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Employing ForteBio Octet platform for the development of a dual-binding potency assay
The document discusses the application of the Fortebio Octet platform in developing dual-binding potency assays for drug analysis, emphasizing its versatility and efficiency in high-throughput settings. It highlights the transformation in assay development for bispecific molecules, detailing the process of using biolayer interferometry to enhance potency testing. Challenges in capturing accurate dual-target potency are addressed, alongside qualitative comparisons to traditional methods like ELISA.
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Introduction to the use of the ForteBio Octet platform for dual-binding potency assay development.
KBI is a Contract Development and Manufacturing Company facilitating drug development and manufacturing.
Explains the roles in analytical development including QC, method development, and challenges faced with sample lots and technology.
Overview of ForteBio Octet technology and assay development considerations for potency assays.
Description of how the Octet platform uses bio-layer interferometry (BLI) to detect binding interactions.
Illustration of a typical sensorgram and the timeline of baseline, loading, association, and wash phases.
Summary of the key considerations for developing assays on the Octet platform.
Discussion of various sensor types available for the Octet platform and their applications.
Exploration of the impact of sensor selection on binding and assay results.
Details the critical steps involved in the development of assays on the Octet instrument.
Pre-development considerations including sensor chemistry, buffer, and loading density scouting.
Exploration of concentration scouting for a secondary molecule in assay development.
Conclusions from single-target potency methods, discussing RSD and potency measurements.
Motivation for developing dual-binding potency assays due to the rise of bispecific molecules.
Steps in evaluating a third molecule for performance in dual-binding assays.
Techniques for troubleshooting issues in dual binding assays involving three molecules.
Analysis of wavelength shifts in the detection process and its implications in assay results.
Observation of signal inversions in assay output and their statistical significance.
Summary of method performance, runtime comparisons, and control antibody results.
Factors affecting intermediate precision across different locations, models, and analysts.
Records the various applications of BLI technology in titer and kinetics assessments.
Thanks to the ForteBio team and contributors involved in the project.
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Employing ForteBio Octet platform for the development of a dual-binding potency assay
- 1. Employing ForteBio Octetplatform 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 1
- 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 2
- 3. Role of AnalyticalDevelopment •Development • Method Development • Method Optimization • PD support (CLD, Upstream and Downstream) • Lots of Samples! •Quality Control • Method Qualification • Stricter Requirements • Fewer Samples 3 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 4
- 5. Octet/BLI 5 • The Octetsends 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 6 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 7
- 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 8
- 9. Sensor Selection Impact 9 ProteinA 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 10
- 11. Assay Development Pre-Development: SensorChemistry, 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. 11
- 12. Assay Development 2. Concentrationscouting of 2nd molecule (antibody) • Holding the first molecule (antigen) constant, a concentration range is evaluated for product association in a concentration dependent manor. 12 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 13 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 14
- 15. Need for aDual 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 15
- 16. Development of aDual 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 16 Molecule X Molecule Y Molecule Z
- 17.
- 18. 3 Molecule Troubleshooting •Molecule Order • Switched 1st and 3rd Molecule 18 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 19 1 or 3 1 or 32
- 20. 3 Molecule Working? •Sensors: Super Streptavidin 20
- 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) 21 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 22 RSquare 0.9964
- 23. Method Conclusions • Afew 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 23
- 24. Intermediate Precision • QualifiedMethod • 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 24 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 25
- 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 26
- 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. 27 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 28
- 29. Conclusions Kinetics/Potency Comparison 29 * Includingstandard 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 Thanksto 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 30