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