improved downstream processing
• Chemically defined media– No animal or human derived products– Robust and reproducible high cell density fermentation
• Simplicity– Significantly improves scale-up and technology transfer