Protein Free Thiol Characterization Using HILIC–Mass Spectrometry

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Solution Overview

Problem

Existing methods for monitoring free thiol groups in therapeutic monoclonal antibodies (mAbs) are inefficient, as they either provide overall thiol levels without site-specific information or are time-consuming, and there is a need for a more accurate and efficient system to characterize intrachain free thiols.

Innovation Solution

A method combining hydrophilic interaction chromatography (HILIC) with mass spectrometry is used to separate and characterize free thiol species in proteins, allowing for quick and confident identification and quantitation of intrachain free thiols.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If labeling strategies are used to measure free thiol groups at the intact level, then overall free thiol level is obtained, but site-specific information is not provided

Engineering Contradiction:

Improvespeed of measurementVSAvoidsite-specific information

Core Design Contradiction:

ProductivityVSLoss of information

Solution Approach 1:

The patent applies segmentation by dividing the intact protein into peptide fragments through enzymatic digestion. This allows the measurement to be performed at the peptide level rather than the intact protein level, thereby obtaining site-specific free thiol information while maintaining measurement efficiency. The mass spectrometer analyzes individual peptide ions to identify which specific cysteine residues contain free thiol groups.

Inventive Principle:

Principle #1Segmentation

2Measurement precision

If labeling strategies are used to measure free thiol groups at the peptide level, then site-specific information is provided, but the process becomes time-consuming

Engineering Contradiction:

Improvesite-specific characterization accuracyVSAvoidmeasurement time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent merges multiple analytical techniques into a unified workflow: enzymatic digestion, hydrophilic interaction chromatography (HILIC) separation, and mass spectrometry detection are combined into a single integrated process. This allows site-specific free thiol characterization to be achieved in a streamlined manner that reduces total analysis time while maintaining high measurement precision through the complementary strengths of each technique.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent utilizes parameter changes in the mass spectrometry detection, specifically monitoring the mass-to-charge ratio to identify characteristic mass shifts that indicate free thiol groups. By detecting these specific mass differences (e.g., +159.99 Da for maleimide labeling), the method achieves rapid and precise site-specific characterization without requiring time-consuming additional steps.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If traditional methods are used to monitor free thiol groups, then overall thiol levels are obtained, but efficiency is reduced

Engineering Contradiction:

Improvemonitoring efficiencyVSAvoidcharacterization accuracy

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent introduces maleimide derivatives as intermediary labeling agents that specifically react with free thiol groups. This intermediary step enables both efficient detection and precise characterization: the maleimide label provides a detectable mass shift for quantitation while also serving as a marker for site-specific identification through mass spectrometry analysis of labeled peptide fragments.

Inventive Principle:

Principle #24Intermediary (Mediator)

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

The method provides site-specific characterization of free thiol groups in proteins, enhancing the accuracy of monitoring and ensuring drug potency and stability.

Implementation Method 1

contacting a sample including a protein and at least one free thiol species thereof to a hydrophilic interaction chromatography (HILIC) system to produce an eluate

Methodology Applied

Scientific EffectHydrophilic interaction chromatography: Chromatography

Implementation Method 2

contacting the eluate to a mass spectrometer to generate a mass measurement of the protein and a mass measurement of the at least one free thiol species

Methodology Applied

Scientific EffectMass spectrometry:

Data Source

PatentUS20250251372A1Methods for characterizing free thiol groups in a protein

Publication Date: 2025.08.07 REGENERON PHARMACEUTICALS INC

AI summary

Described herein are methods of characterizing free thiol species in a protein or peptide. For example, described herein are methods of using hydrophilic interaction chromatography (HILIC) in combination with one or more mass spectrometers to characterize free thiol species in a protein or peptide. Also described herein are methods for characterizing free thiol species in an antibody, such as a therapeutic antibody.