In-Solution Binding Kinetics With Excess-Target Immunoassay
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Solution Overview
Problem
Existing pharmacokinetic models for therapeutic proteins are confounded by interferences in the equilibrium of drug-target interactions, making accurate determination of free drug concentrations challenging, especially in complex matrices like serum or plasma.
Innovation Solution
A method for determining binding affinity and kinetics of a binder to its ligand in buffer or serum/plasma samples using a limited number of measurements, involving a constant excess of one partner and measuring within the linear plateau range of the curve to calculate the KD value without requiring data point linearization or EC50/IC50 calculations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional pharmacokinetic models are used to determine free drug concentrations, then comprehensive drug behavior characterization is achieved, but measurement accuracy is reduced due to interferences in drug-target equilibrium
Solution Approach 1:
The patent extracts the drug from the complex drug-target-equilibrium system by using excess target to drive complete binding, then measures only the unbound drug fraction. This separates the measurement from the equilibrium interference, allowing accurate free drug determination without being confounded by dynamic binding interactions
Solution Approach 2:
The patent introduces an excess target as an intermediary that mediates the measurement process. By adding substantial excess target, the system converts the equilibrium problem into a complete binding scenario where the unbound drug fraction directly reflects the free drug concentration, eliminating equilibrium interference
2Measurement precision
If multiple data points are used for KD value determination through linearization, then measurement precision improves, but device complexity and data analysis requirements increase
Solution Approach 1:
The patent extracts the essential information needed for KD determination by measuring only the unbound fraction of drug in the presence of excess target. This single measurement contains sufficient information to calculate KD without requiring complex multi-point analyses or linearization procedures
Solution Approach 2:
The patent changes the experimental parameter regime by using excess target conditions rather than equimolar mixing. This parameter change transforms the measurement into a direct readout of free drug fraction, simplifying the mathematical relationship and eliminating the need for complex data fitting procedures
3Adaptability or versatility
If conventional ligand binding assays are performed in complex matrices like serum or plasma, then clinical relevance improves, but measurement precision deteriorates due to matrix interferences
Solution Approach 1:
The patent converts the potential harm of matrix complexity into a benefit by using excess target to drive complete binding even in the presence of interfering substances. The robustness of the excess-target approach allows accurate measurements in serum or plasma without being significantly affected by matrix interferences
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables accurate and efficient determination of binding affinity and kinetics with a single data point, providing intrinsic quality control and reducing the need for complex data analysis, suitable for therapeutic proteins in serum or plasma samples.
Implementation Method 1
Drug and target interact in a reversible non-covalent manner governed by the law of mass action
Data Source
AI summary
Herein is reported a method for the determination of the binding affinity of a binder and its ligand comprising the step of determining based on the result of an immunoassay the fraction of free binder in a sample comprising binder, ligand and binder-ligand-complexes for at least two different binder:ligand ratios in the sample, and if the determined fraction of free binder is not comparable for all used binder:ligand ratios then the binder:ligand ratio in the sample is lowered and the sample is re-analyzed by the same immunoassay, and calculating based on the fraction of free binder in the previous step the binding affinity for the binder to its ligand.