# US 12,007,396 B2 · Methods for characterizing low-abundance host cell proteins

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- Number: US 12,007,396 B2
- Document ID: US12007396B2
- Status: Granted
- Kind: B2
- Application number: 17/217,518
- Filed: 2021-03-30
- Published: 2021-09-30
- Granted: 2024-06-11
- Earliest claimed: 2020-03-30
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: I-Hsuan Chen; Hui Xiao; Ning Li
- Web page: https://patentgrep.com/patent/US12007396B2

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- G01N 33/6854: {Immunoglobulins}
- G01N 30/72: Mass spectrometers {(mass spectrometers per se H01J49/00)}
- G01N 2030/8831: {involving peptides or proteins}
- G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material
- 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 present application provides methods and systems to identify host cell protein (HCP) impurities in a sample containing high-abundance proteins. The HCP impurities can be enriched using interacting peptide ligands which have been attached to solid support. The HCP impurities can be eluted from the solid support using solution containing phase transfer surfactants. The isolated HCP impurities can be digested to generate components of the isolated HCP impurities which can subsequently be identified using a mass spectrometer.

## Claims

### Claim 1

A method of identifying at least one host cell protein (HCP) impurity in a sample, comprising:
contacting a sample to a solid support,
wherein said sample includes at least one high-abundance peptide or protein and at least one HCP impurity,
wherein interacting peptide ligands have been attached to said solid support, and
wherein said at least one HCP impurity can bind to said interacting peptide ligands;
washing said solid support using an elution solution to produce an eluate, wherein said elution solution includes at least one surfactant;
subjecting said eluate to an enzymatic digestion condition to generate a peptide digest;
subjecting said peptide digest to acidic conditions to produce an acidified peptide digest;
centrifuging said acidified peptide digest to produce a surfactant-depleted peptide digest; and
subjecting said surfactant-depleted peptide digest to mass spectrometry analysis to identify said at least one HCP impurity.

### Claim 2 (depends on 1)

The method of claim 1, wherein the at least one surfactant is a phase transfer surfactant, an ionic surfactant, an anionic surfactant, a cationic surfactant, or combinations thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one surfactant is sodium deoxycholate, sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium dodecylbenzene sulphonate, or combinations thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein a concentration of the at least one high-abundance peptide or protein is about at least 1000 times, 10,000 times, 100,000 times or 1,000,000 times higher than a concentration of the at least one HCP impurity.

### Claim 5 (depends on 1)

The method of claim 1, wherein the interacting peptide ligands are a library of combinatorial hexapeptide ligands.

### Claim 6 (depends on 1)

The method of claim 1, wherein the at least one HCP impurity is quantified using the mass spectrometer, wherein a detection limit of the at least one HCP impurity is about 0.05-0.1 ppm.

### Claim 7 (depends on 1)

The method of claim 1, wherein the at least one high-abundance peptide or protein is an antibody, a bispecific antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a protein pharmaceutical product, or a drug.

### Claim 8 (depends on 1)

The method of claim 1, wherein an enzyme of the enzymatic digestion condition is trypsin.

### Claim 9 (depends on 1)

The method of claim 1, wherein the mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, wherein the mass spectrometer is coupled to a liquid chromatography system.

### Claim 10 (depends on 1)

The method of claim 1, wherein the mass spectrometer is capable of performing LC-MS (liquid chromatography-mass spectrometry) or a LC-MRM-MS (liquid chromatography-multiple reaction monitoring-mass spectrometry) analyses.

### Claim 11

A system for identifying at least one host cell protein (HCP) impurity in a sample, comprising:
a solid support;
interacting peptide ligands,
wherein said interacting peptide ligands are attached to said solid support, and
wherein said at least one HCP impurity can bind to the interacting peptide ligands;
an elution solution comprising at least one surfactant;
an enzymatic digestion solution;
an acidic solution;
a centrifuge; and
a mass spectrometer capable of identifying said at least one HCP impurity.

### Claim 12 (depends on 11)

The system of claim 11, wherein the at least one surfactant is a phase transfer surfactant, an ionic surfactant, an anionic surfactant, a cationic surfactant, or combinations thereof.

### Claim 13 (depends on 11)

The system of claim 11, wherein the at least one surfactant is sodium deoxycholate, sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium dodecylbenzene sulphonate, or combinations thereof.

### Claim 14 (depends on 11)

The system of claim 11, wherein a concentration of the at least one high-abundance peptide or protein is about at least 1000 times, 10,000 times, 100,000 times or 1,000,000 times higher than a concentration of the at least one HCP impurity.

### Claim 15 (depends on 11)

The system of claim 11, wherein the interacting peptide ligands are a library of combinatorial hexapeptide ligands.

### Claim 16 (depends on 11)

The system of claim 11, wherein a detection limit of the at least one HCP impurity is about 0.05-0.1 ppm.

### Claim 17 (depends on 11)

The system of claim 11, wherein the at least one high-abundance peptide or protein is an antibody, a bispecific antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a protein pharmaceutical product, or a drug.

### Claim 18 (depends on 11)

The system of claim 11, wherein an enzyme of the enzymatic digestion solution is trypsin.

### Claim 19 (depends on 11)

The system of claim 11, wherein the mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, wherein the mass spectrometer is coupled to a liquid chromatography system.

### Claim 20 (depends on 11)

The system of claim 11, wherein the mass spectrometer is capable of performing LC-MS (liquid chromatography-mass spectrometry) or a LC-MRM-MS (liquid chromatography-multiple reaction monitoring-mass spectrometry) analyses.