Enhanced Protein Production

1. Enhanced Protein Production
TAPTAPBOOSTBOOST
TM

2. Degradation
RNA
Gene
Misfolding
folding
polypeptide
Functional
Aggregation
Abnormal Function
Unfolding Protein Response
Secretion
· Translation
· Expression of Chaperones
· Apoptosis
Recombinant Proteins
Protein Secretion
Stability
Functional Recombinant Proteins
Immunogenicity
Alzheimer's disease
Parkinson's disease
Huntington's disease etc.
Impact of Protein Folding
Conformational Diseases
Protein Folding regulates many events in protein production

3. TAPBOOSTTM
(Targeted Protein Boosting Technology)
Proprietary
sequence that
facilitates protein
production
Binding domain
specific for
targeted protein
BSC2
TAPBOOSTERTM
BSC1
TAPBOOSTTM
Technology
TAPBOOSTERTM
ER
Targeted Protein
Golgi

4. Insolubility of expressed proteins is,
however, one of the biggest problems
of Bacterial expression platforms
TAPBOOOSTTM
Technology
significantly enhances solubility of
expressed proteins.
Bacterial Expression Platform
Difficult-to-Express Protein Platform
TAPBOOOSTTM
Technology enhances
active antibody production in mammalian
expression platform.
Market for therapeutic recombinant
protein is huge (>$100 billions) with
tremendous growth potential. However,
difficulties in the production of
therapeutic recombinant proteins make
protein-based therapies the most
expensive treatment in the world.
Antibody Expression Platform
TAPBOOSTTM
Technology
Bacterial
Expression
Antibody
Expression
Tough Protein
Expression
TAPBOOSTTM
Technology
(Targeted Protein Boosting
Technology)
TAPBOOSTTM
Technology

5. Model Protein; IL-13 receptor α2 (IL13Rα2)-Fc fusion protein
The expression levels of IL13Rα2
are poor, and the protein is prone to
misfolding and aggregation.
Cell
IL13Rα2-Fc
ERNu
• The receptor of IL-13, 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
Difficult-to-Express Protein Expression Platform

6. Secreted
IL13Rα2-Fc
mock
IL13Rα2-Fc
Inside Cells
mock
TAPBOOSTTM
enhances the production of IL13Rα2-Fc drastically
Cell
IL13Rα2-Fc
ERNu
Aggregation of
IL13Rα2-Fc
Cell
TAPBOOSTTM
Technology
ERNu
Secreted IL13Rα2-Fc
IL13Rα2-Fc TAPBOOSTERTM
Difficult-to-Express Protein Expression Platform
mock
TAPBOOSTTM
>20x
TAPBOOSTTM

7. Target Specificity
of
TAPBOOSTTM
TAPBOOSTTM
Technology
TAPBOOSTERTM
ER
Targeted Protein
Target Protein
(A or B)
A A A B B B B BTarget Protein

8. Model Protein; Factor VIII-Fc fusion protein
• Factor VIII is the responsible protein for
hemophilia A, disorder that causes serious
bleeds and can lead to degenerative joint
disease.
• The primary treatment for hemophilia A is
replacement therapy utilizing infusions of
clotting factors, mainly Factor VIII.
•Manufacture of Factor VIII is extremely difficult due to protein
folding problem. Therefore, yields are low and often inconsistent.
• The average annual cost for hemophilia treatment in 2008 was
estimated at $155,000, with clotting factor concentrates
accounting for 70-82% of total costs.
Difficult-to-Express Protein Expression Platform

9. Target Protein; Factor VIII-Fc fusion protein
TAPBOOSTTM
enhances the production of Factor
VIII-Fc drastically
Difficult-to-Express Protein Expression Platform
Foldinduction
FVIII Production
0
2
4
6
8
10
12
14
16
18
20
FVIII-Fc
TAPBOOSTTM Foldinduction
FVIII Activity
0
2
4
6
8
10
12
14
16
18
20
22
24
FVIII-Fc
TAPBOOSTTM

10. Antibody Expression Platform
• Monoclonal antibodies are composed
of heavy and light polypeptide chains
bridged through disulfide bonds. Due
to nature of this structural complexity,
monoclonal antibodies are expressed
in mammalian expression systems that
could benefit from an increase in the
yield of fully active antibodies
• The combined sales of 25 actively
marketed mAb therapeutics in 2010
was $43 billion.
Brand Sales
($m)
Market
(%)
Avastin 6973 16.02
Rituxan 6859 15.76
Humira 6548 15.04
Remicade 6520 14.98
Herceptin 5859 13.46
Lucentis 3106 7.14
Erbitux 1791 4.12
Tysabri 1230 2.83
Synagis 1038 2.38
Xolair 750 1.72
Sales of 10 actively marketed monoclonal
antibody therapeutics in 2010
International Journal of Pharmaceutics (2013)

11. Antibody Expression Platform
anti-IL8 Ab
(DP12)
TAPBOOSTTM
enhances the production of all
three tested antibodies
1 2 3
anti-VEGF Ab
(Avastin)
No antibody expressed
antibody without TAPBOOSTTM
1 2 3
anti-TNFα Ab
(Humira)
1 2 3
antibody with TAPBOOSTTM

12. Antibody Expression Platform
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
Antibodies produced by TAPBOOSTTM
hold the activity
Foldinduction
Avastin
TAPBOOSTTM
Foldinduction
Foldinduction
Humira
TAPBOOSTTM
DP12
TAPBOOSTTM

13. Bacterial Expression Platform
Pharma Manufacturing (2012)
Bacterial Expression Platforms
Expression Systems used for
Manufacture of Approved Drugs
Major problem in Bacterial Expression System
Soluble protein
bacteria
Bacterial Genome
plasmid
<Example of Insoluble Protein>
Soluble
Fraction
Insoluble
Fraction
Expressed
protein
Mammalian
E.coli
S. cerevisiae (yeast)
Direct synthesis in
transgenic animals
Other
inclusion body

14. Bacterial Expression Platform
Soluble protein (SP)
Inclusion Body (IB)
bacteria
Bacterial Genome
plasmid
We customized TAPBOOSTTM
technology for
bacterial expression system by using a proprietary
algorithms and intensive search for specific amino
acid sequences involved in protein folding.
SP IB
TAPBOOSTTM
Technology
SP IB
Test Protein A
Test Protein B
Test Protein C

15. Summary
• Any kind of protein can be expressed by TAPBOOSTTM
technology
• Easy implementation into your current bioprocess
• Combine TAPBOOSTTM
with any other technology for synergistic effect
Advantages of TAPBOOSTTM
Increased Productivity by TAPBOOSTTM
(%)Target proteins
TNFRI-Fc (Enbrel) 600%
Factor IX-Fc 320%
Factor FVII-Fc 1000%
IL13Rα2-Fc protein 2000%
Factor VIII-Fc 2100%
Humira (anti-TNFα antibody)
Avastin (anti-VEGFa antibody)
DP12 (anti-IL8 antibody)
600%
420%
730%