Bioprocess International Prague 2006 novel solution in yeast protein expression
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Bioprocess International Prague 2006 novel solution in yeast protein expression
- 2. Novel Solutions to Recombinant Protein Expression in Yeast Dr Stephen Berezenko Bioprocess International Prague, February 2006
- 3. Overview • Issues with Yeast Expression • Misconceptions • Addressing the Issues
- 4. 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
- 5. 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
- 6. Addressing the Issues • Plasmid stability • Protein expression versatility • Expression levels • O-linked glycosylation • Product quality improvements
- 7. 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.
- 9. Mitotically Stable Vector Systems • Whole 2µ plasmids – pJDB219 (Yeast/E. coli shuttle vector) – pSAC35 – Disintegration vector • pDB2244 - Disintegration vector + rHA pDB2244, cirO
- 10. 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%
- 11. Versatility
- 12. 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)
- 13. 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
- 15. 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
- 16. Yeast Strain Family * 0 1 2 3 4 5 D B 1 D S 65 D S 212 D S 569 D S 1101 D 88 D X Y 1 D 540 D 638 D 674 rHAproductivityg/L yap3- hsp150- pmt1- rHA producing yeast strains obtained by aspecific mutagenesis 1,2,7,8-diepoxyoctane (DEO) N-methyl-N'-nitro-N-nitrosoguanidine (NTG) 4-nitroquinoline N-oxide (NQO) Strains obtained by a combination of specific & aspecific mutagenesis DEO NTG NQO NTG NTG * Productivity of monomeric albumin assessed by densitometry / SDS PAGE
- 17. Expression System Performance Competitive yeast systemProtein Delta Saccharomyces cerevisiae 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 Never reported Albumin 4.0 – 4.5 P. pastoris ~2.8 scFv-albumin fusion 5.5 P. pastoris ~0.010 S. cerevisiae 0.009
- 18. 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
- 19. Expression System Performance • High levels of heterologous proteins can be expressed in Saccharomyces cerevisiae
- 20. Glycosylation
- 21. 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
- 22. 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-PMT6 MNT1/KRE2 α1-2 α1-3 α1-2 ER Lumen
- 23. 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
- 24. 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
- 25. 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
- 27. 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
- 28. Translational Read-Through L G L stop A L D F F A R G 34aa S K stop TTA 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 stop TTA GGC TTA TAA TAA GCT TAA TCC .......... C-Terminus Albumin ADH1 Terminator rHA-Adh1p rHA Load FlowTrough Eluate Load FlowTrough Eluate
- 30. ESMS (MaxEntTM) Comparison of RecombuminTM rHA and Pichia-derived rHA 66000 66250 66500 66750 67000 67250 mass0 100 % RecombuminTM 20% Pichia-derived rHA ∆ = 124Da ⇒ Cys34 blocked ?
- 31. Pigmentation of Yeast-Derived Albumin -fermentation control A 20% S. cerevisiae rHA B 5% Pichia rHA C 5% S. cerevisiae rHA
- 32. US Space Shuttle Mission STS-67 rHA crystals
- 33. Crystal Structure of rHA Structure of rHA with five molecules of myristate bound. Curry et al. (1998) Nature Structural Biology 5, 827-835
- 34. 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