PEGS Boston slide strategia
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Protein Expression enhancement
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PEGS Boston slide strategia
- 1. TAPBOOSTTAPBOOST®® Breakthrough Platform to Improve Protein Production
- 2. Misfolded Protein Protein/Ab Stability Protein/Ab Secretion Functional Protein Immunogenicity human cell Polypeptide Protein Folding · Suppression of Translation · Induction/Expression of Chaperones · Cell suicide Platform enhances Protein Production of the Target Proteins by controlling Protein Folding/Cell Quality Control System RNA gene Folded Protein · Degradation of proteins Degradation RNA Protein Folding Cell Quality Control System human cell Total yield of Biologics/Ab TAPBOOSTTAPBOOST®® Protein Folding System and Quality ControlProtein Folding System and Quality Control SystemSystem
- 3. Screening System Step 1: in silico Screening for Protein Domain/Subdomain Misfolding-Prone Protein Degradation Poor Secretion Protein Stability Protein Secretion Step 3 Protein Stability and Secretion Test Develop the technology which controls protein folding/cellular quality control system specifically for an intended target protein GOAL Through the screening, we discovered the amino acid sequence with protein stability and secretion activity First Year Step 2: Library Construction ((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform) DiscoveryDiscovery TAPBOOSTTAPBOOST®®
- 4. Development of Platform Step 4: Construction of Targeting domain (bind to a target protein) Discovered sequence (Effecter Sequence) Targeting Domain • Fc binding sequence: 14 combinations • Antibody binding sequence: 5 combinations • Target specific sequence: 9 combinations • Unfolding protein binding sequence: 5 combinations Flexible Linker / Rigid Liner • 5 combinations Effecter Sequence Signal Sequence • 4 combinations Targeted Protein Second Year Step 5: Enhancement of the Production of Target Protein by PlatforPlatfor mm TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® Golgi ER TAPBOOSTTAPBOOST®® ((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform) Discovery-2Discovery-2
- 5. Discovered sequence Sequence Optimization Deletion of the sequence (49 deletion mutants) Minimum sequence (9 amino acid sequence) Step 6: Identification of the Minimum Sequence Various mutants of the sequence (49 mutations) Step 7: Formulation of the Sequence X1 X2 X3 X4 X5 X6 X7 X8 X9 Formulation of the Amino Acid Sequence ((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform) Discovery-3Discovery-3 TAPBOOSTTAPBOOST®®
- 6. Model Protein; IL-13 receptor α2 (IL13Rα2)-Fc fusion protein CHALLENGE: The expression levels of IL13Rα2 are poor, and the protein is prone to misfolding and aggregation. Cell IL13Rα2-Fc ERNu • Disease Relevance: a key mediator of allergic inflammation Aggregation of IL13Rα2-Fc Only properly folded IL13Rα2-Fc secrets • Fc fusion protein with sIL13Rα2 (sIL13Rα2-Fc) has been developed for the treatment of asthma • Production of sIL13Rα2-Fc using human cells is inefficient due to its instability • Soluble IL13Rα2 (sIL13Rα2) blocks the signal from IL-13 as a decoy receptor Application: IL13RApplication: IL13Rαα2-Fc Protein Case2-Fc Protein Case StudyStudy TAPBOOSTTAPBOOST®®
- 7. 0 500 1000 1500 2000 2500 0 20 40 60 80 100 120 Secreted IL13Rα2-Fc IL13Rα2-Fc Inside Cells Secreted IL13Rα2-Fc IL13Rα2-Fc Inside Cells IL13Rα2-Fc FoldIncrease(%) FoldIncrease(%) IL13Rα2-Fc Case Study - IL13RCase Study - IL13Rαα2-Fc2-Fc ProteinProtein TAPBOOSTTAPBOOST®® enhances the secretion of IL13Rα2-Fc Protein TAPBOOSTTAPBOOST®® 0 500 1000 1500 2000 2500 0 20 40 60 80 100 120 >20x TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
- 8. ER Targeted Protein Targeting Domain was designed to bind only to Protein B Secreted Target Protein (A or B) A A A B B B B BTarget Protein Platform: Target Protein SpecificityPlatform: Target Protein SpecificityTAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® specifically enhances the secretion of the target proteins TAPBOOSTTAPBOOST®® B
- 9. enhances the production of all three tested antibodies Antibody Production Detection: Western Blot Assay using anti-hIgG antibody Platform: Antibody ExamplesPlatform: Antibody ExamplesTAPBOOSTTAPBOOST®® Western (anti-hIgG Ab) TAPBOOSTTAPBOOST®® Target Antibody anti-IL8 Ab (DP12) anti-TNFα Ab (Humira) anti-VEGF Ab (Avastin) antibody Brand Name Production Increase (produced protein) anti-IL8 Ab DP12 6.4 +/- 0.89 anti-TNFα Ab Humira 6.3 +/- 0.33 anti-VEGF Ab Avastin 3.5 +/- 0.27 TAPBOOSTTAPBOOST®®
- 10. Antibody binding affinity for its corresponding epitope Detection: ELISA using rVEGF, rTNFα, or rIL-8 for Avastin, Humira, or DP12, respectively Platform: Antibody ExamplesPlatform: Antibody ExamplesTAPBOOSTTAPBOOST®® Antibodies produced by hold activity TAPBOOSTTAPBOOST®® anti-TNFα Ab (Humira) anti-VEGF Ab (Avastin) anti-IL8 Ab (DP12) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 FoldIncrease Avastin FoldIncrease FoldIncrease Humira DP12 TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® antibody Brand Name Production Increase (produced protein) Production Increase by ELISA anti-VEGF Ab Avastin 3.5 +/- 0.27 4.2 +/- 0.19 anti-TNFα Ab Humira 6.3 +/- 0.33 6.0 +/- 0.82 anti-IL8 Ab DP12 6.4 +/- 0.89 7.3 +/- 0.56
- 11. substantially enhances the production of Factor VIII-Fc Platform: Enzyme ExamplePlatform: Enzyme ExampleTAPBOOSTTAPBOOST®® Foldincrease FVIII Production 0 2 4 6 8 10 12 14 16 18 20 FVIII-Fc Foldincrease FVIII Activity 0 2 4 6 8 10 12 14 16 18 20 22 24 FVIII-Fc Target Protein Production Increase (produced protein) Production Increase by Activity FVIII-Fc 19.2 +/- 0.47 21.3 +/- 0.23 TAPBOOSTTAPBOOST®® 0 2 4 6 8 10 12 14 16 18 20 TAPBOOSTTAPBOOST®® 0 2 4 6 8 10 12 14 16 18 20 22 24 TAPBOOSTTAPBOOST®®
- 12. 0 2 4 6 8 10 12 Foldincrease TNFRI-Fc (IgG1) TNFα Binding Ability (ELISA) TNFRI-Fc (IgG4) Platform: Fc Fusion of hIgG4Platform: Fc Fusion of hIgG4TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® enhances the production of both types of Fc fusion protein TAPBOOSTTAPBOOST®®
- 13. IL13Rα2-FcTNFRI-Fcα1AT-Fc α1ATMT-Fc Original Secretion of Targeted Protein Effect of Maximum Effect may “rescue” problem proteins from misfolding and maximize secretion levels works on difficult proteinsworks on difficult proteinsTAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
- 14. Protein Aggregation Experiment conducted by Major Pharma Partner in Pilot Project • PD group wanted to know whether can also results in lower aggregation. CharacterizationCharacterization TAPBOOSTTAPBOOST®® reduces the aggregation of produced proteins TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
- 15. Target Protein (Conditioned Media) FoldIncrease(%) IL13Rα2-Fc IL13Rα2-Fc Protein Production in CHO Suspension conducted by CRO 0 50 100 150 200 250 300 350 400 450 enhances the production in CHO Suspension Expression System Transition from 293T to CHO suspensionTransition from 293T to CHO suspension systemsystem TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
- 16. PKCActivity(OD450-BG) AT1 receptor -AT1 receptor Angiotensin II (100nM) 5min 10min 15min 30min Angiotensin II receptor type 1 (AT1 receptor) AT1 receptor -AT1 receptor + Angiontensin II Application for Screening R&D – GPCR proteins (POC)Application for Screening R&D – GPCR proteins (POC) enhances the expression of difficult-to-express proteins such as GPCR proteins TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
- 17. Soluble protein (SP) Inclusion Body (IB) bacteria Bacterial Genome plasmid We customized platform for bacterial expression system by using a proprietary algorithms and intensive search for specific amino acid sequences involved in protein folding. Platform Test Protein A Test Protein B Test Protein C SP IBTotal SP IBTotal SP IBTotal SP IBTotal SP IBTotal SP IBTotal Application for Microbial ExpressionApplication for Microbial Expression TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® increases both expression and solubility of the target protein in bacteria TAPBOOSTTAPBOOST®®
- 18. Advantages of SummarySummary Increased Productivity byTarget proteins TNFRI-Fc (Enbrel) 480% Factor IX-Fc 320% Factor FVII-Fc 1000% IL13Rα2-Fc protein 2000% Factor VIII-Fc 2100% Humira Avastin DP12 600% 420% 730% 293T monolayer System 293T Suspension System IL13Rα2-Fc protein 1100% CHO Suspension System IL13Rα2-Fc protein 400% * Optimization process is in progress * * (%)TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® • Any kind of protein can be expressed by platform • Easy implementation into your current bioprocess • Combine with any other technology for synergistic effect TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
Editor's Notes
- Conceptual background on the challenge/opportunity of protein folding in recombinant protein expression systems. TAPBOOST’s solution to these challenges.
- Our efforts at Boston strategics have focused on development of a value-added system using proprietary chaperone technology to optimize/improve folding.
- In order to create a system that produces “clean” and natural untagged sequence we improved upon the original designs. This is now protected by global patents.
- Conclusion: not only larger yields, but less aggregation: larger proportion of monomeric target. The other Steps, transitions to CHO, and scale-up feasibilities are moving along with other partners.
- GPCRs are a major class of therapeutically relevant molecules, representing more than 50% of all screening targets. Here we tested another application for the TapBoost platform- the use in producing difficult to express drug screening targets such as GPCRs.
- Conclusion: not only larger yields, but less aggregation: larger proportion of monomeric target. The other Steps, transitions to CHO, and scale-up feasibilities are moving along with other partners.