Capillary Isoelectric Fractionation for Charge Variant Collection
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Solution Overview
Problem
Existing methods for capillary isoelectric focusing (CIEF) fractionation, such as hyphenated CIEF-MS, face limitations in analyzing charge variants of proteins due to restricted downstream analysis methods, performance compromise with additives, and poor resolution, sensitivity, and operational complexity.
Innovation Solution
A novel icIEF fractionation system that enables semi-automatic separation, visualization, and fractionation of analytes into collection wells, allowing further processing without restrictions, using capillary electrophoresis and chemical mobilizers for selective elution based on isoelectric points.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hyphenated CIEF-MS is used for fractionation, then mass spectrometry analysis can be performed, but downstream analysis methods are restricted and performance is compromised by additives
Solution Approach 1:
The invention extracts the fractionation capability from the hyphenated CIEF-MS system, allowing fractions to be collected and subjected to various downstream analyses independently. This separates the fractionation function from the mass spectrometry detection function, enabling versatile downstream processing without being constrained by MS-specific additives or methods.
Solution Approach 2:
The collected fractions can be used for multiple different downstream analyses including native analysis using ZipChip, mass spectrometry, and other characterization methods. This multi-functional approach allows the same fractionated samples to serve various analytical purposes without being restricted to a single detection method.
2Productivity
If conventional CIEF fractionation is used, then sample analysis can be performed, but resolution and sensitivity are poor
Solution Approach 1:
The invention employs dynamic field asymmetric waveforms during the fractionation process, which allows for enhanced separation resolution and sensitivity. The dynamic field adjustment optimizes the focusing and separation of charge variants, achieving superior resolution compared to conventional static field methods while maintaining efficient analysis throughput.
3Measurement precision
If complex fractionation systems are used, then separation capability is improved, but operational complexity increases
Solution Approach 1:
The system performs automatic fraction collection based on detected analyte peaks, reducing the need for manual intervention. The automated detection and collection process simplifies operation while maintaining high separation resolution, allowing users to benefit from advanced separation capabilities without proportionally increasing operational complexity.
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
Provides high-resolution, sensitive, and efficient fractionation of charge variants, enabling downstream characterization like native analysis using ZipChip, with broad sample matrix compatibility and fast mass spectrometry analysis.
Implementation Method 1
separating, at a second time after the first time, at least a subset of the plurality analytes according to their isoelectric points by applying a voltage across the first running buffer and the second running buffer
Implementation Method 2
separating, at a second time after the first time, at least a subset of the plurality analytes according to their isoelectric points
Implementation Method 3
placing the second end of the capillary into a well including a chemical mobilizer at a third time after the first time to mobilize and selectively elute an analyte from the plurality of analytes from the capillary and into the well
Data Source
AI summary
Embodiments include systems, apparatuses, and methods to efficiently separate analytes in a sample and elute fractions of the separated analytes. In some embodiments, a method includes introducing a sample in a capillary with a first end ionically coupled to a first running buffer and a second end ionically coupled to a second running buffer to form a pH gradient. The method includes applying a voltage between the first running buffer and the second running buffer, to separate a plurality of analytes in the sample. The method includes disposing the second end of the capillary in a collection well including a chemical mobilizer and applying a voltage to elute one or more analytes from the plurality of analytes in the sample, that have been separated, into the collection well. Embodiments include detection methods to monitor separation of analytes, mobilization of analytes, and/or elution of fractions containing analytes.