Online Chromatography-ESI-MS for Low-Sample Protein Characterization

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Solution Overview

Problem

Current methods for characterizing protein biopharmaceuticals face challenges in accurately detecting and quantifying proteins due to similarities in structural and physicochemical properties with their mutated, modified, or cleaved forms, and require large sample amounts for direct analysis.

Innovation Solution

A method combining online chromatography with electrospray ionization mass spectrometry (ESI-MS) is used, where a sample containing the protein is contacted with a chromatographic system using a chromatography resin, washed with a mobile phase to provide an eluent, and then characterized using ESI-MS.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If direct analysis of protein biopharmaceuticals is performed, then large sample amounts are required, but this is undesirable and only possible in selected cases

Engineering Contradiction:

Improvesample amountVSAvoiddetection accuracy

Core Design Contradiction:

Quantity of substanceVSMeasurement precision

Solution Approach 1:

The patent segments the analysis process into two distinct stages: first, chromatographic separation to resolve the protein of interest from impurities and related substances; second, mass spectrometric detection for precise characterization. This segmentation allows the use of small sample amounts because the chromatographic pre-concentration and separation steps enhance the signal-to-noise ratio for the mass spectrometer, eliminating the need for large sample quantities while maintaining high measurement precision

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces chromatography as an intermediary step between the protein sample and the mass spectrometer. The chromatographic system acts as a mediator that concentrates and purifies the protein of interest before it enters the detection system. This intermediary process enables the mass spectrometer to detect and quantify proteins with high precision using minimal sample amounts, as the chromatographic separation enriches the analyte and removes interfering substances

Inventive Principle:

Principle #24Intermediary (Mediator)

2Quantity of substance

If direct analysis of protein biopharmaceuticals is performed, then large sample amounts are required, but isolation is time-consuming and complex

Engineering Contradiction:

Improvesample amountVSAvoidisolation time

Core Design Contradiction:

Quantity of substanceVSLoss of time

Solution Approach 1:

The patent merges the separation and detection functions into a single integrated online chromatography-mass spectrometry system. The chromatographic separation and mass spectrometric detection occur in continuous flow without manual intervention for sample collection and preparation. This merging eliminates the time-consuming isolation steps required in traditional offline methods, as the system automatically separates and detects proteins in real-time, significantly reducing analysis time while maintaining the ability to work with small sample amounts

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent implements continuous flow analysis where the mobile phase continuously carries the protein sample through the chromatographic column and directly into the mass spectrometer. This continuous action eliminates the discrete, time-consuming steps of manual sample collection, transfer, and preparation that characterize traditional isolation methods. The useful action of separation and detection proceeds uninterrupted, reducing total analysis time while enabling work with minimal sample quantities

Inventive Principle:

Principle #20Continuity of useful action

3Measurement precision

If chromatography is used to separate proteins, then resolution is improved, but the system complexity increases due to coupling with mass spectrometer

Engineering Contradiction:

ImproveresolutionVSAvoidsystem complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent employs a mass spectrometer with multi-functionality that can perform both high-resolution mass measurement and structural characterization of proteins. The mass spectrometer serves multiple purposes: it detects the separated proteins from chromatography, determines their molecular weights with high precision, and provides structural information through fragmentation analysis. This multi-functionality consolidates what would otherwise require multiple separate analytical instruments, reducing overall system complexity while maintaining high resolution

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent replaces manual, mechanical sample handling and analysis procedures with an automated online coupled system. The direct online coupling of chromatography to mass spectrometry eliminates the need for manual fraction collection, transfer, and preparation steps. The system uses electronic control and data processing to manage the complex separation and detection processes, substituting mechanical operations with automated electronic systems that reduce operational complexity despite the advanced analytical capabilities

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Measurement precision

If online chromatography with mass spectrometry is used, then sensitivity and resolution are improved, but the device complexity increases

Engineering Contradiction:

ImprovesensitivityVSAvoiddevice complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent introduces a flow splitter as an intermediary device that divides the chromatographic effluent into multiple streams, directing different portions to different detectors simultaneously. This flow splitter enables the system to achieve high sensitivity by concentrating the analyte signal for the mass spectrometer while also providing samples to UV detectors for complementary detection. The intermediary splitter manages the complexity of multi-detector configuration by providing a simple, passive flow distribution mechanism that enhances sensitivity without proportionally increasing device complexity

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

This method enables accurate characterization, identification, and quantification of protein biopharmaceuticals, including impurities and binding stoichiometry, with improved sensitivity and resolution, reducing the need for large sample amounts.

Implementation Method 1

contacting a sample including the protein to a chromatographic system with a chromatography resin, washing said resin using a mobile phase to provide an eluent including the protein

Methodology Applied

Scientific EffectChromatography: Chromatography