Dual Capture ADC Analysis via Protease Digestion

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods struggle to effectively analyze transformations in partner molecules attached to the heavy chain of antibody-drug conjugates (ADCs) due to signal-to-noise issues caused by the large size disparity between the partner molecule and the antibody, making it difficult to assess structural integrity and stability.

Innovation Solution

A dual capture method using affinity beads and proteases to break down the ADC into smaller fragments, allowing for mass spectrometric analysis of the partner molecule attached to the Fc fragment, enabling the detection of transformations such as hydrolysis or oxidation events.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If mass spectrometry is used to analyze the partner molecule in the intact ADC, then the analysis can be performed, but the signal-to-noise ratio is poor due to the large size disparity between the antibody and partner molecule

Engineering Contradiction:

Improvesignal-to-noise ratioVSAvoidmolecular weight disparity

Core Design Contradiction:

Measurement precisionVSWeight of moving object

Solution Approach 1:

The antibody molecule is segmented into F(ab')2 fragments and Fc fragments through proteolytic digestion. The Fc fragment containing the partner molecule is separated from the F(ab')2 fragment, allowing mass spectrometry to analyze the smaller Fc fragment and its attached partner molecule without the signal suppression caused by the large intact antibody structure.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The partner molecule is extracted from the context of the full antibody by selectively isolating the Fc fragment through affinity chromatography. This extraction process removes the bulky F(ab')2 portion, concentrating the partner molecule in a smaller Fc fragment that produces detectable signals in mass spectrometry.

Inventive Principle:

Principle #2Taking out (Extraction)

2Measurement precision

If the partner molecule is attached to the heavy chain Fc fragment, then the ADC structure is stable, but analyzing transformations in the partner molecule becomes difficult due to the large molecular size

Engineering Contradiction:

Improvedetection of transformationsVSAvoidmolecular weight of Fc fragment

Core Design Contradiction:

Measurement precisionVSWeight of moving object

Solution Approach 1:

The Fc fragment is further segmented or analyzed as a discrete unit through affinity-based separation. By capturing the Fc fragment on beads and selectively eluting it, the method creates a purified sample where the partner molecule's transformations can be detected without interference from the rest of the antibody structure.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Affinity beads serve as an intermediary medium to isolate and concentrate the Fc fragment. The beads provide a platform for selective capture and subsequent elution, enabling the Fc fragment and its attached partner molecule to be analyzed in a controlled manner that enhances detection sensitivity.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Ease of operation

If proteolytic digestion is performed to break down the ADC, then the partner molecule can be analyzed, but the process becomes more complex

Engineering Contradiction:

Improveanalysis feasibilityVSAvoidnumber of steps

Core Design Contradiction:

Ease of operationVSDevice complexity

Solution Approach 1:

The proteolytic digestion and affinity capture steps are performed as preliminary actions before mass spectrometry analysis. By pre-processing the ADC to isolate the Fc fragment in this manner, the subsequent mass spectrometry measurement becomes simpler and more reliable, as the complex intact ADC structure has already been broken down and purified.

Inventive Principle:

Principle #10Preliminary action

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 allows for precise analysis of transformations in partner molecules, overcoming the size disparity issue and providing insights into the stability and activity of ADCs over time, thereby improving the assessment of ADC integrity and stability.

Implementation Method 1

digesting the ADC captured on the first beads in a digestion medium comprising a protease that cleaves the antibody into an F(ab')2 fragment and an Fc fragment

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Implementation Method 2

capturing the released Fc fragment having the partner molecule attached thereto on second beads having an affinity for the Fc fragment

Methodology Applied

Scientific EffectAffinity binding:

Implementation Method 3

eluting with an acidic eluent the Fc fragment having the partner molecule attached thereto from the second beads

Methodology Applied

Scientific EffectAcidic elution:

Data Source

PatentUS20240377413A1Dual capture method for analysis of antibody-drug conjugates

Publication Date: 2024.11.14 BRISTOL MYERS SQUIBB CO

AI summary

A method for analyzing whether, and to what extent, a transformation has occurred in the partner molecule component of an antibody-drug conjugate.