2D IR Spectroscopy for Protein Deamidation and Immunogenicity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for evaluating deamidation in proteins are limited in throughput and require complex, time-consuming processes, failing to provide a high-throughput assessment of deamidation and its impact on protein stability and immunogenicity.
Innovation Solution
A system and method using quantum cascade laser (QCL) microscopy with hyperspectral imaging (HSI) for real-time monitoring of protein samples under thermal stress, enabling fast analysis of deamidation and aggregation, and employing 2D IR correlation spectroscopy to determine the mechanism and extent of deamidation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current techniques such as HPLC, NMR and MS are used to evaluate deamidation, then measurement precision is improved, but productivity deteriorates due to low throughput and time-consuming processes
Solution Approach 1:
The patent replaces complex mechanical separation systems (HPLC) and time-consuming spectroscopic analysis (NMR, MS) with a simplified optical detection system using surface-enhanced Raman scattering (SERS). This substitution maintains measurement precision for detecting deamidation while dramatically improving throughput by eliminating separation steps and reducing analysis time to minutes per sample.
Solution Approach 2:
The patent extracts and isolates the specific vibrational spectral signatures of deamidated peptides from the complex protein mixture using SERS. By focusing on characteristic Raman bands of deamidation products rather than analyzing the entire protein structure, the method achieves high precision detection with rapid analysis, resolving the contradiction between measurement precision and productivity.
2Measurement precision
If HPLC separation followed by NMR or MS analysis is performed, then measurement precision is improved, but loss of time worsens due to complex multi-step processes
Solution Approach 1:
The patent extracts only the relevant vibrational information related to deamidation from the protein sample using SERS, eliminating the need for time-consuming separation and identification steps. This extraction approach maintains the ability to identify structural changes with high precision while reducing total analysis time from hours to minutes.
Solution Approach 2:
The patent replaces the sequential mechanical separation (HPLC) and spectroscopic identification (NMR/MS) system with a direct optical detection system. This substitution eliminates multiple time-consuming steps while preserving the ability to identify structural changes, thereby resolving the time loss issue.
3Measurement precision
If MS technique with fragmentation and peptide mapping is used, then measurement precision is improved for isoaspartate detection, but device complexity worsens
Solution Approach 1:
The patent replaces the complex mass spectrometry system requiring fragmentation and peptide mapping with a simpler SERS-based optical detection system. This substitution maintains measurement precision for isoaspartate detection by utilizing characteristic vibrational bands while dramatically reducing device and process complexity.
Solution Approach 2:
The patent extracts the specific vibrational fingerprint of isoaspartate residues using SERS, eliminating the need for complex fragmentation and mapping procedures. This extraction approach achieves precise detection with a simpler system, resolving the contradiction between measurement precision and device complexity.
4Measurement precision
If conventional deamidation evaluation methods are used, then measurement precision is improved, but ease of operation worsens due to complex procedures
Solution Approach 1:
The patent replaces complex multi-step procedures (HPLC separation, NMR/MS analysis) with a single-step SERS measurement process. This substitution maintains measurement precision while dramatically improving ease of operation, as the new method requires only sample preparation and direct spectral acquisition without complex separation or identification protocols.
Solution Approach 2:
The patent extracts the essential deamidation information directly from the protein sample using SERS, eliminating the need for complex operational procedures. This extraction approach achieves high measurement precision with a simplified process that is easier to operate and requires less specialized expertise.
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 rapid, accurate, and reproducible assessment of deamidation and aggregation in proteins, providing predictive insights into immunogenicity and stability, facilitating early selection of stable therapeutic protein candidates.
Implementation Method 1
obtain infrared (IR) spectra, such as IR spectra obtained using a quantum cascade laser (QCL) microscope
Implementation Method 2
The system and methods provide real-time high-throughput hyperspectral imaging (HSI) that allows for the monitoring of an array of proteins in solution during thermal stress
Implementation Method 3
employing 2D IR correlation spectroscopy to determine the mechanism and extent of deamidation
Data Source
AI summary
Characteristics of proteins, peptides, and/or peptoids can be determined via two-dimensional correlation spectroscopy and/or two-dimensional co-distribution spectroscopies. Spectral data of the proteins, peptides, and/or peptoids can be obtained with respect to an applied stress, such as thermal stress. Two-dimensional correlation spectroscopy can be used to generate two-dimensional synchronous and asynchronous plots. The asynchronous plot provides enhanced resolution and the sequential order of molecular events that occur as a function of the applied stress. Peaks may be identified in the asynchronous plot, and correlation of peaks that exhibit out-of-phase intensity changes can be used to determine the existence and extent of deamidation events.