Stimulus responsive polymers for the purification of biomolecules

• US 9,731,288 B2
• Filed: 11/13/2015
• Issued: 08/15/2017
• Est. Priority Date: 05/17/2010
• Status: Active Grant

• Current Assignee: Merck Life Science Germany GmbH
• Original Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Inventors: Jaber, Jad, Hamzik, James, Boudif, Arezki, Zhang, Yu, Soice, Neil, Singh, Nripen, Charkoudian, John, Moya, Wilson
• Primary Examiner(s): CAIN, EDWARD J
• Application Number:
  US14/940,864
• Publication Number:
  US 20160067703A1
• Time in Patent Office: 641 Days
• Field of Search: 424 7835, 5253282, 526310, 521 31
• CPC Class Codes: B01J 41/13 obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds
• CPC Class Codes: B01J 41/14 obtained by reactions only involving unsaturated carbon-to-carbon bonds
• CPC Class Codes: C07K 1/30 by precipitation
• CPC Class Codes: C07K 1/32 as complexes
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C08F 126/02 by a single or double bond to nitrogen
• CPC Class Codes: C08F 26/02 by a single or double bond to nitrogen
• CPC Class Codes: C08F 8/00 Chemical modification by after-treatment graft polymers, block polymers, crosslinking with unsaturated monomers or with polymers C08F251/00 - C08F299/00; of conjugated diene rubbers C08C
• CPC Class Codes: C08F 8/02 Alkylation

Abstract

The present invention provides novel and improved stimulus responsive polymers and methods of using the same for the purification of biomolecules.

Claims

1. A method of separating a target molecule from one or more impurities in a sample, where the method comprises the steps of:

(a) providing a sample comprising a target molecule and one or more impurities;

(b) contacting the sample with a soluble stimulus responsive polymer selected from the group consisting of benzyl modified polyallylamine;

diphenyl modified polyallylamine;

dichlorobenzyl modified polyallylamine;

chlorobenzyl modified polyallylamine;

hexanoic acid and tert-butyl modified polyallylamine;

phenylbenzyl modified polyallylamine;

benzyl modified polyethyleneimine; and

benzyl modified polyvinylamine, wherein the stimulus responsive polymer is capable of binding and precipitating one or more impurities following the addition of a multivalent ion stimulus; and

(c) adding a multivalent ion stimulus selected form phosphate or citrate to the sample to precipitate complex of polymer and one or more impurities out of the sample;

thereby to separate the target molecule from one or more impurities.

2. The method of claim 1, wherein the target molecule is a recombinant protein.

3. The method of claim 1, wherein the target molecule is an antibody.

4. The method of claim 3, wherein the antibody is a monoclonal antibody.

5. The method of claim 1, wherein the one or more impurities is selected from the group consisting of host cell protein, DNA, RNA, viruses, antibody aggregates and cell culture additives.

6. The method of claim 1, wherein the sample comprises a feedstock.

7. The method of claim 1, wherein the sample comprises cell culture media into which the target molecule is secreted.

8. The method of claim 7, wherein the cell culture media is a CHO cell culture media.

9. The method of claim 7, wherein the target molecule is a monoclonal antibody.

10. The method of claim 1, further comprising one or more chromatography steps.

11. The method of claim 1, further comprising one or more filtration steps.

12. The method of claim 1, further comprising the step of removing a residual amount of polymer after step (c), wherein the step comprises the use of a membrane or resin modified with a multivalent ion.

13. The method of claim 12, wherein the multivalent ion is phosphate.