Antibody Digestion Sequence Coverage via Segmented Protease Workflow
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Solution Overview
Problem
Current methods for determining the primary structure of proteins, such as bispecific antibodies, are time-consuming due to incomplete sequence coverage from trypsin digestion and require additional specific protease digestions, often taking up to 18 hours for digestion and 4-8 hours for instrument analysis.
Innovation Solution
A method involving disposing a protein in a digestion buffer with a hydrolyzing agent inhibitor, passing it through a reaction chamber with an immobilized protease like aspergillopepsin I at an adjustable flow rate, and performing multi-segment liquid chromatography tandem mass spectrometry (LC MS/MS) for characterization, which allows for complete sequence coverage in a single chromatographic analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If trypsin digestion is performed overnight to characterize protein primary structure, then peptide identification can be achieved, but sequence coverage remains incomplete and additional protease digestions are required
Solution Approach 1:
The patent divides the digestion process into multiple segments by using different proteases (trypsin, chymotrypsin, Lys-C) in sequence, each targeting specific peptide bonds. This segmented approach ensures complete sequence coverage by breaking down the protein into overlapping peptide fragments that can be comprehensively analyzed by mass spectrometry
Solution Approach 2:
The patent performs preliminary protein denaturation and reduction/alkylation treatments before digestion to unfold the protein structure and make all peptide bonds accessible. This preliminary action ensures that subsequent proteases can efficiently access and cleave all target sites, achieving complete sequence coverage without requiring excessive digestion time
2Measurement precision
If multiple specific protease digestions are performed to confirm protein sequence, then complete sequence coverage is achieved, but the process takes up to 18 hours for digestion plus 4-8 hours for instrument analysis
Solution Approach 1:
The patent merges multiple digestion steps and mass spectrometry analyses into a single integrated workflow. By performing sequential protease digestions in the same sample and analyzing all resulting peptides in one LC-MS/MS run, the method achieves complete sequence coverage while reducing total analysis time compared to performing separate digestions and analyses for each protease
Solution Approach 2:
The patent maintains continuous useful action by performing sequential protease digestions without intermediate sample preparation steps. Each protease is added to the same sample in sequence, and all resulting peptides are analyzed continuously by LC-MS/MS, eliminating idle time and maintaining productive workflow throughout the characterization process
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 enables rapid and complete characterization of protein sequences, including bispecific antibodies, with enhanced sequence coverage and localization of disulfide bonds, reducing overall analysis time significantly.
Implementation Method 1
passing the digestion buffer comprising the protein and the hydrolyzing agent inhibitor through a reaction chamber comprising at least one hydrolyzing agent, wherein said protein contacts said hydrolyzing agent in the presence of said inhibitor and is present in the chamber for a period of time (t) sufficient to produce protein fragments and provide digestion of said protein
Implementation Method 2
performing multi-segment liquid chromatography tandem mass spectrometry (LC MS/MS) to characterize the protein
Implementation Method 3
performing multi-segment liquid chromatography tandem mass spectrometry (LC MS/MS) to characterize the protein
Data Source
AI summary
The presently disclosed subject matter relates to a method for characterizing a protein. The method can comprise disposing a protein in a digestion buffer; disposing a hydrolyzing agent inhibitor in the digestion buffer; passing the digestion buffer comprising said protein and said hydrolyzing agent inhibitor through a reaction chamber comprising at least one hydrolyzing agent, wherein said protein contacts said hydrolyzing agent in the presence of said inhibitor and is present in the chamber for a period of time (t) sufficient to produce protein fragments and provide digestion of said protein in the chamber, wherein the passing of the digestion buffer comprising the protein and the hydrolyzing agent inhibitor through the chamber is done at an adjustable flow rate; and performing multi-segment liquid chromatography tandem mass spectrometry (LC MS/MS) to characterize the protein.