# US 12,000,839 B2 · Methods for characterizing disulfide bonds

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- Number: US 12,000,839 B2
- Document ID: US12000839B2
- Status: Granted
- Kind: B2
- Application number: 18/154,401
- Filed: 2023-01-13
- Published: 2023-09-07
- Granted: 2024-06-04
- Earliest claimed: 2019-01-16
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Shunhai Wang
- Web page: https://patentgrep.com/patent/US12000839B2

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- G01N 1/28: Preparing specimens for investigation {including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q}(mounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 {; laboratory gas handling apparatus B01L5/00})
- 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 33/6815: {containing sulfur, e.g. cysteine, cystine, methionine, homocysteine}
- G01N 33/6854: {Immunoglobulins}
- G01N 2030/027: {Liquid chromatography}
- G01N 2030/8831: {involving peptides or proteins}
- G01N 2458/15: Non-radioactive isotope labels, e.g. for detection by mass spectrometry
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Compositions and methods for analyzing disulfide bonds are provided. An exemplary method includes preparing peptide standards having no disulfide bonds, scrambled disulfide bond peptide standards, and native disulfide bond peptide standards according to the sequence of the region of the protein drug product that includes the disulfide bond, digesting a sample of protein drug product into peptides, separating the protein drug product peptides, analyzing the protein drug product peptides and the peptide standards, identifying scrambled and native disulfide bond peptides by retention time, and quantifying the level of scrambled disulfide bond peptides.

## Claims

### Claim 1

A method of producing a protein drug product, comprising:
producing the protein drug product in a cell culture;
identifying scrambled disulfide bonds of the protein drug product, wherein identifying the scrambled disulfide bonds of the protein drug product comprises: preparing peptide standards comprising regions of the protein drug product containing one or more disulfide bonds, wherein the disulfide bonds in the peptide standards are formed through an oxidation reaction that comprises exposing the peptide standard to zinc; and
modifying one or more cell culture, purification or excipient conditions to reduce the amount of crossed hinge disulfide bonds of the protein drug product to less than 1.0%.

### Claim 2 (depends on 1)

The method of claim 1, wherein the one or more cell culture, purification or excipient conditions that are modified are selected from the group consisting of temperature, pH, oxygen levels, reactive oxygen species, surfactants, or combinations thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the protein drug product is selected from the group consisting of an antibody, a fusion protein, recombinant protein, or a combination thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein preparing the peptide standards comprises using a peptide concentration of from 0.1 μg/mL to 0.8 μg/mL.

### Claim 5 (depends on 1)

The method of claim 1, wherein the disulfide bonds in the peptide standards are formed through an oxidation reaction that comprises oxidation by air or chemical.

### Claim 6 (depends on 1)

The method of claim 1, wherein the disulfide bonds in the peptide standards are formed through an oxidation reaction that comprises oxidation with Cu2+.

### Claim 7 (depends on 6)

The method of claim 6, wherein the molar ratio of peptide:Cu2+ for formation of disulfide bonds is 5:1.

### Claim 8 (depends on 1)

The method of claim 1, wherein a first peptide standard comprises a first scrambled disulfide bond, and a second standard comprises a second and different scrambled disulfide bond.

### Claim 9 (depends on 8)

The method of claim 8, wherein the first peptide standard and the second peptide standard have different liquid chromatography retention times.

### Claim 10 (depends on 1)

The method of claim 1, wherein identifying the scrambled disulfide bonds of the product drug product further comprises:
digesting a sample of the protein drug product into peptides;
analyzing a sample containing the protein drug product peptides comprising one or more disulfide bonds and the peptide standards comprising one or more disulfide bonds using an LC-MS2 system; and
comparing retention times of the sample containing protein drug product peptides comprising one or more disulfide bonds with the retention times of the peptide standards comprising one or more disulfide bonds.

### Claim 11 (depends on 10)

The method of claim 10, wherein peptides detected at the retention time of the first peptide standard indicates that the scrambled disulfide bonds of the first peptide standard are present in the protein drug product, and peptides detected at the retention time of the second peptide standard indicates that the scrambled disulfide bonds of the second peptide standard are present in the protein drug product.

### Claim 12 (depends on 10)

The method of claim 10, wherein digesting the sample comprises tryptic digestion or dual-enzyme digestion.

### Claim 13 (depends on 1)

The method of claim 1, wherein the scrambled disulfide bonds are selected from the group consisting of crossed disulfide bonds, crisscrossed disulfide bonds, and intra-chain disulfide bonds.

### Claim 14 (depends on 1)

The method of claim 1, wherein the one or more disulfide bonds are in the hinge region of an antibody.

### Claim 15 (depends on 1)

The method of claim 1, wherein the method further comprises:
quantifying the amount of one or more disulfide bonds present in the protein drug product.

### Claim 16 (depends on 3)

The method of claim 3, wherein the protein drug product is a fusion protein.

### Claim 17 (depends on 16)

The method of claim 16, wherein the fusion protein is an Fc fusion protein.

### Claim 18 (depends on 1)

The method of claim 1, wherein the protein drug product has less than 30% scrambled disulfide bonds.