# US 10,119,944 B2 · Materials for hydrophilic interaction chromatography and processes for preparation and use thereof for analysis of glycoproteins and glycopeptides

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- Number: US 10,119,944 B2
- Document ID: US10119944B2
- Status: Granted
- Kind: B2
- Application number: 14/576,682
- Filed: 2014-12-19
- Published: 2015-07-23
- Granted: 2018-11-06
- Earliest claimed: 2013-12-24
- Assignees: Waters Technologies Corporation
- Applicants: Waters Technologies Corporation
- Inventors: Matthew A. Lauber; Stephan M. Koza; Pamela C. Iraneta; Kevin D. Wyndham
- Web page: https://patentgrep.com/patent/US10119944B2

## CPC

- C08F 230/085: {the monomer being a polymerisable silane, e.g. (meth)acryloyloxy trialkoxy silanes or vinyl trialkoxysilanes}
- B01D 15/305: {Hydrophilic interaction chromatography [HILIC]}
- B01J 20/286: Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J 20/3212: {consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds}
- B01J 20/3272: {Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds}
- C08L 33/14: of esters containing halogen, nitrogen, sulfur, or oxygen atoms in addition to the carboxy oxygen
- G01N 30/06: Preparation
- B01J 2220/82: Shaped bodies, e.g. monoliths, plugs, tubes, continuous beds
- G01N 2030/027: {Liquid chromatography}
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 1

A porous material comprising a copolymer comprising at least one hydrophilic monomer and a first poly-amide bonded phase and a second poly-amide bonded phase, wherein:
the average pore diameter is greater than or equal to about 200 Å;
the porous material comprises:
a porous or nonporous organic-inorganic hybrid core comprising an aliphatic bridged silane,
at least one hydrophilic monomer grafted to the core,
and the first and the second poly-amide bonded phase bonded to the at least one hydrophilic monomer and forming a chemically modified chromatographic surface wherein there is a distinct transition from the core to the chromatographic surface;
and wherein the second poly-amide bonded phase is derived from N,N-methylenebisacrylamide, N,N-ethylenebisacrylamide, N,N-propylenebisacrylamide, N,N-butylenebisacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide, or 1,4-bis(acryloyl)piperazine.

### Claim 2 (depends on 1)

The porous material of claim 1, wherein the porous material comprises a porous particle that comprises said copolymer.

### Claim 3 (depends on 1)

The porous material of claim 1, wherein the porous material comprises a porous monolith that comprises said copolymer.

### Claim 4 (depends on 1)

The porous material of claim 1, wherein said hydrophilic monomer is 3-methacryloxypropyltrichlorosilane, 3-methacryloxypropylmethyldichlorosilane, 3-methacryloxypropyldimethylchlorosilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyldimethylmethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyldimethylethoxysilane, 3-acryloxypropyltrichlorosilane, 3-acryloxypropylmethyldichlorosilane, 3-acryloxypropyldimethylchlorosilane 3-acryloxypropyltrimethoxysilane, 3-acryloxypropylmethyldimethoxysilane, 3-acryloxypropyldimethylmethoxysilane, 3-acryloxypropyltriethoxysilane, 3-acryloxypropylmethyldiethoxysilane, 3-acryloxypropyldimethylethoxysilane, styrylethyltrichlorosilane, styrylethylmethyldichlorosilane, styrylethyldimethylchlorosilane, styrylethyltrimethoxysilane, styrylethylmethyldimethoxysilane, styrylethyldimethylmethoxysilane, styrylethyltriethoxysilane, styrylethylmethyldiethoxysilane, styrylethyldimethylethoxysilane, vinyltriethoxysilane, vinyltrimethoxysilane, N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane, (3-acryloxypropyl) trimethoxysilane, O-(methacryloxyethyl)-N-(triethoxysilylpropyl)urethane, N-(3-methacryloxy-2-hydroxypropyl)-3-aminopropyl triethoxysilane, methacryloxy methyltriethoxysilane, methacryloxymethyl trimethoxysilane, methacryloxypropy methyldiethoxysilane, methacryloxypropyl methyldimethoxysilane, methacryl oxypropyltris (methoxyethoxy)silane, 3-(N-styrylmethyl-2-aminoethylamino) propyltrimethoxysilane hydrochloride,

### Claim 5 (depends on 1)

The porous material of claim 1, wherein said first poly-amide bonded phase is derived from acrylamide, divinylbenzene, styrene, ethylene glycol dimethacrylate, 1-vinyl-2-pyrrolidinone and tert-butylmethacrylate, methacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide, N,N′-ethylenebisacrylamide, N,N′-methylenebisacrylamide, butyl acrylate, ethyl acrylate, methyl acrylate, 2-(acryloxy)-2-hydroxypropyl methacrylate, N,N-bis(2-cyanoethyl)acrylamide, N-acryloyltris(hydroxymethyl)aminomethane, 3-(acryloxy)-2-hydroxypropyl methacrylate, trimethylolpropane triacrylate, trimethylolpropane ethoxylate triacrylate, tris[(2-acryloyloxy)ethyl]isocyanurate, acrylonitrile, methacrylonitrile, itaconic acid, methacrylic acid, trimethylsilylmethacrylate, N-[tris(hydroxymethyl)methyl]acrylamide, (3-acrylamidopropyl)trimethylammonium chloride, [3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide inner salt,

### Claim 6 (depends on 1)

The porous material of claim 1, wherein the first poly-amide bonded phase is present in 35 to 99 mole % of the poly-amide bonded phases.

### Claim 7 (depends on 1)

The porous material of claim 1, wherein the second poly-amide bonded phase is present in 65 to 1 mole % of the poly-amide bonded phases.

### Claim 8 (depends on 1)

The porous material of claim 1, wherein the porous material has a median pore diameter of about 100 Å to about 1000 Å.

### Claim 9 (depends on 1)

The porous material of claim 1 wherein the porous material has a nitrogen content of said material is from about 0.5% N to about 20% N as measured by combustion analysis.

### Claim 10 (depends on 1)

A method for removing or isolating a component from a mixture comprising:
contacting the mixture with a chromatographic material comprising the porous material according to claim 1, to thereby remove or isolate the component from the mixture.

### Claim 11 (depends on 1)

A hydrophilic interaction chromatography cartridge comprising the porous material according to claim 1.