Employing ForteBio Octet platform for the development of a dual-binding potency assay and as a highly versatile tool for early stage product analysis

1. Employing ForteBio Octet platform for the
development of a dual-binding potency assay
and as a highly versatile tool for early stage
product analysis
Carson Cameron, MS
Analytical Development Research Associate
KBI Biopharma
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2. Who is KBI
• About KBI: A Contract Development and Manufacturing
Company
• KBI helps partners accelerate and optimize drug
development and manufacturing programs with an
extensive suite of expert development and manufacturing
services in an agile, client-friendly partnering environment
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3. Role of Analytical Development
•Development
• Method Development
• Method Optimization
• PD support (CLD, Upstream
and Downstream)
• Lots of Samples!
•Quality Control
• Method Qualification
• Stricter Requirements
• Fewer Samples
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Challenge: Technology that can be utilized in a Q.C.
and in a high-throughput fashion
Answer: ForteBio Octet

4. Overview
1. ForteBio Octet: Bio-Layer Interferometry
2. Assay Development Considerations
3. Single-Target Relative Potency Assay
4. Dual-Binding Relative Potency Assay
5. Supplementary Octet Capabilities
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5. Octet/BLI
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• The Octet sends white light
through the biosensor tip.
There is a shift in the
pattern of the returning
light based on how much is
bound to the tip.

6. Example Sensorgram
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Baseline
30 – 60 sec.
Loading
~ 3-5 minutes
Baseline/
Wash
60 – 120 sec.
Association
2 – 10+ minutes
Y Y

7. Overview
1. ForteBio Octet: Bio-Layer Interferometry
2. Assay Development Considerations
3. Single-Target Relative Potency Assay
4. Dual-Binding Relative Potency Assay
5. Supplementary Octet Capabilities
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8. Octet Sensor Selection
• Ni (NTA)
• Protein A
• SSA (Super Streptavidin)
• AHC (Anti-Human Fc Capture)
• AMC (Anti-Mouse Fc Capture)
• Anti-Human IgG Quantitation
• Aminopropylsilane
• Amine Reactive
• Anti-Human Fab
• Anti-GST
• Anti-Penta-HIS
• Anti-His
• Protein G
• Protein L
• Streptavidin
• High Precision Streptavidin
• Kits:
• Anti-CHO HCP detection kit
• Residual Protein A detection kit
• Immunogenicity Assay Kit
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9. Sensor Selection Impact
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Protein A Sensors
Fc Capture

10. Overview
1. ForteBio Octet: Bio-Layer Interferometry
2. Assay Development on the Octet instrument
3. Single-Target Relative Potency Assay
4. Dual-Binding Relative Potency Assay
5. Supplementary Octet Capabilities
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11. Assay Development
Pre-Development: Sensor Chemistry, Buffer/Matrix, Dilutions
1. Load Density Scouting
• Sensor loading is evaluated using an antigen/protein to ensure
sufficient protein is loaded on the sensor, but not overloaded.
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12. Assay Development
2. Concentration scouting of 2nd
molecule (antibody)
• Holding the first molecule (antigen) constant, a concentration
range is evaluated for product association in a concentration
dependent manor.
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For typical antibodies, assay development would be done => method
optimization and assessment (accuracy, linearity, etc.)

13. Single Target Conclusions
• Qualified 5 single target potency methods using different
antibody/antigen pairs
• 2 titer methods using the same method development in that was used
for qualified methods using antibody/glycoprotein pairs
• Frequently achieve ≤10% RSD for sample triplicates and 90 – 110%
Potency
• Release method for activity much faster than typical ELISA or SPR
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R2
≥ 0.99

14. Overview
1. ForteBio Octet: Bio-Layer Interferometry
2. Assay Development on the Octet instrument
3. Single-Target Relative Potency Assay
4. Dual-Binding Relative Potency Assay
5. Supplementary Octet Capabilities
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15. Need for a Dual Binding Potency
• New Bispecific molecules and fusion proteins drive the
need for dual binding potency assays
• BLI technology reduces impact of non-specific binding
when compared to an ELISA, though development is not as
direct as a single binding potency assay
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16. Development of a Dual Binding Potency
3. Addition of 3rd
molecule
• Evaluate the 3rd
molecule at multiple concentrations and
identify the concentration that produces the largest
wavelength shift but keeps concentration dependent signal
from previous step
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Molecule X
Molecule Y
Molecule Z

17. Addition of 3rd
Molecule
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1
2
3
Trouble!

18. 3 Molecule Troubleshooting
• Molecule Order
• Switched 1st
and 3rd
Molecule
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1
2
3

19. 3 Molecule Troubleshooting
• Sensors:
• Ni (NTA), Fc Capture, Streptavidin
• 1st
and 3rd
Molecules (antigens)
• His-Tagged and/or Biotinylated of the following
• Full Length Molecule (Transmembrane)
• Extracellular Domain only
• Partial Sequence (ECD and part of Transmembrane portion)
• Non-Tagged antigens
• Pre-incubating Fc Fusion with the 3rd
Molecule
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1 or 3
1 or 32

20. 3 Molecule Working?
• Sensors: Super Streptavidin
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21. Negative Wavelength Shift
• Spectrophotometer in the instrument covers a range of 550-850 nm, a
wavelength is adjusted to cover one of the cycles
• Peak location is determined by the optical thickness (n*d)
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Small change in optical thickness Large change in optical thickness
4 peaks in the window 5 peaks in the window
G. Kraus and G. Gauglitz Application and comparison of algorithms for the evaluation of interferograms,
Fresenious J. Anal. Chem. (1994) 349: 399 - 402

22. Inversion of Signal
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R Square 0.9964

23. Method Conclusions
• A few months of trouble shooting
• Run time = 25 minutes per read (2 columns)
• < 1 hour for Standard Curve triplicate + 2 samples
• vs. ELISA Run time = Overnight + 6 Hrs.
• An equivalent method has been qualified
• Negative Control Antibodies (targeting either 1 or 3)
showed 0% dual-target potency
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24. Intermediate Precision
• Qualified Method
• 2 Locations, 2 Octet Models, 2 different days
• N=6 /analysts (n=12 total) at the nominal load
• Criteria: ≤ 20% RSD & 70% - 130% relative Potency
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RSD 9.0% Average Relative
Potency
105.6 %

25. Overview
1. ForteBio Octet: Bio-Layer Interferometry
2. Assay Development on the Octet instrument
3. Single-Target Relative Potency Assay
4. Dual-Binding Relative Potency Assay
5. Supplementary Octet Capabilities
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26. Additional BLI Uses
Titer
• ELISA Titer replacement: just like Single Target Potency
• Example: antigen + Antibody (Std. Curve and unknowns)
• Protein A Titer reads in as little as 2 minutes
• KBI frequently tests 500 samples/day
• Have tested ~2000 samples/day
• Great for High-Throughput CLD Clone ranking
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27. Additional BLI Uses
Kinetics
• It has been suspected that certain steps in the purification process may be
responsible for loss of binding between antibody and antigen. Testing the protein
that has been through different purification methods, it can be determined which
process is deteriorating activity and which process better preserves binding
activity.
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Association Dissociation
Octet® Method Biacore® Method
Sample KD (nM)
Fold difference
from Control Sample KD (nM)
Fold difference
from Control
Octet Reference Material 18.2 NA Biacore Reference Material 26.0 NA
Process 1 36.7 2.0 Process 1 94.9 3.7
Process 2 27.5 1.5 Process 2 41.8 1.6
Process 3 20.0 1.1 Process 3 36.9 1.4
Process 4 17.2 0.9 Process 4 24.8 1.0
Process 5 21.4 1.2 Process 5 50.5 1.9

28. Additional BLI Uses
• Resin Screening Tool
• To determine the resin which purifies the most active
species
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29. Conclusions
Kinetics/Potency Comparison
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* Including standard curve tested in triplicate (Potency). 2 Samples may be run
simultaneously (Kinetics).
** 8 hr. run time accounts for standard curve and samples tested in triplicate
(Potency). 26 samples tested at 7 concentrations plus a blank (Kinetics).
*** 3 samples per plate, 2 plates per day not including overnight coating.

30. Acknowledgements
• Major Thanks to ForteBio!
• AD Team
• James Smedley III, Ph.D.
• Nathan Oien, Ph.D.
• Brendan Peacor, Ph.D.
• Srilaxmi Sarikonda
• Kyle Jones
• Teena Jagadish
• Analyst II – Melissa Ragan
• SPR Data – Spandana Kankanala
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