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

Steve.Berezenko@aventis.com