Purification of biological substances

• US 20030201230A1
• Filed: 05/23/2003
• Published: 10/30/2003
• Est. Priority Date: 02/22/1999
• Status: Active Grant

• Current Assignee: Smartflow Technologies Inc.
• Original Assignee: NCSRT, Inc.
• Inventors: Kopf, Henry B.
• Granted Patent: US 6,946,075 B2
• Time in Patent Office: Days
• US Class Current: 210/656
• CPC Class Codes: B01D 15/1871 placed in series
• CPC Class Codes: B01D 15/361 Ion-exchange
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: C07K 1/18 Ion-exchange chromatography
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 1/34 by filtration, ultrafiltration or reverse osmosis
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12M 47/12 Purification C12M47/04 takes precedence

Abstract

A process and apparatus for purifying one or more target substances from a source liquid, employing one or more cross-flow filter elements, and one or more types of chromatography resins, in combination, to provide purification with advantageous yield, product purity, and cost- and time-efficiency.

Claims

1. A process for purifying one or more target substances from a source liquid, said process comprising:

contacting the source liquid with a chromatography resin;

incubating the source liquid with the chromatography resin for a sufficient contact time to allow the resin to bind a desired fraction of one or more target substances;

recirculating the chromatography resin in a cross-flow filter wherein the following steps are performed;

concentrating the chromatography resin and separating contaminants from the chromatography-resin-bound target substance by diafiltration;

eluting the target substance from the chromatography resin; and

separating the target substance from the chromatography resin by diafiltration;

recovering the target substance; and

optionally concentrating the target substance

wherein the chromatography resin comprises porous chromatography resin particles that are functionalized with affinity ligands, with the affinity ligands being located on chains extending from the particle surface.

2. The process of claim 1, further comprising an initial clarification of the source liquid.

3. The process of claim 2, wherein the initial clarification is carried out by cross-flow filtration to remove particulate matter.

4. The process of claim 2, wherein the initial clarification is carried out by cross-flow filtration to remove interfering substances.

5. The process of claim 1, wherein the eluted target substance is concentrated and diafiltered in a cross-flow filter module.

6. The process of claim 1, further comprising subjecting permeate generated by the concentration and diafiltration of the chromatography resin, prior to elution, to further purification steps.

7. The process of claim 1, further comprising contacting the eluted target substance with a second chromatography resin for further purification.

8. The process of claim 1, wherein the chromatography resin comprises one or more ligand species selected from the group consisting of:

Protein A;

Protein A analogs;

antibodies;

bacterial antigens;

viral antigens; and

dyes.

9. The process of claim 1, wherein the target substance is an immunoglobulin.

10. The process of claim 1, wherein the source liquid comprises one or more liquids selected from the group consisting of serum, plasma, plasma fractions, whole blood, milk, colostrum, whey, cell fluids, tissue culture fluids, plant cells fluids, plant cell homogenates, and tissue homogenates.

11. The process of claim 1, wherein the source liquid is selected from the group consisting of plasma, whole blood, serum and tissue culture.

12. The process of claim 1, wherein the target substance is selected from the group consisting of vaccines, clotting factors, immunoglobulins, antigens, antibodies, proteins, glycoproteins, peptides, sugars, carbohydrates, and enzymes.

13. The process of claim 1, wherein the chromatography resin particles comprise organic copolymer, and the chains are bound to the organic copolymer by graft polymerization.

14. The process of claim 1, wherein the chromatography resin comprises resin particles including a core of a matrix material, copolymerized from oligoethyleneglycol, glycidylmethacrylate and pentaerythroldimethacrylate, to which is grafted polymerized chains of acrylamide derivatives that are from about 15 to about 50 units in length.

15. The process of claim 14, where said resin particles have a diameter of from about 10 to about 100 micrometers.

16. The process of claim 15, wherein the affinity ligands on the chains of the resin particles bind at least one of the target species selected from the group consisting of proteins, nucleic acids, viruses, sugars, carbohydrates, immunoglobulins, clotting factors, glycoproteins, peptides, antibodies, antigens, and hormones.

17. A process for purifying an immunoglobulin from a source liquid, said process comprising:

contacting the source liquid with a chromatography resin, wherein the chromatography resin comprises Protein A linked to rigid, non-porous spherical beads;

incubating the source liquid with the chromatography resin for a sufficient contact time to allow the resin to bind a desired fraction of the immunoglobulin;

recirculating the chromatography resin in a cross-flow filter wherein the following steps are performed;

concentrating the chromatography resin and separating contaminants from the chromatography-resin-bound immunoglobulin by diafiltration;

eluting the immunoglobulin from the chromatography resin; and

separating the immunoglobulin from the chromatography resin by diafiltration;

recovering the immunoglobulin; and

optionally concentrating the immunoglobulin

wherein the chromatography resin comprises porous chromatography resin particles that are functionalized with affinity ligands, with the affinity ligands being located on chains extending from the particle surface.

18. The process of claim 17, wherein the source liquid is selected from the group consisting of tissue culture fluid, plant cell culture fluid, plant cell homogenates and raw human plasma.

19. The process of claim 17, wherein the step of incubating the source liquid with the chromatography resin is performed by recirculating the source liquid and chromatography resin between a cross-flow filter module and a reservoir containing the source liquid and chromatography resin.

20. A purification apparatus, for separating and concentrating a target substance from a source liquid, said apparatus comprising:

a first reservoir constructed and arranged for holding a solid-phase chromatography resin material, and for selectively flowing liquid into and out of said first reservoir;

a solid-phase chromatography resin material disposed in said first reservoir;

a first cross-flow filtration module for separating liquids into permeate and retentate streams, provided with means for flowing liquid in and permeate and retentate liquid streams out of said first cross-flow filtration module;

a second reservoir constructed and arranged for capturing and holding the permeate liquid stream, and for selectively flowing liquid into and out of said second reservoir;

a second cross-flow filtration module for concentrating a liquid stream, provided with means for flowing liquid in and permeate and retentate liquid streams out of said second cross-flow filtration module;

a collection vessel constructed and adapted for capturing the concentrated liquid stream from the second cross-flow filtration module; and

conduit, valve and pump means constructed and arranged for;

providing make-up liquids to the first and second reservoirs;

selectively flowing a source liquid to the first reservoir charged with a chromatography resin to form a slurry;

incubating the source liquid with the chromatography resin by recirculating the slurry from the first reservoir to the first cross-flow filtration module and returning both the permeate and retentate liquid streams to the first reservoir;

recirculating the slurry in a cross-flow filter in a flow pathway adapted for;

concentrating the slurry and separating contaminants from the slurry by diafiltration;

eluting the target substance from the chromatography resin; and

separating the target substance from the chromatography resin by diafiltration;

capturing the target substance in a second reservoir;

concentrating the target substance by flowing it through the second cross-flow filtration module; and

recovering the concentrated target substance in the collection vessel

wherein the chromatography resin comprises porous chromatography resin particles that are functionalized with affinity ligands, with the affinity ligands being located on chains extending from the particle surface.

21. An apparatus according to claim 20, wherein the first and second reservoirs are provided with thermal jackets to maintain appropriate process temperatures.

22. An apparatus according to claim 20, wherein the chromatography resin comprises resin particles including a core of a matrix material, copolymerized from oligoethyleneglycol, glycidylmethacrylate and pentaerythroldimethacrylate.

23. An apparatus according to claim 22, wherein the chromatography resin further comprises Protein A.

24. A method of purification of a liquid containing a target substance, comprising the steps of contacting the liquid with a chromatography resin to bind the target substance thereto, and cross-flow filtering the target substance-bound chromatography resin under elution conditions to recover a filtrate comprising the target substance, wherein the chromatography resin comprises porous chromatography resin particles that are functionalized with affinity ligands, with the affinity ligands being located on chains extending from the particle surface.

25. The method of claim 24, wherein the liquid is clarified prior to said contacting with the chromatography resin.

26. The method of claim 25, wherein said clarification removes contaminants adverse to purification of the liquid.

27. The method of claim 24, wherein said cross-flow filtering comprises diafiltering the target substance-bound chromatography resin against a buffer.

28. The method of claim 24, wherein said cross-flow filtering comprises diafiltering the target substance from the chromatography resin after elution disengagement of the target substance from the chromatography resin.

29. The method of claim 24, wherein the cross-flow filtering produces a permeate and said permeate is further purified.

30. The method of claim 29, wherein further purification of the permeate comprises contacting thereof with a second chromatography resin to bind a selected species of the permeate and cross-flow filtering the selected species-bound second chromatography resin under conditions producing a filtrate containing said selected species as a purification product.

31. The method of claim 24, wherein said filtrate comprising the target substance is further purified.

32. The method of claim 31, wherein further purification of the filtrate comprising the target substance comprises contacting thereof with a second chromatography resin to bind the target substance, and cross-flow filtering the target substance-bound second chromatography resin under elution conditions producing a filtrate containing further purified target substance as a purification product.

33. The method of claim 24, wherein the step of contacting the liquid with a chromatography resin to bind the target substance thereto, comprises adding the liquid to a recirculating loop of chromatography resin using a cross-flow filter prior to imposition of said elution conditions.

34. The method of claim 33, wherein the cross-flow filter is constructed, arranged and operated to discharge permeate liquid at substantially the same rate as said liquid is added for said contacting.

35. The method of claim 24, wherein said contacting of the liquid and the chromatography resin comprises addition of liquid to an agitated constant volume reservoir having chromatographic resin therein.

36. The method of claim 24, wherein said cross-flow filtering is conducted using a cross-flow filter selected from the group consisting of one or more of:

hollow fiber filters;

spiral filters;

plate and frame filters;

cassette filters;

tubular filters;

stirred cells; and

ceramic filters.

37. The method of claim 24, wherein the chromatography resin comprises resin particles including a core of a matrix material, copolymerized from oligoethyleneglycol, glycidylmethacrylate and pentaerythroldimethacrylate.

38. The method of claim 24, wherein the chromatographic resin comprises at least one ligand selected from the group consisting of:

Protein A;

antibodies; and

antigens.

39. The method of claim 24, carried out to effect a separation selected from the group consisting of:

separating the liquid to produce a vaccine or vaccine component;

separating plasma or a plasma fraction into its constituent parts;

separating clostrum into its constituent parts;

separating milk into its constituent parts;

separating whey into its constituent parts;

separating a fermentation fluid into its constituent parts;

separating insect cell culture fluid into its constituent parts;

separating plant cell culture fluid into its constituent parts;

separating viral culture fluid into its constituent parts;

separating an immunoglobulin from an immunoglobulin-containing culture of bacteria, yeast, fungus, insect cells, plant cells or animal cells;

separating an immunoglobulin from serum;

separating an immunoglobulin from plasma or a plasma fraction;

separating an immunoglobulin from whole blood;

separating an immunoglobulin from milk;

separating an immunoglobulin from clostrum;

separating an immunoglobulin from whey;

separating an immunoglobulin from ascites fluid;

separating a clotting factor from whole blood;

separating a clotting factor from plasma;

separating a clotting factor from serum;

separating a clotting factor from a clotting factor-containing culture of bacteria, yeast, fungus, insect cells, plant cells or animal cells;

separating a clotting factor from milk;

separating a clotting factor from whey;

separating a clotting factor from clostrum;

separating a clotting factor from ascites fluid;

separating a protein from a protein-containing culture of bacteria, yeast, fungus, insect cells, plant cells, or animal cells;

separating an antigen from an antigen-containing culture of bacteria, yeast, fungus, insect cells, or animal cells;

separating an antigen from a viral culture containing same;

separating a hormone from a hormone-containing culture of bacteria, yeast, fungus, insect cells, plant cells, or animal cells;

separating a hormone from serum;

separating a hormone from plasma or a plasma fraction;

separating a hormone from whole blood;

separating a hormone from plasma;

separating a hormone from serum;

separating a hormone from milk;

separating a hormone from whey;

separating a hormone from clostrum;

separating a hormone from ascites fluid;

separating a hormone from tissue;

separating a glycoprotein from a viral culture;

separating a glycoprotein from a glycoprotein-containing culture of bacteria, yeast, fungus, insect cells, plant cells, or animal cells;

separating an enzyme from serum;

separating an enzyme from plasma or a plasma fraction;

separating an enzyme from whole blood;

separating an enzyme from plasma;

separating an enzyme from serum;

separating an enzyme from milk;

separating an enzyme from whey;

separating an enzyme from clostrum;

separating an enzyme from ascites fluid;

separating an enzyme from tissue;

separating a glycoprotein from a viral culture;

separating a glycoprotein from a glycoprotein-containing culture of bacteria, yeast, fungus, insect cells, plant cells, or animal cells;

separating a glycoprotein from serum;

separating a glycoprotein from plasma or a plasma fraction;

separating a glycoprotein from whole blood;

separating a glycoprotein from plasma;

separating a glycoprotein from serum;

separating a glycoprotein from milk;

separating a glycoprotein from whey;

separating a glycoprotein from clostrum;

separating a glycoprotein from ascites fluid; and

separating a glycoprotein from tissue.

42. The method of claim 24, wherein two or more resins are used to purify one or more target substances simultaneously.

43. The method of claim 42, wherein the ligands are antigens and the targets are antibodies.

44. The method of claim 24, wherein the chromatography resin comprises resin particles, wherein at least some of the resin particles have multiple ligands thereon, whereby different target substances may be bound by a single resin particle.

40. A method of separating a liquid in a separation system comprising a bioreactor, a chromatographic resin reservoir, a first cross-flow filtration module, a second cross-flow filtration module, and a third cross-flow filtration module, said method comprising clarifying the perfusate of the bioreactor in the first cross-flow filtration module to yield a permeate, flowing the permeate to the chromatographic resin reservoir and flowing chromatographic resin and permeate to the second cross-flow filtration module for concentration, diafiltration and elution to yield an eluate, and flowing the eluate to the third cross-flow filtration module for concentration and diafiltration therein, wherein the chromatographic resin comprises porous chromatographic resin particles that are functionalized with affinity ligands, with the affinity ligands being located on chains extending from the particle surface.

41. The method of claim 40, wherein said bioreactor is of a type selected from the group consisting of hollow fiber bioreactors and cell culture bioreactors.

45. A method of purifying at least one target substance from a source liquid containing a bioactive contaminant, comprising the steps of:

contacting the source liquid with a chromatography resin;

incubating the source liquid with the chromatography resin for a sufficient contact

time to bind to the chromatography resin at least one target substance from the source liquid;

recirculating the chromatography resin in a cross-flow filter wherein the following steps are performed;

concentration and diafiltration of the chromatography resin;

elution of the target substance from the chromatography resin; and

separation of the target substance from the chromatography resin by diafiltration;

recovering the target substance; and

optionally concentrating the target substance;

further comprising inactivating the bioactive contaminant during said method

wherein the chromatography resin comprises porous chromatography resin particles that are functionalized with affinity ligands, with the affinity ligands being located on chains extending from the particle surface.

46. The method of claim 45, wherein the bioactive contaminant comprises a bioactive species selected from the group consisting of bacteria, yeasts, viruses and immunoantigens.

47. The method of claim 45, wherein the bioactive contaminant comprises a virus.

48. The method of claim 45, wherein the bioactive contaminant comprises HIV.

49. The method of claim 45, wherein said inactivation comprises contacting the bioactive contaminant with an inactivating agent.

50. The method of claim 45, wherein said inactivation comprises contacting the bioactive contaminant with an inactivating agent as an initial step of the method.

51. The method of claim 45, wherein said inactivation comprises contacting the bioactive contaminant with an inactivating agent during the incubation of the source liquid with the chromatography resin.

52. The method of claim 45, wherein said inactivation comprises contacting the bioactive contaminant with an inactivating agent during cross-flow filtration processing.

53. The method of claim 49, wherein said inactivating agent comprises a microbead-bound inactivating agent species.

54. The method of claim 49, wherein said inactivating agent comprises a microbead-bound capture agent for the bioactive contaminant.

55. The method of claim 49, wherein said inactivating agent comprises radiation.

56. The method of claim 49, wherein said inactivating agent comprises an agent selected from the group consisting of iodine, chlorine, solvent-detergent compositions, and ultraviolet radiation.

57. A process for purifying one or more target substances from a source liquid containing said target substances together with one or more viral species, such process comprising:

contacting the source liquid with a viral inactivation agent for the one or more viral species;

concentrating the target substance(s) in the source liquid by tangential flow filtration in a tangential flow filter comprising a membrane effective to concentrate at least 90% by weight of the target substance(s) in such filtration;

contacting the source liquid with a chromatography resin;

incubating the source liquid with the chromatography resin for a sufficient contact time to bind a desired fraction of the target substance(s);

recirculating the chromatography resin in a cross-flow filter wherein the following steps are performed;

concentrating the chromatography resin and separating contaminants from the chromatography-resin-bound target substance by diafiltration in a filter comprising a membrane effective to retain the resin and pass species that are not bound to the resin;

eluting the target substance from the chromatography resin by contact with an elution membrane effective to retain the resin and pass the target substance(s);

separating the target substance from the chromatography resin by diafiltration;

recovering the target substance(s); and

optionally concentrating the target substance(s),

wherein the chromatography resin comprises porous chromatography resin particles that are functionalized with affinity ligands, with the affinity ligands being located on chains extending from the particle surface.

58. A process for purifying one or more target substances from a source liquid containing said target substances, such process comprising:

contacting the source liquid with a chromatography resin;

recirculating the chromatography resin in at least one cross-flow filter wherein the following steps are performed;

concentrating the target substance and chromatography resin in the source liquid by tangential flow filtration;

diafiltering the chromatography resin in a filter comprising a membrane effective to retain the resin and pass species that are not bound to the resin;

eluting the target substance from the chromatography resin by contact with an elution membrane effective to retain the resin and pass the target substance(s);

separating the target substance from the chromatography resin by diafiltration recovering the target substance(s); and

optionally concentrating the target substance(s),

wherein the chromatography resin comprises porous chromatography resin particles that are functionalized with affinity ligands, with the affinity ligands being located on chains extending from the particle surface.

59. The process of claim 58, wherein the source liquid contains one or more pathogenic organisms, further comprising contacting the source liquid with a viral inactivation agent for the one or more pathogenic organisms.

60. The process of claim 58, comprising concentrating the target substance(s) by contact with a concentration membrane effective to yield a concentrate comprising at least 90% by weight target substance(s).

62. The method of claim 58, wherein the chromatography resin comprises resin particles including a core of a matrix material, copolymerized from oligoethyleneglycol, glycidylmethacrylate and pentaerythroldimethacrylate.

63. The method of claim 58, wherein the chromatography resin comprises resin particles including a core of a matrix material, copolymerized from oligoethyleneglycol, glycidylmethacrylate and pentaerythroldimethacrylate, wherein said resin particles have a diameter in a range of from about 10 to about 100 micrometers, and said chains comprise chains of acrylamide derivatives.

61. A method of manufacturing universal plasma from blood containing serological Group A and/or Group B antibodies, comprising contacting the blood containing serological Group A and/or Group B antibodies with a chromatography resin comprising corresponding Group A and/or Group B antigen, and recovering an antibodies-depleted blood product as the universal plasma, wherein the chromatography resin comprises porous chromatography resin particles that are functionalized with said corresponding Group A and/or Group B antigen, with the corresponding Group A and/or Group B antigen being located on chains extending from the particle surface.