# US 10,246,506 B2 · Methods for reducing levels of protein-contaminant complexes and aggregates in protein preparations by treatment with electropositive organic additives

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- Number: US 10,246,506 B2
- Document ID: US10246506B2
- Status: Granted
- Kind: B2
- Application number: 14/555,224
- Filed: 2014-11-26
- Published: 2015-05-28
- Granted: 2019-04-02
- Earliest claimed: 2012-05-31
- Assignees: Agency for Science, Technology and Research
- Applicants: Agency for Science, Technology and Research
- Inventors: Peter Gagnon
- Web page: https://patentgrep.com/patent/US10246506B2

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 1/145: {by extraction or solubilisation}
- C07K 1/20: Partition-, reverse-phase or hydrophobic interaction chromatography
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Methods for reduction of aggregate levels in antibody and other protein preparations through treatment with low concentrations of electropositive organic additives (e.g., ethacridine, chlorhexidine, or polyethylenimine) in combination with ureides (e.g., urea, uric acid, or allantoin) or organic modulators (e.g., nonionic organic polymers, surfactants, organic solvent or ureides). Some aspects of the invention relate to methods for reducing the level of aggregates in conjunction with clarification of cell culture harvest. It further relates to the integration of these capabilities with other purification methods to achieve the desired level of final purification.

## Claims

### Claim 1

A method of reducing the aggregate content of a sample, wherein the sample comprises a target antibody and aggregates comprising nucleic acid contaminants associated with the target antibody, the method comprising:
(a) contacting the sample with an electropositive organic additive at a non-zero amount of 0.1% weight/volume or less, and an undissolved allantoin, thereby forming a mixture comprising allantoin and precipitated nucleic acid contaminants, wherein allantoin is supersaturated in the mixture and the electropositive organic additive comprises ethacridine or polyethyleneimine; and
(b) removing the undissolved allantoin and precipitated nucleic acid contaminants from the sample,
wherein the sample, after (b) comprises the target antibody and a reduced amount of the aggregates, wherein the sample is derived from a cell culture or cell culture supernatant, and the target antibody is a recombinant antibody.

### Claim 2

A method of reducing the aggregate content of a sample, wherein the sample comprises a target antibody and aggregates comprising nucleic acid contaminants associated with the target antibody, the method comprising:
(a) contacting the sample having a conductivity value of 5 mS/cm or higher with an electropositive organic additive at a non-zero amount of 0.1% weight/volume or less, and an undissolved allantoin, thereby forming a mixture comprising allantoin and precipitated nucleic acid contaminants, wherein allantoin is supersaturated in the mixture and the electropositive organic additive comprises ethacridine or polyethyleneimine; and
(b) removing the undissolved allantoin and precipitated nucleic acid contaminants from the sample,
wherein the sample, after (b) comprises the target antibody and a reduced amount of the aggregates, wherein the sample is derived from a cell culture or cell culture supernatant, and the target antibody is a recombinant antibody.

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein allantoin is present in the mixture in a range of from about 0.5% to about 2% weight/volume.

### Claim 4 (depends on 1, 2)

The method of claim 1 or 2, wherein the undissolved allantoin is added to the sample before the electropositive organic additive.

### Claim 5 (depends on 2)

The method of claim 2, wherein the antibody comprises an antibody fragment.

### Claim 6 (depends on 1, 2)

The method of claim 1 or 2, wherein the amount of the electropositive organic additive in the mixture is 0.01% or less weight/volume.

### Claim 7 (depends on 1, 2)

The method of claim 1 or 2, wherein the amount of the electropositive organic additive in the mixture is 0.001% or less weight/volume.

### Claim 8 (depends on 1, 2)

The method of claim 1 or 2, wherein the amount of the electropositive organic additive in the mixture is from 0.02% to 0.03%.

### Claim 9 (depends on 1)

The method of claim 1, wherein two or more electropositive organic additives are present in the mixture.

### Claim 10 (depends on 9)

The method of claim 9, wherein the total concentration of the two or more electropositive organic additives in the mixture is 0.1% or less weight/volume.

### Claim 11 (depends on 10)

The method of claim 10, wherein the total concentration of the two or more electropositive organic additives in the mixture is 0.01% or less weight/volume.

### Claim 12 (depends on 11)

The method of claim 11, wherein the total concentration of the two or more electropositive organic additives in the mixture is 0.001% or less weight/volume.

### Claim 13 (depends on 3)

The method of claim 3, wherein allantoin is present in the mixture at a concentration in range of from about 0.56% weight/volume to about 1% weight/volume.

### Claim 14 (depends on 3)

The method of claim 3, wherein allantoin is present in the mixture at a concentration in range of from about 1% weight/volume to about 2% weight/volume.

### Claim 15 (depends on 1, 2)

The method of claim 1 or 2, wherein allantoin is present at a concentration in range of from about 2% weight/volume to about 5% weight/volume.

### Claim 16 (depends on 1, 2)

The method of claim 1 or 2, wherein allantoin is present in the mixture at a concentration in range of from about 5% weight/volume to about 10% weight/volume.

### Claim 17 (depends on 1, 2)

The method of claim 1 or 2, wherein allantoin is present in the mixture at a concentration in range of from about 10% weight/volume to about 15% weight/volume.

### Claim 18 (depends on 2)

The method of claim 2, wherein the conductivity of the sample is adjusted to a level that is higher than necessary to counteract substantial loss of the antibody.

### Claim 19 (depends on 2)

The method of claim 2, wherein the conductivity of the sample is adjusted to greater than 20 mS/cm.

### Claim 20 (depends on 2)

The method of claim 2, wherein the conductivity of the sample is adjusted up to greater than 25 mS/cm.

### Claim 21 (depends on 18)

The method of claim 18, wherein the conductivity of the sample is adjusted before addition of the electropositive organic additive.

### Claim 22 (depends on 1)

The method of claim 1, wherein the sample comprises polyethylene glycol or wherein polyethylene glycol is added to the sample.

### Claim 23 (depends on 22)

The method of claim 22, wherein the polyethylene glycol is at, or is added to a concentration in a range of from about 0.01% to about 20% weight/volume.

### Claim 24 (depends on 22)

The method of claim 22, wherein the polyethylene glycol is at, or is added to a concentration in range of from about 0.01 to about 1%.

### Claim 25 (depends on 22)

The method of claim 22, wherein the polyethylene glycol is at, or is added to a concentration in range of from about 1% to about 5%.

### Claim 26 (depends on 22)

The method of claim 22, wherein the polyethylene glycol is at, or is added to a concentration in range of from about 5% to about 10%.

### Claim 27 (depends on 22)

The method of claim 22, wherein the polyethylene glycol is at, or is added to a concentration in range of from about 10% to about 20%.

### Claim 28 (depends on 1)

The method of claim 1, wherein a surfactant is present in the mixture.

### Claim 29 (depends on 28)

The method of claim 28, wherein the surfactant comprises a nonionic surfactant.

### Claim 30 (depends on 29)

The method of claim 29, wherein the nonionic surfactant comprises one selected from the group consisting of Tween and Triton.

### Claim 31 (depends on 28)

The method of claim 28, wherein the surfactant comprises a zwitterionic surfactant.

### Claim 32 (depends on 31)

The method of claim 31, wherein the zwitterionic surfactant comprises one selected from the group consisting of CHAPS, CHAPSO, and octaglucoside.

### Claim 33 (depends on 28)

The method of claim 28, wherein the surfactant is present in the mixture at a non-zero concentration less than 1%.

### Claim 34 (depends on 28)

The method of claim 28, wherein the surfactant is present in the mixture at a non-zero concentration less than 0.1%.

### Claim 35 (depends on 28)

The method of claim 28, wherein the surfactant is present in the mixture at a non-zero concentration less than 0.01%.

### Claim 36 (depends on 1)

The method of claim 1, wherein a chelating agent is present in the mixture.

### Claim 37 (depends on 36)

The method of claim 36, wherein the chelating agent is positively charged.

### Claim 38 (depends on 37)

The method of claim 37, wherein the positively charged chelating agent comprises tris(2-aminoethyl)amine or deferoxamine.

### Claim 39 (depends on 36)

The method of claim 36, wherein the chelating agent is present in the mixture at a non-zero concentration of less than 100 mM.

### Claim 40 (depends on 36)

The method of claim 36, wherein the chelating agent is present in the mixture at a non-zero concentration of less than 50 mM.

### Claim 41 (depends on 36)

The method of claim 36, wherein the chelating agent is present in the mixture at a non-zero concentration of less than 20 mM.

### Claim 42 (depends on 36)

The method of claim 36, wherein the chelating agent is present in the mixture at a non-zero concentration of less than 10 mM.

### Claim 43 (depends on 36)

The method of claim 36, wherein the chelating agent is present in the mixture at a non-zero concentration of less than 5 mM.

### Claim 44 (depends on 1)

The method of claim 1, wherein the contacting of the sample with the undissolved allantoin comprises contacting the sample with allantoin in an amount of greater than 0.5% (w/v).