LC-MS/MS Disulfide Bond Characterization for Monoclonal Antibodies

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for analyzing disulfide bonds in monoclonal antibodies are time-consuming and labor-intensive, and fail to distinguish between native and scrambled disulfide bonds, which can affect the stability and immunogenicity of the antibodies.

Innovation Solution

The development of peptide standards with known disulfide bond patterns, specifically parallel and crossed disulfide bonds, which are used in liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantify scrambled disulfide bonds in monoclonal antibodies, allowing for the modification of cell culture and purification conditions to reduce scrambled bonds to less than 30%.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional methods are used to analyze disulfide bonds, then the analysis can be performed, but the process is time-consuming and labor-intensive

Engineering Contradiction:

Improveanalysis speedVSAvoidtime required for analysis

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent replaces conventional manual analysis methods with automated liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems. This substitution of mechanical/manual processes with automated instrumental analysis significantly reduces analysis time and labor requirements while maintaining or improving detection accuracy for disulfide bond characterization.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent utilizes changes in mass-to-charge ratio (m/z) parameters detected by mass spectrometry to distinguish between native and scrambled disulfide bonds. By monitoring specific m/z transitions and retention times, the system achieves rapid, automated identification of disulfide bond types without requiring lengthy manual procedures.

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If conventional methods are used to analyze disulfide bonds, then the analysis can be performed, but the methods fail to distinguish between native and scrambled disulfide bonds

Engineering Contradiction:

Improvedistinction accuracyVSAvoidinformation about disulfide bond type

Core Design Contradiction:

Measurement precisionVSLoss of information

Solution Approach 1:

The patent segments the disulfide bond analysis into distinct detectable features: native disulfide bonds produce specific m/z transitions and retention times that are differentiated from scrambled disulfide bonds. By separating and detecting these distinct spectral signatures, the method achieves precise identification of disulfide bond types that conventional methods miss.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces peptide standards containing known disulfide bond patterns as intermediaries for comparison. These standards serve as reference markers that enable the detection system to distinguish between native and scrambled disulfide bonds in sample peptides through comparative analysis of retention times and mass spectrometry signals.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If scrambled disulfide bonds are present in monoclonal antibodies, then the antibodies can be produced, but the stability and immunogenicity are affected

Engineering Contradiction:

Improveantibody stabilityVSAvoidimmunogenicity

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent implements feedback control by using LC-MS/MS analysis to detect scrambled disulfide bonds in the antibody product. The detection results provide feedback that enables monitoring and assessment of antibody quality, allowing for identification of batches with harmful scrambled bonds and adjustment of production conditions to improve reliability and reduce immunogenicity.

Inventive Principle:

Principle #23Feedback

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 efficient characterization of disulfide bonds, improving the stability and reducing immunogenicity of monoclonal antibodies by accurately identifying and minimizing scrambled disulfide bonds, thereby enhancing the quality control of protein drug products.

Implementation Method 1

analyzing a sample containing protein drug product peptides and the peptide standards using a liquid chromatography tandem mass spectrometry system (LC-MS2 system)

Methodology Applied

Scientific EffectLiquid chromatography: Chromatography

Implementation Method 2

liquid chromatography tandem mass spectrometry system (LC-MS2 system)

Methodology Applied

Scientific EffectMass spectrometry:

Implementation Method 3

the oxidation reaction is performed using Cu2+

Methodology Applied

Scientific EffectOxidation: Oxidation

Data Source

PatentUS12000839B2Methods for characterizing disulfide bonds

Publication Date: 2024.06.04 REGENERON PHARMACEUTICALS INC

AI summary

Compositions and methods for analyzing disulfide bonds are provided. An exemplary method includes preparing peptide standards having no disulfide bonds, scrambled disulfide bond peptide standards, and native disulfide bond peptide standards according to the sequence of the region of the protein drug product that includes the disulfide bond, digesting a sample of protein drug product into peptides, separating the protein drug product peptides, analyzing the protein drug product peptides and the peptide standards, identifying scrambled and native disulfide bond peptides by retention time, and quantifying the level of scrambled disulfide bond peptides.