Novel Solutions to Recombinant Protein Expression in Yeast

Dr Stephen Berezenko

Bioprocess International

Prague, February 2006

Overview

• Issues with Yeast Expression• Misconceptions• Addressing the Issues

Issues with Yeast Expression

“S. cerevisiae glycosylation isn’t the same as highereukaryotes”

– 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

Addressing the Issues

• Plasmid stability• Protein expression versatility• Expression levels• O-linked glycosylation• Product quality improvements

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, one approved

Gerngross, T. (2004) Nature Biotechnology 22, 1409-1414

8m3 working volume fermentation vessel

Nottingham, U.K.

Plasmid Stability

Mitotically Stable Vector Systems

• Whole 2µ plasmids– pJDB219 (Yeast/E. coli shuttle vector)– pSAC35 – Disintegration vector

• pDB2244 - Disintegration vector + rHA

pDB2244, cirO

Plasmid Stability

• Chemostat experiments• Fill and draw mode operation

– Harvest 90% culture use remainder as inoculum

• Stable over 256 generations– rHA titre and yx/s unchanged– Plasmid stability 100%

Versatility

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– >50 protein variants

expressed

• Fibronectin & fragments• Insulin• Apolipoprotein A1• Pro-urokinase & ATF

• PAI-2• A. niger glucose

oxidase• Fab’ & scFv• Growth hormone• Interferon α-2b• Transferrin• Lactoferrin

Expression levels

Based on Albumin Expression

• Albumin titres increased by a number of approaches

– Molecular biology• Non specific chemical mutagenesis• Specific gene deletion and insertion

– Fermentation• Media optimisation• Tight RQ control algorithms• Control pH

Yeast Strain Family

*

0

1

2

3

4

5

DB1

DS65DS212DS569DS110

1

D88

DXY1

D540

D638

D674

rHA

prod

uctiv

ity 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

DEONTG

NQO

NTG

NTG

* Productivity of monomeric albumin assessedby densitometry / SDS PAGE

Expression System Performance

Competitive yeast systemProtein Delta Saccharomycescerevisiae expression (g.L-1) Yeast Titre (g.L-1)

hGH 1.3 P. pastoris 0.011

P. pastoris 0.049

S. cerevisiae ~0.0015

S. cerevisiae ~0.0015

S. cerevisiae 1.3

Transferrin(N413Q, N611Q)

~3.0 P. pastoris Neverreported

Albumin 4.0 – 4.5 P. pastoris ~2.8

scFv-albumin fusion 5.5 P. pastoris ~0.010

S. cerevisiae 0.009

Enhanced Productivity

• General properties of the system

Secreted Intracellular

Albumin 4.5 g/L WC *

Transferrin (N413Q, N611Q) ~3.0 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

• High levels of heterologous proteins can be expressed in Saccharomyces cerevisiae

Glycosylation

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

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)

α1-3

S/T

MNN1

PMT1-PMT6MNT1/KRE2

α1-2

α1-3

α1-2

ER Lumen

Mannosylated rHA

• Approx. 0.7% of rHA binds to Con A– Due to O-glycosylation with mannose– Average of 3-5 moles/mole– Linkages α-1,2 and α-1,3. No evidence

of branching– Twelve potential sites of modification

identified• Tryptic peptide mapping of Con A eluate and

sequence and mass analysis of peptides

Mannosylated rHA cont.

• Reduction in m-rHA– New yeast strain, pmt1– Improved downstream process

• pmt1 mutant– Shorter glycoforms

• Additional chromatography steps– Reduced the amount of Con A binding

material five fold

Improvements in Product Quality

Mannosylated rHA– Reduced approx. 5-fold in final product– Reactivity with AE subjects’ antibodies

reduced by a factor of between 4 to >20– Combined reduction in reactivity of

Recombumin >20-fold

Protein Quality Improvements

Downstream Process Improvement through Expression Strain Modifications

YAP3

yap3

rHA monomer

45kDa fragment

• 45kDa N-terminal fragment

• Observed in Pichia sp, Klyveromyces sp and Hansenula sp

• Heterogeneous carboxy-terminus

– most common terminus Leu407 or Val409

Phe-Gln-Asn-Ala-Leu-Leu-Val-Arg-Tyr-Thr-Lys-Lys

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

• Estimated translational read-through– 0.002% (w/w) rHA-Adh1p fusion

L G L stop stop A stopTTA GGC TTA TAA TAA GCT TAA TCC ..........

C-Terminus Albumin ADH1 Terminator

rHA-Adh1p rHA

Load

Flow

Tro

ugh

Elua

te

Load

Flow

Tro

ugh

Elua

te

Saccharomyces cerevisiaeversus

Pichia pastoris

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

?

Pigmentation of Yeast-Derived Albumin-fermentation control

A 20% S. cerevisiae rHAB 5% Pichia rHAC 5% S. cerevisiae rHA

US Space Shuttle Mission STS-67

rHA crystals

Crystal Structure of rHA

Structure of rHA with five molecules of myristate bound.

Curry et al. (1998) Nature Structural Biology 5, 827-835

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