Protein purification methods

Patent metadata

Field	Value
Publication number	US-9988419-B2
Application number	US-201414766131-A
Country	US
Kind code	B2
Filing date	Feb 5, 2014
Priority date	Feb 6, 2013
Publication date	Jun 5, 2018
Grant date	Jun 5, 2018

Abstract

A method for the purification of a desired protein from a protein preparation includes conditioning the protein preparation by treatment with soluble organic multivalent ions, immobilized organic multivalent ions, or both, optionally in the presence of supersaturated allantoin, thereby removing at least 90% of chromatin, then (1) precipitating the desired protein with a nonionic organic polymer in the presence of non-protein-precipitating salts at greater than physiological concentration to provide a precipitate of the desired protein; or (2) precipitating the desired protein with a nonionic organic polymer in the absence of non-precipitating salts at greater than physiological concentration to provide a precipitate and subsequently washing the precipitate with a nonionic organic polymer in the presence of non-protein-precipitating salts at greater than physiological concentration.

First claim

What is claimed is:

1. A method for the purification of an antibody from a protein preparation comprising:
conditioning the protein preparation by contacting the preparation with ethacridine or an electronegative organic additive selected from the group consisting of heptanoic acid, octanoic acid, octenoic acid, nonanoic acid, nonenoic acid, and decanoic acid, thereby removing at least 90% of chromatin, then
(1) precipitating the antibody with polyethylene glycol (PEG) in the presence of non-protein-precipitating salts at a concentration in a range from about 0.2 M to about 2.0 M to provide a precipitate comprising the antibody; or
(2) precipitating the antibody with PEG at a concentration of non-protein-precipitating salts less than 0.15 M to provide a precipitate and subsequently washing the precipitate with the non-ionic organic polymer in the presence of the non-protein-precipitating salts at a concentration in a range from about 0.2 M to about 2.0 M;
wherein the PEG has a polymer size in a range of 1000 to 12,000 Daltons (D), the non-protein-precipitating salts are selected from the group consisting of sodium acetate, potassium acetate, sodium chloride, potassium chloride, and combinations thereof, and the precipitating steps or washing step is optionally carried out in a substantial absence of a protein-precipitating salt.

2. The method of claim 1 , wherein the electronegative organic additive is octanoic acid.

3. The method of claim 1 , wherein the conditioning comprises contacting the preparation with allantoin at a supersaturating concentration in a range of 0.6 to 50%, 0.7 to 20%, 0.8 to 10%, 0.9 to 5%, or 1 to 2% (w/v).

4. The method of claim 1 , wherein the PEG comprises a polymer size in a range of 2000 to 8,000 D, or 3000 to 6000 D, or a polymer size of 1500 D, 3500 D, 4000 D, or 6000 D.

5. The method of claim 1 , wherein the non-protein-precipitating salts comprise sodium chloride.

6. The method of claim 1 , wherein a net salt concentration of the non-protein precipitating salts in the protein preparation after step (1) is in a range selected from the group consisting of 200 mM to 2 M, 300 mM to 1.5 M, 400 mM to 1 M and 500 mM to 800 mM.

7. The method of claim 1 , further comprising washing the precipitate of step (1) with the non-ionic organic polymer in the presence of the non-protein-precipitating salts at a concentration of at least 0.2M, or washing the precipitate with one or more precipitating salts at a concentration sufficient to maintain the antibody in a precipitated state.

8. The method of claim 1 , wherein the protein precipitating salt is selected from the group consisting of sodium sulfate, potassium sulfate, ammonium sulfate, sodium phosphate, potassium phosphate, ammonium phosphate, sodium citrate, potassium citrate, ammonium citrate, and combinations thereof.

9. The method of claim 1 , wherein the antibody is a naturally occurring protein, a recombinant protein, a monoclonal antibody, an IgG antibody, or an IgM antibody.

10. The method of claim 1 , where the protein preparation comprises a bodily fluid, milk, plasma, serum, or a cell culture harvest.

Assignees

• Agency Science Tech & Res

Inventors

• Gagnon Peter Stanley

Classifications

• B01D15/36

  involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K1/303 Primary

  by salting out · CPC title

• C07K1/30

  by precipitation · CPC title

• C07K16/2863

  against receptors for growth factors, growth regulators · CPC title