Polypeptide Quantitation via Engineered Mutation Mass Spectrometry
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Solution Overview
Problem
Current methods for quantitating therapeutic proteins, such as ligand binding assays and liquid chromatography tandem mass spectrometry (LC-MS/MS), face challenges in accurately measuring therapeutic proteins in both pre-clinical and clinical samples due to limited linear dynamic range, cross-reactivity, and the need for surrogate peptides that are not universally applicable across species.
Innovation Solution
A method involving digestion of a sample containing a polypeptide with an engineered mutation in the antibody heavy chain constant region to produce a peptide fragment, which is then analyzed by mass spectrometry to quantify the polypeptide, utilizing specific enzymes like trypsin, Asp-N, or Glu-C to generate fragments like TTPPVLDSDGSFFLVSK, avoiding interference from endogenous human peptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ligand binding assays are used for quantitation of therapeutic proteins, then sensitivity and throughput are improved, but linear dynamic range is limited and cross-reactivity occurs
Solution Approach 1:
The patent replaces ligand binding assays (biological/mechanical system) with mass spectrometry-based detection (physical/analytical system). This substitution enables accurate quantitation across a broader linear dynamic range while maintaining high sensitivity, as mass spectrometry directly measures peptide abundance without relying on antibody binding characteristics that limit dynamic range and cause cross-reactivity.
2Productivity
If surrogate peptides are used in LC-MS/MS for quantification of therapeutic proteins, then quantification capability is improved, but accuracy deteriorates when surrogate peptide sequences are present in human proteome
Solution Approach 1:
The patent introduces unique amino acid substitutions at specific local positions within the constant region of the therapeutic protein to create peptide sequences that are distinctive to the therapeutic protein and absent from the human proteome. This localized modification ensures that the surrogate peptide can be accurately detected by mass spectrometry without interference from endogenous human peptides, thereby maintaining both quantification capability and accuracy.
3Adaptability or versatility
If universal quantification methods are developed for both pre-clinical and clinical samples, then adaptability is improved, but method complexity increases
Solution Approach 1:
The patent develops a universal mass spectrometry-based quantification method that can accurately measure therapeutic proteins in both pre-clinical (non-human) and clinical (human) samples. By targeting the constant region of the therapeutic protein and introducing unique substitutions, the method achieves species-independent detection, eliminating the need for separate quantification approaches for different sample types and reducing overall method complexity.
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 accurate and sensitive quantitation of therapeutic proteins in both non-human and human samples, overcoming the limitations of existing methods by using engineered mutations to create unique peptide fragments that are not present in native human or animal proteomes, thus reducing cross-reactivity and improving assay specificity.
Implementation Method 1
digestion of a sample containing a polypeptide with an engineered mutation in the antibody heavy chain constant region to produce a peptide fragment, which is then analyzed by mass spectrometry
Implementation Method 2
analyzed by mass spectrometry to quantify the polypeptide
Data Source
AI summary
Provided are methods for quantitating an amount of a polypeptide that comprises a portion of an antibody present in a sample (e.g., a plasma or serum sample) wherein the antibody comprises a constant region (e.g., a heavy chain or light chain constant region) that comprises an engineered mutation.