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TAPBOOSTTAPBOOST®®
Breakthrough Platform to Improve Protein Production
Misfolded Protein
Protein/Ab StabilityProtein/Ab SecretionFunctional ProteinImmunogenicity
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 Control Protein Folding System and Quality Control SystemSystem
Screening System
Step 1: in silico Screening for Protein Domain/Subdomain
Misfolding-Prone Protein
DegradationPoor Secretion
Protein Stability Protein Secretion
Step 3Protein 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
((TaTargeted rgeted PProtein rotein BoostBoosting Platform) ing Platform) DiscoveryDiscovery
TAPBOOSTTAPBOOST®®
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
PlatforPlatformm
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
Golgi
ER
TAPBOOSTTAPBOOST®® ((TaTargeted rgeted PProtein rotein BoostBoosting Platform) ing Platform) Discovery-2Discovery-2
Discovered sequence
Sequence Optimization
Deletion ofthe 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
((TaTargeted rgeted PProtein rotein BoostBoosting Platform) ing Platform) Discovery-3Discovery-3
TAPBOOSTTAPBOOST®®
Model Protein; IL-13 receptor 2 (IL13R2)-Fc fusion protein
CHALLENGE: The expression levels of IL13R2 are poor, and the protein is prone to misfolding and aggregation.
Cell
IL13R2-Fc
ERNu
• Disease Relevance: a key mediator of allergic inflammation
Aggregation of IL13R2-Fc
Only properly folded IL13R2-Fc secrets
• Fc fusion protein with sIL13R2 (sIL13R2-Fc) has been developed for the treatment of asthma
• Production of sIL13R2-Fc using human cells is inefficient due to its instability
• Soluble IL13R2 (sIL13R2) blocks the signal from IL-13 as a decoy receptor
Application: IL13RApplication: IL13Rαα2-Fc Protein Case 2-Fc Protein Case StudyStudy
TAPBOOSTTAPBOOST®®
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SecretedIL13R2-Fc
IL13R2-FcInside Cells
Secreted IL13R2-Fc IL13R2-Fc Inside Cells
IL13R2-Fc
Fold
Incr
ease
(%)
Fold
Incr
ease
(%)
IL13R2-Fc
Case Study - IL13RCase Study - IL13Rαα2-Fc 2-Fc ProteinProteinTAPBOOSTTAPBOOST®®
enhances the secretion of IL13Rα2-Fc ProteinTAPBOOSTTAPBOOST®®
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TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
ER
Targeted ProteinTargeting Domain was designed to bind only to Protein B
SecretedTarget 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 proteinsTAPBOOSTTAPBOOST®®
B
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-TNF Ab(Humira)
anti-VEGF Ab(Avastin)
antibody Brand NameProduction Increase
(produced protein)anti-IL8 Ab DP12 6.4 +/- 0.89
anti-TNFα Ab Humira 6.3 +/- 0.33anti-VEGF Ab Avastin 3.5 +/- 0.27
TAPBOOSTTAPBOOST®®
Antibody binding affinity for its corresponding epitopeDetection: 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-TNF Ab(Humira)
anti-VEGF Ab(Avastin)
anti-IL8 Ab(DP12)
00.5
11.5
22.5
33.5
44.5
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Fold
Incr
ease
Avastin
Fold
Incr
ease
Fold
Incr
ease
Humira DP12TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
antibody Brand NameProduction Increase
(produced protein)
Production Increaseby ELISA
anti-VEGF Ab Avastin 3.5 +/- 0.27 4.2 +/- 0.19anti-TNFα Ab Humira 6.3 +/- 0.33 6.0 +/- 0.82
anti-IL8 Ab DP12 6.4 +/- 0.89 7.3 +/- 0.56
substantially enhances the production of Factor VIII-Fc
Platform: Enzyme ExamplePlatform: Enzyme ExampleTAPBOOSTTAPBOOST®®
Fold
incr
ease
FVIII Production
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FVIII-Fc
Fold
incr
ease
FVIII Activity
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FVIII-Fc
Target ProteinProduction Increase (produced protein)
Production Increaseby Activity
FVIII-Fc 19.2 +/- 0.47 21.3 +/- 0.23
TAPBOOSTTAPBOOST®®
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TAPBOOSTTAPBOOST®®
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TAPBOOSTTAPBOOST®®
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Fold
incr
ease
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 proteinTAPBOOSTTAPBOOST®®
IL13R2-FcTNFRI-Fc1AT-Fc 1ATMT-Fc
Original Secretion of Targeted Protein
Effect of
MaximumEffect
may “rescue” problem proteins from misfolding and maximize secretion levels
works on difficult proteinsworks on difficult proteinsTAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
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 proteinsTAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
Target Protein(Conditioned Media)
Fold
Incr
ease
(%)
IL13Rα2-Fc
IL13Rα2-Fc
Protein Production in CHO Suspension conducted by CRO
050
100150200250300350400450
enhances the production in CHO Suspension Expression System
Transition from 293T to CHO suspension Transition from 293T to CHO suspension systemsystem
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
PK
C A
ctiv
ity (O
D45
0-B
G)
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 proteinsTAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
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 bacteriaTAPBOOSTTAPBOOST®®
Advantages of
SummarySummary
Increased Productivity byTarget proteins
TNFRI-Fc (Enbrel) 480%
Factor IX-Fc 320%Factor FVII-Fc 1000%
IL13R2-Fc protein 2000%
Factor VIII-Fc 2100%
HumiraAvastin
DP12600%420%
730%
293T monolayer System
293T Suspension System
IL13R2-Fc protein 1100%CHO Suspension System
IL13R2-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®®