# US 5,710,251 A · Purification of bacterially expressed human interleukin-10

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- Number: US 5,710,251 A
- Document ID: US5710251A
- Status: Granted
- Kind: A
- Application number: 08/495,558
- Filed: 1995-12-18
- Granted: 1998-01-20
- Earliest claimed: 1993-03-05
- Assignees: Schering Corporation
- Inventors: Gary Vellekamp; Susan Cannon-Carlson; John Tang
- Web page: https://patentgrep.com/patent/US5710251A

## CPC

- C07K 14/5428: {IL-10}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Disclosed is a method for purifying Interleukin-10 (IL-10). The method is comprised of subjecting an IL-10 containing solution to cation exchange chromatography, anion exchange chromatography, hydroxyapatite chromatography, and gel filtration chromatography. The present invention is also comprised of a process for separating different IL-10 dimers present in a protein fraction from each other by subjecting the protein fraction to hydroxyapatite chromatography. The present invention is also comprised of a process for separating variants of a protein differing in an N-terminal amino acid sequence present in a protein fraction from each other by subjecting the protein fraction to hydroxyapatite chromatography.

## Claims

### Claim 1

A method for purifying bacterially expressed human Interleukin-10 (IL-10) contained within a solution comprising:
(a) applying a solution containing IL-10 to a cation exchange chromatography column thereby obtaining fractions containing IL-10;
(b) applying the IL-10-containing fractions from step (a) to an anion exchange chromatography column thereby obtaining fractions containing IL-10;
(c) applying the IL-10-containing fractions from step (b) to a hydroxyapatite chromatography chromatography thereby obtaining fractions containing a single isolated dimer of IL-10.

### Claim 2 (depends on 1)

The method of claim 1 wherein the cation exchange chromatography column from step (a) is comprised of sulfonate exchange groups attached to a support matrix.

### Claim 3 (depends on 2)

The method of claim 2 wherein the support matrix is agarose.

### Claim 4 (depends on 1)

The method of claim 1 wherein the anion exchange chromatography column is comprised of quaternary amino ethyl exchange groups attached to a support matrix.

### Claim 5 (depends on 4)

The method of claim 4 wherein the support matrix is agarose.

### Claim 6 (depends on 1)

The method of claim 1 further comprising applying the IL-10-containing fractions obtained from step (c) of claim 1 to a gel filtration chromatography column to obtain dimeric IL-10 substantially free of high and low molecular weight impurities.

### Claim 7 (depends on 6)

The method of claim 6 wherein the material in the gel filtration chromatography column has a fractionation range of from 1 to 600 kDa.

### Claim 8 (depends on 1)

The method of claim 1 wherein prior to step (a) the bacterially expressed human IL-10 is extracted from bacteria in inclusion bodies, denatured and refolded into biologically active human IL-10.

### Claim 9

A method for separating non-acetylated homodimers of Interleukin-10 (IL-10) from acetylated IL-10 homodimers and from acetylated IL-10 heterodimers contained within a solution comprising:
applying the solution to an anion exchange chromatography column under conditions in which the non-acetylated homodimers are separated from the acetylated homodimers and from the acetylated heterodimers.