# US 10,501,525 B2 · Methods for purification of antibodies using aliphatic alcohols

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- Number: US 10,501,525 B2
- Document ID: US10501525B2
- Status: Granted
- Kind: B2
- Application number: 14/423,802
- Filed: 2013-08-27
- Published: 2015-08-13
- Granted: 2019-12-10
- Earliest claimed: 2012-08-28
- Assignees: Novartis AG
- Applicants: Alois Jungbauer; Peter Satzer; Anne-Luise Tscheliessnig
- Inventors: Alois Jungbauer; Peter Satzer; Anne-Luise Tscheliessnig
- Web page: https://patentgrep.com/patent/US10501525B2

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 1/30: by precipitation
- C07K 16/065: {Purification, fragmentation}
- 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

This disclosure relates to methods for isolating antibodies from cell-free culture supernatant.

## Claims

### Claim 1

A method for isolating a protein from a cell culture supernatant, the method comprising:
a) combining a cell culture supernatant with a divalent cation salt under conditions suitable for the precipitation of impurities in the supernatant to produce a primary supernatant;
b) combining the primary supernatant with an aliphatic alcohol under conditions suitable for forming a protein-containing precipitate and isolating the protein-containing precipitate;
c) re-suspending the protein-containing precipitate in a buffer comprising a divalent cation salt under conditions suitable for the precipitation of impurities therefrom to produce a protein-containing solution; and,
d) combining the protein-containing solution with an aliphatic alcohol under conditions suitable for forming a purified protein-containing precipitate and isolating the purified protein-containing precipitate.

### Claim 2 (depends on 1)

The method of claim 1 wherein the aliphatic alcohol is ethanol.

### Claim 3 (depends on 1)

The method of claim 1 wherein the conditions of step a) comprise a phosphate concentration of less than or equal to about 6 mM.

### Claim 4 (depends on 1)

The method of claim 1 wherein the conditions of step c) comprise a phosphate concentration of less than about 6 mM.

### Claim 5 (depends on 1)

The method of claim 1 wherein the conditions in each of the steps comprise a pH of about 6.5 to about 8.5.

### Claim 6 (depends on 1)

The method of claim 1 wherein the divalent cation salt is CaCl2.

### Claim 7 (depends on 1)

The method of claim 1 wherein the conditions of steps a) and c) comprise a conductivity of about 8-80 mS/cm.

### Claim 8 (depends on 1)

The method of claim 1 carried out at a temperature of from about −10° C. to about 4° C.

### Claim 9 (depends on 1)

The method of claim 1 wherein the conditions of steps b) and d) are selected from the group consisting of a pH of about 6.5, a final concentration (v/v) of aliphatic alcohol of about 25%, and a temperature of about −10° C.

### Claim 10 (depends on 1)

The method of claim 1 wherein the protein is an antibody.

### Claim 11 (depends on 10)

The method of claim 10, the method comprising:
a) combining a cell-free culture supernatant with CaCl2 at about pH 8.5 and removing any resulting precipitate to produce a primary supernatant;
b) combining the primary supernatant with ethanol from a stock solution of less than about 50% over a time period of about 60 minutes or a feeding rate of about 30 μl/min or less at about pH 6.5 and isolating the antibody-containing precipitate;
c) re-suspending the antibody-containing precipitate in a buffer comprising CaCl2 at about pH 8.5 and removing any resulting precipitate to produce an antibody-containing solution; and,
d) combining the antibody-containing solution with ethanol from a stock solution of less than about 50% over a time period of about 60 minutes or a feeding rate of about 30 μl/min or less at about pH 6.5 and isolating the purified antibody-containing precipitate.

### Claim 12 (depends on 1)

The method of claim 1 wherein the cell culture supernatant is a cell-free culture supernatant.

### Claim 13

A method for isolating a protein from a cell culture supernatant, the method comprising:
a) combining a cell culture supernatant comprising the protein with an aliphatic alcohol under conditions suitable for forming a protein-containing precipitate and isolating the protein-containing precipitate;
b) re-suspending the protein-containing precipitate in a buffer comprising a divalent cation salt under conditions suitable for the precipitation of impurities therefrom to produce a protein-containing solution; and,
c) combining the protein-containing solution with an aliphatic alcohol under conditions suitable for forming a purified protein-containing precipitate and isolating the purified protein-containing precipitate.

### Claim 14 (depends on 13)

The method of claim 13 wherein the protein is an antibody.

### Claim 15 (depends on 14)

The method of claim 14, the method comprising:
a) combining a primary supernatant derived from a cell-free culture supernatant with ethanol from a stock solution of less than about 50% over a time period of about 60 minutes or a feeding rate of about 30 μl/min or less at about pH 6.5 and isolating the antibody-containing precipitate;
b) re-suspending the antibody-containing precipitate in a buffer comprising CaCl2 at about pH 8.5 and removing any resulting precipitate to produce an antibody-containing solution; and,
c) combining the antibody-containing solution with ethanol from a stock solution of less than about 50% over a time period of about 60 minutes or a feeding rate of about 30 μl/min or less at about pH 6.5 and isolating the purified antibody-containing precipitate.

### Claim 16 (depends on 13)

The method of claim 13 wherein the cell culture supernatant is a cell-free culture supernatant.

### Claim 17 (depends on 13)

The method of claim 13 wherein the aliphatic alcohol is ethanol.

### Claim 18 (depends on 13)

The method of claim 13 wherein the divalent cation salt is CaCl2.

### Claim 19 (depends on 13)

The method of claim 13 wherein the conditions of step b) comprise a phosphate concentration of less than 6 mM.

### Claim 20 (depends on 13)

The method of claim 13 wherein the conditions in each of the steps comprise a pH of about 6.5 to about 8.5.