employing fortebio octet platform for the development of a dual-binding potency assay

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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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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http://www.kbibiopharma.com/about-us/company-overview/














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

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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Octet/BLI5

• 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.

Example Sensorgram6

Baseline

30 – 60 sec.

Loading

~ 3-5 minutes

Baseline/ Wash

60 – 120 sec.

Association

2 – 10+ minutes

Y Y

Overview


2. Assay Development Considerations




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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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Sensor Selection Impact9

Protein A Sensors

Fc Capture

Overview


2. Assay Development on the Octet instrument




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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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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.)

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

Overview






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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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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 XMolecule Y

Molecule Z

Addition of 3rd Molecule 17

1

2

3

Trouble!

3 Molecule Troubleshooting

•Molecule Order

• Switched 1st and 3rd Molecule

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3

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 31 or 32

3 Molecule Working?

• Sensors: Super Streptavidin

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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

Inversion of Signal22

R Square 0.9964

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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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 %

Overview






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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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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 DissociationOctet® 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

Additional BLI Uses

• Resin Screening Tool

• To determine the resin which purifies the most active species

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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.

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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employing fortebio octet platform for the development of a dual-binding potency assay

Health & Medicine