Quantitative NMR Protein Concentration Determination
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Solution Overview
Problem
Existing methods for determining the concentration and molecular-specific parameters of proteins or peptides are inaccurate and imprecise due to reliance on experimentally derived properties, harsh conditions, and interference from formulation components.
Innovation Solution
A novel quantitative nuclear magnetic resonance (NMR) spectroscopy method that involves denaturing the protein, using a diffusion filter to eliminate resonances of water and formulation components, and comparing the data to a reference standard for accurate concentration determination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (UV extinction coefficient, gravimetric analysis, chromogenic methods, amino acid analysis) are used to determine protein concentration, then concentration measurement is performed, but accuracy and precision deteriorate due to reliance on experimentally derived properties, harsh conditions, and interference from formulation components
Solution Approach 1:
The patent replaces conventional chemical and physical measurement methods (UV spectroscopy, gravimetric analysis, chromogenic methods, amino acid analysis) with nuclear magnetic resonance (NMR) spectroscopy. This substitution eliminates reliance on experimentally derived properties such as extinction coefficients and differential refractive index increments, providing direct absolute concentration measurements based on fundamental NMR signal intensities that are independent of protein structure and formulation components.
Solution Approach 2:
The patent changes the measurement parameter from indirect optical or chemical properties to direct NMR signal intensity. By utilizing the intrinsic magnetic properties of nuclei and the quantitative relationship between NMR signal area and number of nuclei, the method achieves accurate concentration determination without requiring calibration curves or knowledge of protein-specific parameters.
2Measurement precision
If NMR spectroscopy is used to measure absolute concentrations of large proteins in complex matrices, then concentration measurement is performed, but accuracy and precision deteriorate due to intense peaks from salts, buffers, surfactants, and water that dominate the spectrum
Solution Approach 1:
The patent extracts or removes the interfering signals from salts, buffers, surfactants, and water from the NMR spectrum through selective suppression techniques. This allows the protein signals to be clearly observed and integrated without interference, enabling accurate concentration determination even in complex therapeutic formulations.
Solution Approach 2:
The patent introduces an internal reference standard as an intermediary substance with known concentration and distinct NMR signals. This reference standard serves as a mediator to calibrate the NMR measurement system and provide a basis for calculating absolute protein concentration, compensating for any remaining interference from formulation components.
3Stability of the object's composition
If protein secondary, tertiary, and quaternary structure is maintained during NMR measurement, then native structure is preserved, but resonance linewidths broaden making integration and concentration determination difficult
Solution Approach 1:
The patent changes the physical state parameter of the protein from folded (native) to unfolded (denatured) by applying denaturing conditions during NMR measurement. This parameter change transforms the broad resonances characteristic of structured proteins into sharp resonances suitable for accurate integration and quantification, while the denaturation is reversible and does not permanently damage the protein.
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
This method provides clean spectra with well-resolved resonances, enabling precise and accurate determination of protein or peptide concentration, which is essential for therapeutic applications.
Implementation Method 1
acquire a qNMR spectrum with a diffusion filter to eliminate the resonances of water and the other formulation components
Implementation Method 2
Nuclear magnetic resonance (NMR) spectroscopy is a quantitative technique that has been used to measure the concentration of compounds
Data Source
AI summary
Methods for determining protein and/or peptide concentration or molecular parameter, such as the extinction coefficient, and uses thereof.