PEGS Boston slide strategia
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The document describes TAPBOOST, a breakthrough protein production platform that enhances protein folding and secretion. It works by controlling the protein folding system and cellular quality control mechanisms in the host cell. The platform can increase yields of various target proteins including antibodies, enzymes, and difficult-to-express proteins. It has been shown to boost production levels by over 2000% for some proteins while also reducing aggregation. The platform works in mammalian, bacterial, and yeast expression systems and provides advantages such as increased productivity for any target protein and easy implementation into existing bioprocesses.
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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.