Time-Resolved Intrinsic Fluorescence for Co-eluting Protein Quantification
Solution Overview
Problem
Current methods for monitoring proteins in chromatography processes struggle to accurately distinguish and quantify co-eluting protein species in real-time, leading to suboptimal product purity and yield due to limitations in existing detection technologies such as UV absorbance, mass spectrometry, and fluorescence intensity measurements.
Innovation Solution
A time-resolved intrinsic fluorescence (TRIF) method and apparatus that uses fluorescence lifetime measurements, combined with high-bandwidth digitization and pulsed excitation, to deconvolve and quantify co-eluting protein species without the need for extrinsic labels, enabling simultaneous measurement of fluorescence intensity and decay times at fast acquisition rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If UV absorbance or fluorescence intensity measurements are used to monitor proteins in chromatography, then real-time measurement capability is achieved, but the ability to distinguish and quantify co-eluting protein species is lost
Solution Approach 1:
The patent transitions from measuring only fluorescence intensity (one dimension) to measuring fluorescence lifetime in addition to intensity (adding a second dimension). This dimensional expansion enables differentiation of co-eluting proteins that have identical intensity profiles but distinct lifetime characteristics, resolving the contradiction between real-time measurement and species discrimination capability
Solution Approach 2:
The patent changes the measurement parameter from static fluorescence intensity to dynamic fluorescence lifetime decay characteristics. By measuring how fluorescence intensity changes over time after excitation pulse termination, the system extracts additional information (lifetime decay curves) that provides discriminatory power for distinguishing co-eluting protein species while maintaining real-time measurement capability
2Measurement precision
If sophisticated curve-fitting algorithms and multi-wavelength absorbance spectroscopy are used to resolve co-eluting proteins, then measurement precision improves, but device complexity and computational requirements increase
Solution Approach 1:
The patent replaces complex computational curve-fitting algorithms with direct measurement of fluorescence lifetime decay parameters. Instead of using sophisticated mathematics to deconvolve overlapping absorbance spectra, the system measures inherently different lifetime characteristics of each protein species, providing a more straightforward measurement approach that reduces computational complexity while improving resolution capability
Solution Approach 2:
The patent introduces fluorescence lifetime as an intermediary parameter that mediates between the excitation source and the detector. This intermediary measurement dimension provides direct discriminatory information about protein species without requiring complex spectral analysis, simplifying the overall detection system while enhancing the ability to resolve co-eluting proteins
3Measurement precision
If narrow peak fractionation is used to achieve higher product purity, then measurement precision improves, but productivity decreases due to yield loss
Solution Approach 1:
The patent implements real-time feedback by continuously monitoring fluorescence lifetime decay characteristics during chromatographic elution. This feedback enables dynamic adjustment of fraction collection decisions, allowing the system to achieve high purity through intelligent pooling strategies rather than overly narrow fractionation, thereby maintaining both purity and yield
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 approach allows for real-time, accurate quantification of individual protein species even when they fully co-elute, improving process control and product purity by providing a linear and proportional signal across a wide range of concentrations, thus enhancing yield and reducing contamination risks.
Implementation Method 1
Each protein species has an intrinsic fluorescence decay signature. The method comprises: addressing the mixture with one or more pulses of light, wherein the light has a wavelength in the 240-295 nm range, preferably in the 250-280 nm range
Implementation Method 2
taking a series of measurements of the fluorescence intensity of the mixture at a series of time points where the time interval between a fluorescence measurement and a preceding light pulse is recorded
Data Source
AI summary
A method of quantifying the concentration of a protein of interest, or the concentration of a conformational state of the protein of interest, in a mixture, wherein the protein of interest or conformational state has an intrinsic fluorescence decay signature. The method comprises: addressing the mixture with one or more pulses of light, wherein the light has a wavelength in the 240-295 nm range, preferably in the 250-280 nm range, further preferably wherein the laser light has a wavelength of 266 nm. The method further comprises: taking a series of measurements of the fluorescence intensity of the mixture at a series of time points where the time interval between a fluorescence measurement and a preceding light pulse is recorded. The series of measurements comprises measurements for which the time intervals differ from each other by less than a nanosecond, and where the difference between largest and smallest time intervals is at least 10 nanoseconds (ns) and/or a sufficient time to detect a decay of the fluorescence intensity towards a baseline level, such that the series of measurements defines a fluorescence decay curve. The method further comprises quantifying the concentration of a protein of interest or conformational state of the protein of interest in the sample by reference to the fluorescence decay curve.