# US 10,246,484 B2 · Method for purifying recombinant protein

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- Number: US 10,246,484 B2
- Document ID: US10246484B2
- Status: Granted
- Kind: B2
- Application number: 15/034,821
- Filed: 2014-11-03
- Published: 2016-10-06
- Granted: 2019-04-02
- Earliest claimed: 2013-11-06
- Assignees: Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd.
- Applicants: Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd.
- Inventors: Hui Hu; Yunbin Zhu; Meiling Zang
- Web page: https://patentgrep.com/patent/US10246484B2

## CPC

- C07K 1/18: Ion-exchange chromatography
- C07K 16/065: {Purification, fragmentation}
- C07K 16/32: against translation products of oncogenes
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- 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

A method is provided for purifying a recombinant protein from a mixture comprising the recombinant protein and related proteins, comprising the steps of: A. using a first equilibrating buffer in a first conductivity and pH to make the recombinant protein bind to an ion exchange medium; B. using a second equilibration buffer in a second conductivity and pH to continually equilibrate the ion exchange medium bound to the protein; C. using a washing liquid in a third conductivity and a gradually increasing pH to wash the ion-exchange medium, and eluting the first category-related proteins; D. using a first eluent in a fourth conductivity and pH to elute the ion exchange medium, and eluting the target recombinant protein; and E. using a second eluent in a fifth conductivity and pH to continually elute the ion exchange medium, and eluting the second category-related proteins.

## Claims

### Claim 1

A method for purifying a recombinant protein from a mixture comprising the recombinant protein and its related proteins, which comprises the following steps performed sequentially:
A. binding the recombinant protein to an ion exchange medium using a first equilibration buffer, wherein the first equilibration buffer is at a first conductivity and pH;
the ion exchange medium is a cation exchange medium;
B. equilibrating the protein-bound ion exchange medium continually using a second equilibration buffer, wherein the second equilibration buffer is at a second conductivity and pH;
the conductivity of the second equilibration buffer is less than that of the first equilibration buffer, but the pH of both are the same;
C. washing the ion exchange medium using a wash buffer which has different pH, so as to elute a first class of the related proteins from the ion exchange medium, wherein the wash buffer is at a third conductivity and a gradually increased pH;
the conductivity of the wash buffer is less than that of the first equilibration buffer, but the pH of the wash buffer is greater than that of the first and/or second equilibration buffer;
D. washing the ion exchange medium using a first elution buffer, so as to elute the target recombinant protein from the ion exchange medium, wherein the first elution buffer is at a fourth conductivity and pH;
the pH of the first elution buffer is greater than that of the wash buffer, but the conductivity of both are essentially the same; and
E. then washing the ion exchange medium using a second elution buffer, so as to elute a second class of related proteins from the ion exchange medium, wherein the second elution buffer is at a fifth conductivity and pH;
the pH and/or conductivity of the second elution buffer are/is greater than that of the first elution buffer.

### Claim 2 (depends on 1)

The method of claim 1, characterized in that, the cation exchange medium is a filler having a functional group SO3− that is bound to different substrates.

### Claim 3 (depends on 1)

The method of claim 1, characterized in that, the elution of the first class of the related proteins and the target protein is achieved by changing the pH of the wash buffer and the first elution buffer.

### Claim 4 (depends on 3)

The method of claim 3, characterized in that, the change of pH of the wash buffer and the first elution buffer is achieved by adjusting the mixed ratio of two salt-containing buffers that have different pH.

### Claim 5 (depends on 4)

The method of claim 4, characterized in that, the change of pH of the wash buffer and the first elution buffer is achieved by changing 25% Na2HPO4 pH7.5+75% Na2HPO4 pH9.3-9.4 to 15% Na2HPO4 pH7.5+85% Na2HPO4 pH9.3-9.4.

### Claim 6 (depends on 1)

The method of claim 1, characterized in that, in steps A-D, the pH is gradually increased.

### Claim 7 (depends on 1)

The method of claim 1, characterized in that, the recombinant protein and its related proteins have different PI, wherein the PI of the first class of the related proteins is less than that of the recombinant protein, and the PI of the recombinant protein is less than that of the second class of related proteins.

### Claim 8 (depends on 1)

The method of claim 1, characterized in that, the first class of the related proteins is an acidic variant of the recombinant protein, which is defined as a substance which has a less retention time than the target protein on CEX-HPLC; the second class of the related protein is a basic variant of the recombinant protein, which is defined as a substance which has a greater retention time than the target protein on CEX-HPLC.

### Claim 9 (depends on 1)

The method of claim 1, characterized in that, the recombinant protein is an antibody.

### Claim 10 (depends on 9)

The method of claim 9, characterized in that, the antibody is selected from the group consisting of trastuzumab, pertuzumab and other HER2 antigen-binding recombinant antibodies.

### Claim 11 (depends on 1)

The method of claim 1, characterized in that, the method further comprises subjecting the mixture comprising the recombinant protein and its related proteins to one or more purification steps, either before, during, or after the ion exchange chromatography, so as to obtain a homogeneous preparation of the recombinant protein.

### Claim 12

A method for purifying a recombinant anti-HER2 antibody from a mixture comprising the recombinant anti-HER2 antibody and its related proteins, which comprises the following steps performed sequentially:
a). binding the recombinant protein to a cation exchange medium using a first equilibration buffer, wherein the first equilibration buffer is at a first conductivity and pH;
b). then equilibrating the protein-bound cation exchange medium using a second equilibration buffer, wherein the second equilibration buffer is at a second conductivity and pH;
c). washing the ion exchange medium using a wash buffer which has different pH, so as to elute the first class of the related proteins from the cation exchange medium, wherein the wash buffer is at a third conductivity and a gradually increased pH;
d). washing the ion exchange medium using a first elution buffer, so as to elute the target recombinant protein from the cation exchange medium, wherein the first elution buffer is at a fourth conductivity and pH; and
e). washing the ion exchange medium using a second elution buffer, so as to elute the second class of the related proteins from the cation exchange medium, wherein the second elution buffer is at a fifth conductivity and pH;
the conductivity of the wash buffer is less than that of the first equilibration buffer, but the pH of the wash buffer is greater than that of the first and/or second equilibration buffer; the pH of the first elution buffer is greater than that of the wash buffer, and the conductivity of the first elution buffer is basically the same as that of the wash buffer; the pH and conductivity of the second elution buffer are/is greater than that of the first elution buffer.

### Claim 13 (depends on 12)

The method of claim 12, characterized in that, the cation exchange medium is a filler having a functional group SO3− which is bound to different substrates.

### Claim 14 (depends on 13)

The method of claim 13, characterized in that, the cation exchange medium is at least one selected from the group consisting of carboxy-methyl-cellulose, sulphopropyl immobilized on agarose, sulphopropyl immobilized on polystyrene/divinyl benzene, sulfonyl immobilized on agarose, and sulphopropyl immobilized on hydrophilic polyacrylamides.

### Claim 15 (depends on 12)

The method of claim 12, characterized in that, the first equilibration buffer is a salt-containing buffer, of which the buffer is 10-50 mmol/L acetic acid buffer, the salt is 40-60 mmol/L sodium chloride or ammonium sulfate, the pH is 4-6, and the conductivity is 8-13 ms/cm; the second equilibration buffer is a salt-free buffer, of which the buffer is 10-50 mmol/L acetic acid buffer, the pH is the same as that of the first equilibration buffer, and the conductivity is 1-2 ms/cm.

### Claim 16 (depends on 12)

The method of claim 12, characterized in that, the change of pH of the wash buffer and the first elution buffer is achieved by adjusting the mixed ratio of two salt-containing buffers that have different pH, and achieved by changing 25% Na2HPO4 pH7.5+75% Na2HPO4 pH9.3-9.4 to 15% Na2HPO4 pH7.5+85% Na2HPO4 pH9.3-9.4.

### Claim 17 (depends on 12)

The method of claim 12, characterized in that, the recombinant anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab and other HER2 antigen-binding recombinant antibodies.

### Claim 18 (depends on 12)

The method of claim 12, characterized in that, the method further comprises subjecting the mixture comprising the recombinant anti-HER2 antibody and its related proteins to one or more purification steps, either before, during, or after the cation exchange chromatography, so as to obtain a homogeneous preparation of the recombinant anti-HER2 antibody.