Kinetics on the Octet Systems: What Lies Beneath the Curves

1 Kinetics on the Octet Systems: what lies Beneath the Curves Pui Seto Scientist ForteBio, A Division of Pall Life Sciences Basic Kinetics : what can the sensorgram tell us? Background on BLI technology An ideal sensorgram Recognizing non-ideal behavior More complex models Using more complex models to optimize Kinetics assay Label-free, real-time analysis BioLayer Interferometry (BLI). A layer of molecules attached to the tip of an optic fiber creates an interference pattern at the detector. BioLayer Interferometry (BLI). A layer of molecules attached to the tip of an optic fiber creates an interference pattern at the detector. Any change in the number of molecules bound causes a measured shift in the pattern Octet Systems and Features LOD (direct): LOD (direct): (multi-step): Sub-ng/mL (multi-step): Sub-ng/mL.

2 Inside the Octet Classic system ForteBio Biosensors and Assay Kits Biosensor Biosensor Functionality AHQ Anti-Human IgG Fc AMQ Anti-Murine IgG Fv FAB Anti-Human Fab CH1. ProA Protein A. ProG Protein G. ProL Protein L. SA Streptavidin HIS Anti-Penta-HIS. NTA Ni-Tris NTA. GST Anti-GST. AMC Anti-Murine IgG Fc Capture AHC Anti-Human IgG Fc Capture AR2G Amine Reactive 2nd Gen Custom biosensors can be APS Aminopropylsilane made to specifications by SSA Super Streptavidin Fortebio Kits/Methods Dip and Read Immunogenicity Dip and Read Residual Protein A. Dip and Read HCP Custom Assay Kinetics Application: Drug Discovery Process # of Samples per run 10's -1000's 100K 3M 10K 100s 100s Research: Primary Lead Characterization Secondary Screening Target ID Screening & Optimization & Hit Validation & Validation >150 Daltons Antibody Applications: 1) Antigen generation - identify high producers 2) Serum titering 3) Screening for antigen specific antibodies 4) Quantitation of hybridoma supernatants 5) Off-rate ranking 6) Epitope binding and domain mapping 7) Apparent affinity measurement KD.

3 8) Antibody sandwich pair identification 9) Assay development 10) Speed up humanization workflow 11) Identify high producers for manufacturing BLI Platform Capabilities Quantitation Direct Sandwich ELISA. mg/mL to sub-ng/mL. Kinetics Label-free ka, kd, KD. Proteins Peptides, Oligos Small molecules Kinetics : what can a sensorgram tell us? Before we launch into simple Kinetics theory, we should keep in mind that: Curve shapes should only be described by the rate constants and the analyte concentration If there are any other influences at play, then the Kinetics constants which you calculate can be meaningless Don't over interpret data An Ideal Sensorgram A classic' 1:1 binding curve Association Dissociation Response 1:1 Binding Both the association and dissociation phases ka follow a path described by a single A+B AB.

4 Exponential function kd what are Req, Rmax and KD? Most Curves , if the binding is left for long enough, will reach a point where the rates of association and dissociation are the same. This is the equilibrium binding level (Req). There is a fixed amount of ligand on the sensor surface, so there must be a maximum possible amount of sample binding at equilibrium. This is the saturating or maximum binding level (Rmax). The affinity constant KD is defined by the ratio of rate constants kd / ka The relationship between Req, Rmax and KD. Saturation (Rmax) is achieved if conc = 100x KD. 100. %age of Rmax Equilibration (Req) at 50%. saturation if conc = KD. 50. Detection limit around KD. Time ka The simple 1:1 Interaction: A+B AB. kd The relationship between Req, Rmax and KD. Saturation (Rmax) is achieved if conc = 100x KD.

5 KD =. [A] [B] = k d 100. [AB] k a %age of Rmax Equilibration (Req) at 50%. saturation if conc = KD. ka = M-1s-1. 50 kd = s-1. Detection limit around KD. Time ka The simple 1:1 Interaction: A+B AB. kd A Closer Look at Affinity Constant KD. This is a VERY important relationship: KD =. [A] [B] = k d [AB] k a Relationship shows that the Affinity (KD) is equal to the ratio of the rate constants So, ANY set of rate constants which have the same ratio will yield the same KD. For example: kd 10-3 10-6. KD = = 1nM = etc ka 106 103. KD and Rate Constants This sensorgram shows the same concentration of 3 different analytes binding to the same immobilized ligand. They have the same KD but very different rate constants, and would look identical in an end-point assay such as ELISA. KD =. [A] [B] = k d [AB] k a Response Time Dissociation is a simple decay process, and is independent of sample concentration kd % complex Time to 50%.

6 Dissociated per dissociation second 1 100 s 10 s 1 x 10-2 1 s 1 x 10-3 min 1 x 10-4 h 1 x 10-5 h 1 x 10-6 8 days Slow off-rates need a long time to fit accurately! Recognizing Non-ideal Behavior Recognizing Non-ideal Behavior Non ideal behavior arises where the interaction proceeds through multiple binding sites. Often a slower phase, never reaching equilibrium Often has portion which Heterogeneous Binding never dissociates Response 1:1 Binding Time Here, the Curves show more than one phase, both in the association and dissociation Curves . Basically, what this means is that there are more than one binding event going on. The Most Likely Cause of Heterogeneity Heterogeneity can come from the original samples or from the way that the ligand has been immobilised. Direct coupling methods like amine coupling will usually tend to give rise to heterogeneity due to their potentially random orientation.

7 Capture approaches are better for Kinetics , since they use specific orientation. Heterogeneous binding can also be observed if concentrations well above the KD are used, where non-specific binding can become more significant. Mass transport (MT) Limited Binding More difficult to recognise than Heterogeneity The Curves are slower than expected and can be limited by the speed at which the analyte diffuses to the surface. km ka Asolution Asurface + B AB. -k m kd To reduce MT effects, it is best to consider the binding as a supply and demand' process. If demand for binding outweighs supply to the surface (by diffusion), then it will be that diffusion which defines the observed rates. So, to minimise MT effects, limit consumption and speed up supply by: Reducing the level of immobilised ligand (whilst still maintaining the required sensitivity) and increase the stir rate of the sample plate Solutions For Correcting Non-ideal Binding Heterogeneity Reduce sample concentration range and use capture approach Concentrations far above KD often unmask heterogeneous, non-specific sites Use capture approach for Kinetics experiment, , AHC, or AMC biosensor Try using a new lot of ligand or analyte Mass Transport fit Try to reduce sample depletion and increase sample supply Increase shake speed'.

8 Decrease ligand level Complex Binding system More Complex Reaction Schemes Here, deviation from 1:1 binding is a function of the type of interaction, rather than some experimental artifact . The 2 most common complex reaction schemes are: Heterogeneous ligand Bivalent analyte Let's look at these on the next few of slides .. Heterogeneous Ligand k a1. A + B1 A B1. Assumes 2 independent k d1. ligand binding sites k a2. A + B2 A B2. k d2. A A. B1 B2. This is an example of parallel' interactions, where the formation of AB1 and AB2 proceed independently of each other, Octet software can calculate both KDs Bivalent Analyte Assumes the bivalent analyte can form the 'bridged' AB2 complex. k a1. This causes a slower dissociation A +B AB. k d1. than expected and is purely an artifact of the surface interaction.

9 K a2. AB +B A B2. k d2. Formation of Formation of AB complex AB2 complex This is an example of a linked' interaction, where the formation of AB2 cannot proceed before the formation of AB, and AB cannot dissociate before the dissociation of AB2. Also, Octet software will calculate KD1 but not KD2 due to unknown concentration of surface AB complex Use Capture Approach To Deal With Bivalent Analyte Avidity issue, typically seen with antibody as Antibody capture approach analyte, slow off-rate observed can be used for bivalent analyte, avoiding avidity Target molecule Y Bivalent molecule Bivalent molecule Target molecule Sensor surface Y Anti Human capture sensor surface Complex Curves 1:1 Homogeneous Curves Summary It's the shape of sensorgrams with respect to time that give us the kinetic information Curves should only be influenced by ka, kd and concentration An understanding of the relationships between ka, kd, Req, Rmax and KD can help understand the processes and design our Kinetics assays Using the more complex models available in the Octet analysis software can help troubleshoot more complex Curves and optimize the design of Kinetics experiments ForteBio offers different chemistry biosensors for off-the-shelf use Octet can use up to 16 biosensors for scouting experiments, and each sensor is independent Contact Info Pui Seto ForteBio - A Division of Pall Life Sciences, Pall Corp Email: office phone: + or call 888- Octet -75.