TitrationAnalysis: a tool for high throughput binding kinetics data analysis for multiple label-free platforms

Abstract

Label-free techniques including Surface Plasmon Resonance (SPR) and Biolayer Interferometry (BLI) are biophysical tools widely used to collect binding kinetics data of bimolecular interactions. To efficiently analyze SPR and BLI binding kinetics data, we have built a new high throughput analysis tool named the TitrationAnalysis. It can be used as a package in the Mathematica scripting environment and ultilize the non-linear curve-fitting module of Mathematica for its core function. This tool can fit the binding time course data and estimate association and dissociation rate constants ( k _a and k _d respectively) for determining apparent dissociation constant ( K _D ) values. The high throughput fitting process is automatic, requires minimal knowledge on Mathematica scripting and can be applied to data from multiple label-free platforms. We demonstrate that the TitrationAnalysis is optimal to analyze antibody-antigen binding data acquired on Biacore T200 (SPR), Carterra LSA (SPR imaging) and ForteBio Octet Red384 (BLI) platforms. The k _a , k _d and K _D values derived using TitrationAnalysis very closely matched the results from the commercial analysis software provided specifically for these instruments. Additionally, the TitrationAnalysis tool generates user-directed customizable results output that can be readily used in downstream Data Quality Control associated with Good Clinical Laboratory Practice operations. With the versatility in source of data input source and options of analysis result output, the TitrationAnalysis high throughput analysis tool offers investigators a powerful alternative in biomolecular interaction characterization.

Keywords: Biolayer Interferometry; Surface Plasmon Resonance; antibody binding; high-throughput kinetics analysis; non-linear curve fitting.

Figure 1.. The TitrationAnalysis tool can batch process sensorgram fitting and automatically generate reports.

This figure shows an overall schematic of the installation and execution of the TitrationAnalysis tool. Panel A shows the installation of KineticsToolkit, the overall package containing TitrationAnalysis tool. Panel B shows the input commands to execute the TitrationAnalysis tool and the output at the end of TitrationAnalysis tool execution. Panel C shows an example of PDF report pages automatically generated after fitting analysis. Panel D shows the available modules that can be called within the TitrationAnalysis tool to import and analyze data collected on different instruments.

Figure 2.. Flowchart of TitrationAnalysis tool data importing and results exporting.

Figure 3.. Summary of information and preferences to be provided for each sensorgram prior to automatic fitting.

Figure 4.. The TitratonAnalysis tool can do automatic baseline alignment and analyte concentration range down-selection before fitting.

Fitting process of a set of simulated regenerative titration data ( A– D) and a set of simulated non-regenerative titration data ( E– H) is shown. Panels A and E show the titration data prior to baseline alignment. The color of each titration cycle and the corresponding analyte concentration in nanomolar is shown in the legend. Panels B and F show the titration after automatic baseline alignment. Panels C and G show the automatic selection of a subset of the analyte concentrations that best approximate the dose response linear range, highlighted as points in red in dose response curves, matching kinetics traces in red in panels B and F. Panels D and H show the resulting fitted sensorgram overlaid on top of underlying data points. Titration data were simulated using Equation 1 and Equation 3 with k _a = 1×10 ⁵ (M ^-1 s ^-1), k _d = 1×10 ^-4 (s ^-1) and R _max = 100 (RU).

Figure 5.. Comparison of fitted sensorgrams obtained using commercial software with the TitrationAnalysis tool fitted sensorgrams.

Each panel from A to F shows the binding of AE.A244 gp120 to mAb CH31. Data collected on Biacore T200 are compared in A (Biacore T200 evaluation software) and B ( TitrationAnalysis); data collected on high-throughput SPR _i platform Carterra LSA are compared in C (Carterra Kinetics software) and D ( TitrationAnalysis); data collected on Octet RED384 are compared in E (ForteBio Data Analysis software) and F ( TitrationAnalysis).

Figure 6.. Comparison of fitted sensorgrams for replicate measurements.

Panels A– D show fitted sensorgrams using TitrationAnalysis tool for replicates of AE.A244 gp120 binding to mAb CH31. All data were collected on Carterra LSA.