Antibody Chain Pairing via ECD and UVPD Disulfide Cleavage
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Solution Overview
Problem
Current methods for determining antibody chain pairing, especially in polyclonal antibody responses, are limited by the inability to accurately separate and sequence heavy and light chains due to intramolecular and intermolecular disulfide bonds, leading to incomplete sequence coverage and reliance on B-cell sequencing and hybridoma methods.
Innovation Solution
The development of top-down and middle-down proteomics approaches using electron capture dissociation (ECD) and ultraviolet photodissociation (UVPD) for efficient disulfide bond cleavage, enabling accurate mass measurement and sequence determination of intact antibody chains and fragments, thereby facilitating direct determination of chain pairing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bottom-up proteomics methods are used for antibody sequencing, then sequencing capability is improved, but chain pairing information is lost
Solution Approach 1:
The patent segments the antibody analysis into two distinct approaches: bottom-up proteomics for sequencing and top-down/middle-down proteomics for pairing determination. By dividing the overall analysis workflow, each method can optimize for its specific function while preserving complementary information.
Solution Approach 2:
The patent implements a nested analytical strategy where bottom-up sequencing results are integrated with top-down mass measurement data. The sequencing information from digested peptides is nested within the broader context of intact chain mass and pairing data, creating a comprehensive analysis framework.
2Measurement precision
If top-down and middle-down approaches are used for direct chain pairing determination, then pairing accuracy is improved, but sequence coverage is limited due to disulfide bonds
Solution Approach 1:
The patent segments the disulfide bond constraints by using enzymatic digestion to break peptide bonds within chains, converting the intact chain problem into smaller peptide fragments that can be sequenced while preserving disulfide-linked pairings for mass measurement.
Solution Approach 2:
The patent introduces enzymatic digestion as an intermediary step that partially breaks down the antibody structure. This mediator allows access to sequence information while maintaining the disulfide bond connections that encode pairing information, enabling both types of data to be obtained.
3Reliability
If B-cell sorting techniques are used to ensure single mAb production, then chain pairing ambiguity is reduced, but productivity and representation of pAb response are significantly decreased
Solution Approach 1:
The patent creates a molecular copy or fingerprint of the pairing information through mass spectrometry measurements. Instead of physically isolating single B-cells, the method captures pairing data through mass spectral signatures that serve as unique identifiers for chain combinations.
Solution Approach 2:
The patent replaces the mechanical B-cell sorting and single-cell isolation process with a mass spectrometry-based analytical system. This substitution eliminates the need for physical cell manipulation while obtaining equivalent pairing information through molecular mass measurements.
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
These methods provide complete sequence coverage of antibody chains, reducing reliance on B-cell sequencing and hybridoma methods, and enable accurate identification of chain pairing in intact antibodies and fragments, enhancing the efficiency of antibody discovery and analysis.
Implementation Method 1
dissociating by a first dissociation method, a light chain of the intact antibody from a heavy chain of the intact antibody to generate a first type of product ions of the light chain
Implementation Method 2
dissociating by a second dissociation method, the light chain of the intact antibody from the heavy chain of the intact antibody to generate a second type of product ions of the light chain
Data Source
AI summary
Methods are described for using a combination of mass spectroscopic and proteomic approaches for identifying the specific pairing of heavy and light chains for an intact antibody, an antibody fragment, a mixture of intact antibodies, or a mixture of antibody fragments.