Purification of biological products by constrained cohydration chromatography

Patent metadata

Field	Value
Publication number	US-9809639-B2
Application number	US-201214124706-A
Country	US
Kind code	B2
Filing date	Jun 1, 2012
Priority date	Jun 8, 2011
Publication date	Nov 7, 2017
Grant date	Nov 7, 2017

Abstract

Materials and methods for use of constrained cohydration agents in the purification of biological materials such as antibodies, viruses, cells, and cellular organelles in connection with convective chromatography, fluidized bed or co-precipitation applications.

First claim

The invention claimed is:

1. A method for purification of a hydrated target species of biological origin in a sample comprising the steps of contacting the sample with a hydrated surface of an undissolved material and contacting the sample with a constraining agent in an amount sufficient to cause more than 50% to substantially all of the target species to be retained at the hydrated surface of the undissolved material, wherein
(i) the undissolved material comprises particles,
(ii) a surface of the particles includes charged moieties,
(iii) the target species is retained exclusively by constrained cohydration under conditions providing a substantial absence of a direct chemical interaction between the target species and the hydrated surface, and
(iv) the conditions that provide the substantial absence of a direct chemical interaction comprise pH, conductivity and salt concentration of buffers delivered to a column of the particles, wherein said conditions discourage direct interaction between the hydrated target species and the charged moieties, and wherein the particles are optionally a convective chromatography material.

2. The method of claim 1 , wherein the hydrated target species is a virus.

3. The method of claim 1 , wherein the sample is a virus-containing solution derived from cell culture.

4. The method of claim 1 , wherein the surface of the undissolved material has one or more polar chemical moieties comprising a hydroxyl group.

5. The method of claim 1 , wherein the constraining agent comprises a non-ionic organic polymer.

6. The method of claim 5 , wherein the non-ionic organic polymer comprises polyethylene glycol.

7. The method of claim 6 , wherein the polyethylene glycol has an average polymer weight between 100 and 10,000 D or between 600 and 8,000 D.

8. The method of claim 6 , wherein the polyethylene glycol is provided at a concentration between approximately 2% and 50% (w/v).

9. The method of claim 6 , wherein the polyethylene glycol is provided at a concentration between approximately 2% and 25% (w/v) with the polyethylene glycol having an average polymer weight above about 6,000 D.

10. The method of claim 6 , wherein the polyethylene glycol is provided at a concentration between approximately 15% and 50% (w/v) with the polyethylene glycol having an average polymer weight below about 6,000 D.

11. The method of claim 6 , wherein the polyethylene glycol is provided at a concentration between approximately 2% and 25% (w/v) with the polyethylene glycol having an average polymer weight between about 4,000 D and about 8000 D.

12. The method of claim 1 , wherein the particles comprise microparticles.

13. The method of claim 1 , wherein the particles comprise magnetic particles.

14. The method of claim 1 , wherein the particles comprise metal-core particles having a polymer coating.

15. The method of claim 1 , wherein the particle size is (a) between about 100 nm and about 500 μm, or (b) between about 100 nm and about 50 μm, or (c) between about 100 nm and about 4 μm, or (d) between about 100 nm and about 3 μm, or (e) between about 100 nm and about 1 μm, or (f) between about 200 nm and about 2 μm, or (g) between about 200 nm and about 500 nm, or (h) 500 nm and about 1 μm, or (i) between about 5 μm and about 50 μm.

16. The method of 1 , wherein the step of contacting the target sample with the particles occurs prior to the step of contacting the sample with the constraining agent.

17. The method of claim 1 , comprising the additional steps of separating the particles with the target species associated with the hydrated surface of the particles from the liquid phase and dissociating the target species from the particles.

18. The method of claim 17 , wherein the step of dissociating the target species consists of washing the particles with a solution where the solution contains the constraining agent in an amount insufficient to retain the target species at the hydrated surface of the particles.

19. The method of claim 17 , wherein an agent which effects dissociation of the target species from the hydrated surface of the particles comprises a surfactant.

20. The method of claim 1 , wherein the constraining agent is added to the sample and the particles over a period of time from about one minute to about five hours; between 2 min and 15 min; between 2 min and 30 min; between 10 min and 30 min; between 2 min and 2 hours; or between about 2 min and 1 hour.

21. The method of claim 1 , wherein the particles are separated from the liquid component of the mixture by filtration.

22. The method of claim 21 , wherein the separated particles are washed with a solution comprising the constraining agent.

23. The method of claim 17 , wherein the step of dissociating is carried out in a single step of adding a dissociating buffer to the particles.

24. The method of claim 1 , wherein the method is performed prior to or after a method for fractionating the target species from other materials is performed and the method for fractionating comprises anion exchange chromatography.

25. The method of claim 1 , wherein the particles are a convective chromatography material and the convective chromatography material comprises a monolith.

26. The method of claim 25 , wherein the monolith is comprised of polymethacrylate.

27. The method of claims 25 , wherein the monolith has an average channel size between about 1 micron and 200 micron.

28. The method of claim 27 , wherein the channel size is between about 1 micron and 5 microns or between about 10 micron and 20 microns, or between about 50 micron and 200 microns.

29. The method of claim 25 , wherein the monolith is chemically modified to increase its capacity for the hydration of its surface.

30. The method of claim 1 , wherein the particles are a convective chromatography material and the convective chromatography material is hydroxylated.

31. The method of claim 30 , wherein (i) the convective chromatography material includes charged moieties and (ii) wherein said conditions suspend direct interaction between the target species and such charged moieties during at least a portion of the step of contacting the sample with the constraining agent so that during that portion the target species is retained at the hydrated surface exclusively by constrained cohydration.

32. The method of claim 1 , wherein the particles are the convective chromatography material and the step of contacting the sample with the constraining agent is performed by in-line dilution prior to contacting the sample with the convective chromatography.

33. The method of claim 32 , wherein the convective chromatography material is equilibrated with the constraining agent prior to contacting of the sample with the convective chromatography material.

34. The method of claim 32 , comprising the additional steps of washing the hydrated convective chromatography material with a solution comprising the constraining agent in an amount sufficient to remove contaminants not retained at the hydrated surface of the undissolved material, and subsequently dissociating the target species from the convective chromatography material.

35. The method of claim 34 , wherein the step of dissociating the target species comprises washing the conv

Assignees

• Gagnon Peter Stanley
• Agency Science Tech & Res

Inventors

• Gagnon Peter Stanley

Classifications

• C07K14/755

  Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)} · CPC title

• B01D15/08 Primary

  Selective adsorption, e.g. chromatography · CPC title

• B01D15/1807

  using counter-currents, e.g. fluidised beds · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C07K16/065

  Purification, fragmentation · CPC title