Poly-Amide HILIC Stationary Phases for Wide-Pore Glycoform Separation
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Solution Overview
Problem
Existing hydrophilic interaction chromatography (HILIC) methods face challenges in efficiently separating and analyzing large biomolecules, such as glycoproteins, due to limited availability of wide-pore materials and poor resolution, leading to issues like on-column aggregation and low sensitivity in chromatographic detection of glycan moieties.
Innovation Solution
Development of poly-amide bonded HILIC stationary phases with larger pore diameters (≥200 Å) and novel chromatographic methods for high-resolution separation of large biomolecules, including glycoproteins, using materials with hydrophilic monomers and poly-amide bonded phases, which provide desirable retentivity and selectivity for glycans while minimizing interference from other analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional HILIC stationary phases with smaller pore diameters are used, then the chromatographic resolution for small molecules is improved, but the separation efficiency for large biomolecules deteriorates due to restricted mass transfer and on-column aggregation
Solution Approach 1:
The patent applies porous materials with specifically engineered large pore diameters (≥200 Å) to enable efficient mass transfer and accommodate large biomolecules like glycoproteins while maintaining chromatographic resolution. The porous structure allows analytes to access internal surface area without restricted diffusion, resolving the contradiction between resolution and separation efficiency for large molecules.
Solution Approach 2:
The patent changes the critical parameter of pore diameter from conventional small sizes to large sizes (≥200 Å), fundamentally altering the mass transfer characteristics and enabling simultaneous achievement of high resolution and efficient separation for large biomolecules by matching pore dimensions to analyte size.
2Reliability
If wide-pore HILIC materials are used to improve separation of large biomolecules, then the mass transfer and resolution for glycoproteins are improved, but the availability and selection of suitable materials are limited
Solution Approach 1:
The patent employs composite materials combining poly-amide bonded phases with porous support structures of ≥200 Å pore diameter. This composite approach creates stationary phases that integrate the hydrophilic interaction capabilities of poly-amide bonding with the enhanced mass transfer properties of large pore structures, expanding material availability for glycoprotein separation.
Solution Approach 2:
The patent systematically develops and applies porous materials with large pore diameters (≥200 Å) as the foundation for HILIC stationary phases, ensuring adequate pore size is built into the material structure itself, thereby providing a versatile platform for separating various large biomolecules including glycoproteins, antibodies, and other proteinaceous compounds.
3Ease of operation
If conventional HILIC phases are used for glycan analysis, then the method is simple, but the sensitivity for detecting glycan moieties is low due to on-column aggregation
Solution Approach 1:
The patent uses porous materials with large pore diameters (≥200 Å) to prevent on-column aggregation of glycoproteins and glycans during chromatographic separation. The adequate pore space eliminates aggregation issues while maintaining method simplicity, thereby achieving high detection sensitivity for glycan moieties without complicating the analytical procedure.
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
The solution achieves high-resolution separation of large biomolecules, including glycoproteins, with improved chromatographic efficiency and sensitivity, effectively resolving protein and peptide glycoforms by reducing on-column aggregation and enhancing detection sensitivity.
Implementation Method 1
Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) is a variant of normal phase liquid chromatography... The stationary phase of HILIC is a polar and hydrophilic phase which results in enhanced retention for polar analytes
Implementation Method 2
A mechanism of separating analytes in HILIC can be a combination of partitioning, ion exchange and reverse-phase chromatography
Implementation Method 3
The stationary phase of HILIC is a polar and hydrophilic phase which results in enhanced retention for polar analytes... optimized wide-pore HILIC phases are not available
Data Source
AI summary
The invention relates to poly-amide bonded hydrophilic interaction chromatography (HILIC) stationary phases and novel HILIC methods for use in the characterization of large biological molecules modified with polar groups, known to those skilled in the art as glycans. The invention particularly provides novel, poly-amide bonded materials designed for efficient separation of large biomolecules, e.g. materials having a large percentage of larger pores (i.e. wide pores). Furthermore, the invention advantageously provides novel HILIC methods that can be used in combination with the stationary phase materials described herein to effectively separate protein and peptide glycoforms by eliminating previously unsolved problems, such as on-column aggregation of protein samples, low sensitivity of chromatographic detection of the glycan moieties, and low resolution of peaks due to restricted pore diffusion and long intra/inter-particle diffusion distances.