Antibody Sequencing via Cathepsin Cleavage
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Solution Overview
Problem
Current methods for antibody sequencing, such as bottom-up and middle-down approaches, face challenges in accuracy and practicality due to the limitations of existing mass spectrometry instrumentation, which struggle with fragmenting large polypeptides and achieving complete coverage, necessitating the development of alternative methods that yield more suitable protein fragments for industry-standard LC-MS/MS instruments.
Innovation Solution
The use of cathepsin L and cathepsin D enzymes to cleave antibodies into specific fragments, such as VL and VH fragments, which are then analyzed by mass spectrometry, allowing for improved sequencing and alignment of antibody fragments, even in the native state without denaturation, to overcome the limitations of existing methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If middle-down approaches are used to generate larger antibody fragments (e.g., F(ab')2, Fc), then software challenges are reduced, but the fragments are difficult to completely fragment using common MS instrumentation
Solution Approach 1:
The patent applies segmentation by using two different proteases (IdeS and pepsin) with different cleavage specificities to break down the antibody into multiple distinct fragment types (F(ab')2, Fc, Fd, VL-CL). This multi-enzyme approach creates a comprehensive fragment library that can be analyzed by MS, resolving the contradiction between reducing software complexity and achieving complete fragmentation coverage.
2Measurement precision
If bottom-up analysis with multiple proteases is used to generate short peptides, then complete sequence coverage can be achieved, but a large number of mass spectra must be generated and assembled
Solution Approach 1:
The patent uses IdeS to perform partial cleavage at the hinge region, generating larger fragments (F(ab')2, Fc) rather than complete digestion into small peptides. This partial action reduces the number of spectra and assembly complexity while still achieving sufficient sequence coverage for antibody characterization, balancing measurement precision with data processing complexity.
3Ease of operation
If IdeS is used to cleave at the hinge region, then F(ab')2 and Fc/2 fragments are generated, but the fragments are about 25 kDa and about 200 amino acids in length which are difficult to fragment completely
Solution Approach 1:
The patent merges two enzymatic cleavage systems: IdeS (which cleaves at the hinge region) and pepsin (which cleaves in the CH1 domain). This combination produces a mixture of fragment sizes including smaller Fd and VL-CL fragments alongside the larger F(ab')2 and Fc fragments. The smaller fragments generated by pepsin are more amenable to complete MS fragmentation, thereby improving overall fragmentation efficiency while maintaining the operational simplicity of enzymatic cleavage.
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 enables efficient and accurate de novo sequencing of antibodies by generating fragments suitable for standard LC-MS/MS instruments, achieving high coverage and sequence determination, particularly for the challenging CDR regions, and can be optimized for specific conditions to maximize cleavage efficiency.
Implementation Method 1
the cathepsin L and/or D cleaves the antibody between the VL and CL and/or between the VH and CH regions to create VL and/or VH antibody fragments and CL and/or CH antibody fragments
Implementation Method 2
after the cleavage, one or more antibody fragments are analyzed by mass spectrometry
Data Source
AI summary
This disclosure relates to new methods for antibody characterization sequencing, such as middle down antibody characterization and sequencing, for example, for de novo antibody sequencing, identifying known antibodies in a sample, or verifying the sequence of antibodies in a sample. In some embodiments, the methods involve exposing antibodies to cathepsin D, cathepsin L, and/or cathepsin D and L, followed by mass spectrometry and sequence identification and deconvolution.