BIO Philadelphia yeast expression 2005
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Transcript of BIO Philadelphia yeast expression 2005
Novel Solutions to Yeast Recombinant Protein Expression
Dr Stephen Berezenko
Bio 2005 Philadelphia
June 21 2005
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
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
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
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
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
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.
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
Albumin Fusion Technology
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
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
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
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
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)
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
Mitotically Stable Vector Systems
• Whole 2µ plasmids– pJDB219 (Yeast/E. coli shuttle vector)– pSAC35 – Disintegration vector
• pDB2244 - Disintegration vector + rHA
pDB2244, cirO
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
Mitotically Unstable Vector Systems
• YEp – Yeast Episomal plasmids– YEp24, YEp13, pJDB207 (Yeast/E. coli
shuttle vectors)– Highly unstable – in cir+ yeast strains
YEp13, cir+
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
Host Strain Improvement Programme
• Plate assay for increased albumin expression– in vivo– Semi-quantitative
Mutants -Increased rHA expression
Parental strain
Control -Non-rHA producing
Selection Cycle
Chemically mutate
Plate screen
Shake FlaskFermentation
Cure and Retransform
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
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
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
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
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
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
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
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
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
?
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
Stephen Berezenko