# US 2022/0034852 A1 · Materials for hydrophilic interaction chromatography and processes for preparation and use thereof for analysis of glycoproteins and glycopeptides

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- Number: US 2022/0034852 A1
- Document ID: US20220034852A1
- Status: Published application
- Kind: A1
- Application number: 17/370,589
- Filed: 2021-07-08
- Published: 2022-02-03
- 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/US20220034852A1

## 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 20/28078: {Pore diameter}
- 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 20/3219: {involving a particular spacer or linking group, e.g. for attaching an active group}
- B01J 20/3272: {Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds}
- C08F 220/54: Amides {, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide}
- C08F 220/56: Acrylamide; Methacrylamide
- C08F 222/385: {Monomers containing two or more (meth)acrylamide groups, e.g. N,N'-methylenebisacrylamide}
- C08F 230/08: containing silicon
- C08L 33/14: of esters containing halogen, nitrogen, sulfur, or oxygen atoms in addition to the carboxy oxygen
- G01N 30/06: Preparation
- G01N 30/7233: {interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04)}
- G01N 2030/027: {Liquid chromatography}
- G01N 2030/525: {surface properties, e.g. porosity}
- 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 at position 1

1-49. (canceled)

### Claim 50

A method for analyzing a glycosylated proteinaceous sample, comprising contacting the sample with a chromatographic material in the presence of a mobile phase eluent to thereby analyze the sample, wherein the chromatographic material is a porous material which comprises at least one hydrophilic monomer and a poly-amide bonded phase.

### Claim 51 (depends on 50)

The method of claim 50, wherein an average pore diameter of the porous material is greater than or equal to about 200 Å.

### Claim 52 (depends on 50)

The method of claim 50, wherein an average pore diameter of the porous material is ranges from about 200 Å to about 450 Å.

### Claim 53 (depends on 50)

The method of claim 50, wherein the porous material has an average pore diameter of about 300 Å.

### Claim 54 (depends on 50)

The method of claim 50, wherein the mobile phase eluent is a high organic eluent.

### Claim 55 (depends on 50)

The method of claim 50, wherein the mobile phase eluent comprises acetonitrile, isopropanol, n-propanol, methanol, ethanol, butanol, water, or a mixture thereof.

### Claim 56 (depends on 50)

The method of claim 50, wherein this mobile phase eluent comprises acetonitrile and water.

### Claim 57 (depends on 50)

The method of claim 50, wherein an ion pairing agent is added to the mobile phase eluent.

### Claim 58 (depends on 57)

The method of claim 57, wherein the ion pairing agent is selected from the group consisting of trifluoroacetic acid, heptafluorobutyric acid, pentafluoropropionic acid, nonafluoropentanoic acid, acetic acid, propanoic acid, and butanoic acid.

### Claim 59 (depends on 57)

The method of claim 57, wherein the ion pairing agent is trifluoroacetic acid.

### Claim 60 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is derived from a glycoprotein.

### Claim 61 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is derived from a glycosylated monoclonal antibody.

### Claim 62 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is derived from an antibody drug conjugate.

### Claim 63 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is prepared by reduction, enzymatic digestion, denaturation, fragmentation, chemical cleavage, or a combination thereof.

### Claim 64 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is prepared by denaturation.

### Claim 65 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is denatured using a denaturant.

### Claim 66 (depends on 65)

The method of claim 65, wherein denaturant is guanidine hydrochloride.

### Claim 67 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is prepared by enzymatic digestion.

### Claim 68 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is derived from a glycosylated monoclonal antibody and wherein the monoclonal antibody is fragmented with an immunoglobulin degrading enzyme.

### Claim 69 (depends on 68)

The method of claim 68, wherein the immunoglobulin degrading enzyme is IdeS (IgG-degrading enzyme of Streptococcus pyogenes).

### Claim 70 (depends on 50)

The method of claim 50, wherein the glycosylated proteinaceous sample is prepared by reduction such that disulfide bonds in the glycosylated proteinaceous sample are reduced into two thiol groups.