Native Mass Spectrometry for Intact Antibody-Drug Conjugate Analysis
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Solution Overview
Problem
Current methods for measuring the mass of intact antibody-drug conjugates (ADCs) are inadequate due to protein denaturation and time-consuming processes, particularly in high-throughput applications, as existing techniques fail to maintain the native structure of ADCs during mass spectrometry analysis.
Innovation Solution
A method involving the use of size-exclusion chromatography coupled with mass spectrometry, where ADCs are maintained in non-denaturing conditions and introduced into a mass spectrometer in a volatile salt solution, allowing for direct and rapid determination of the intact mass without denaturation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If rp-HPLC desalting is used to measure ADC mass, then the mass of constituent fragments can be determined, but the ADC dissociates into heavy and light chains and protein structure is destroyed
Solution Approach 1:
The invention changes the physical and chemical parameters of the mass spectrometry analysis by using native conditions (aqueous buffer, ambient or low temperature, no denaturing agents) instead of traditional denaturing conditions. This allows the ADC to maintain its intact quaternary structure while still enabling mass measurement through native electrospray ionization
Solution Approach 2:
The invention replaces the mechanical/chemical separation process of rp-HPLC with a direct mass spectrometry measurement approach. Instead of physically separating and then measuring fragments, the invention directly measures the intact ADC mass in solution, substituting a chemical separation mechanism with a direct physical measurement mechanism
2Stability of the object's composition
If nanospray ESI-MS is used to measure intact ADC mass, then protein structure is preserved, but the analysis process is very time consuming and not amenable to high throughput
Solution Approach 1:
The invention introduces dynamic flow conditions by using a continuous flow native electrospray system instead of static nanospray. The ability to adjust flow rates and maintain continuous sample introduction enables higher throughput while preserving native structure through controlled dynamic conditions rather than static slow analysis
3Extent of automation
If standard ESI is used with heated sheath and desolvation gases, then ionization efficiency is improved, but protein structure is disrupted and denaturation occurs
Solution Approach 1:
The invention changes the temperature parameter from high (heated sheath and desolvation gases) to ambient or low temperature. This parameter change maintains sufficient ionization efficiency for mass measurement while eliminating the thermal denaturation that occurs with standard ESI heating conditions
Solution Approach 2:
The invention uses volatile salts as an intermediary substance that facilitates ionization in the electrospray process without requiring high temperature. The volatile salts enable efficient ion formation at ambient or low temperatures, acting as a mediator between the sample and the mass spectrometer detector without causing thermal denaturation
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
Enables the precise and rapid measurement of the intact mass of ADCs, maintaining their native structure and improving analysis efficiency, overcoming the limitations of previous methods by preventing denaturation and reducing analysis time.
Implementation Method 1
The present invention provides a method for detecting a mass of a non covalently associated antibody-drug conjugate compound. The method includes the steps of providing a non covalently associated and non denatured antibody-drug conjugate compound in a volatile salt and free of a non-volatile salt
Implementation Method 2
introducing the antibody-drug conjugate compound into a mass spectrometer; and directly establishing the mass of the antibody-drug conjugate compound by mass spectrometry
Data Source
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AI summary
The present invention provides methods and systems for the rapid determination of the intact mass of non-covalently associated antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. By analyzing the antibody-drug conjugate (ADC) using native desalting conditions, the intact-bivalent structure of the ADC, which ordinarily would decompose as a consequence of denaturing chromatographic conditions typically used for LCMS, is maintained. The mass of the desalted ADC is subsequently determined using desolvation and ionization ESI-MS conditions. The methods described herein provide for direct measurement of the intact mass of an ADC conjugated at interchain cysteine residues. The methods described herein also provide for the relative quantitation of the individual ADC species.