Purification of biological substances

• US 6,596,172 B1
• Filed: 11/06/2000
• Issued: 07/22/2003
• Est. Priority Date: 02/22/1999
• Status: Expired due to Fees

• Current Assignee: Smartflow Technologies Inc.
• Original Assignee: Henry B. Kopf
• Inventors: Kopf, Henry B.
• Primary Examiner(s): Therkorn, Ernest G.
• Application Number:
  US09/707,203
• Time in Patent Office: 988 Days
• Field of Search: 210/635, 210/650, 210/651, 210/656, 210/659, 210/653, 210/654, 210/655, 210/198.2, 530/413, 530/414, 530/417, 530/387.1
• US Class Current: 210/635
• 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:

(a) contacting the source liquid with one or more chromatography resins;

(b) maintaining the contact for a sufficient period of time and under conditions suitable for the resins to bind a predetermined fraction of the target substance(s);

(c) circulating the chromatography resin(s) in a cross-flow filter wherein one or more of the following steps is performed;

(i) separating unbound substances from the chromatography-resin-bound target substance(s) by concentration and/or diafiltration;

(ii) eluting the target substance(s) from the chromatography resin(s); and

(iii) separating the target substance(s) from the chromatography resin(s) by concentration and/or diafiltration;

(d) recovering the target substance(s); and

(e) optionally concentrating and/or diafiltering the target substance(s).

7. The method according to any of claims 1, 2, 3, 4, 5 or 6, further comprising initially clarifying the source liquid.

8. The process of claim 7, wherein the initial clarifying is accomplished using cross-flow filtration to remove particulate matter.

9. The process of claim 7, wherein the initial clarifying is accomplished using cross-flow filtration to remove interfering substances.

10. The process of claim 7, wherein the initial clarifying is accomplished using a centrifuge.

11. The process of claim 7, wherein the initial clarifying is accomplished using a depth filter.

12. The process of claim 7, wherein the initial clarifying is accomplished using a membrane filter.

13. The process of claim 1 or 2, wherein the eluted target substance(s) are concentrated and/or diafiltered in a cross-flow filter module.

14. The process of claim 1 or 2, further comprising subjecting permeate generated by the concentration and diafiltration of the chromatography resin(s), prior to elution, to further purification steps.

15. The process of claim 1 or 2, further comprising contacting the eluted target substance(s) with a second chromatography resin(s) for further purification.

16. The process of claim 1 or 2, wherein the chromatography resin(s) comprises one or more ligand species selected from the group consisting of:

Protein A;

Protein A analogs;

antibodies;

bacterial antigens;

viral antigens; and

dyes.

18. The method according to any of claims 1-6, wherein step (b) is accomplished by mixing the source liquid and chromatography resin(s) in a reservoir and/or in a mixing tube comprising the source liquid and chromatography resin(s).

19. The method according to any of claims 1-6, 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, cell culture fluids, tissue culture fluids, and tissue homogenates.

20. The process of claim 1 or 2, wherein the target substance(s) are selected from the group consisting of vaccines, clotting factors, immunoglobulins, antigens, antibodies, proteins, glycoproteins, peptides, enzymes and ions.

21. The method according to any of claims 1-6, wherein the chromatography resin(s) are selected from the group consisting of affinity resins and ion-exchange resins.

22. The process of claim 21, where the affinity resin comprises rigid, non-porous cellulose beads coupled to affinity ligands.

23. The process of claim 22, wherein the affinity resin comprises one or more of more affinity ligands are selected from the group consisting of Protein A and Protein A analogs.

24. The method of claim 1 or 2, wherein the step of contacting the liquid with the chromatography resin(s) to bind the target substance(s) thereto, comprises adding the liquid to a circulating loop of chromatography resin(s) using a cross-flow filter.

26. The method of claim 24 or 25, 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.

27. The method according to any of claims 1-6 wherein said contacting of the liquid and the chromatography resin(s) comprises addition of liquid to an agitated constant volume reservoir comprising chromatographic resin therein.

28. The method according to any of claim 1-6 wherein said cross-flow filtering is conducted using one or more cross-flow filters selected from the group consisting of:

hollow fiber filters;

spiral filters;

plate and frame filters;

cassette filters;

tubular filters;

stirred cells; and

ceramic filters.

29. The method according to any of claims 1-6 wherein the resin comprises one or more compatible characteristics selected from the group consisting of:

spherical shape;

irregular shape;

porous character;

non-porous character;

compressible character; and

non-compressible character.

30. The method according to any of claims 1-6 carried out to effect a separation selected from the group consisting of:

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

(b) separating a clotting factor from ascites fluid;

(c) separating a clotting factor from clostrum;

(d) separating a clotting factor from milk;

(e) separating a clotting factor from plasma;

(f) separating a clotting factor from serum;

(g) separating a clotting factor from whey;

(h) separating a clotting factor from whole blood;

(i) separating a fermentation fluid into its constituent parts;

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

(k) separating a glycoprotein from a viral culture;

(l) separating a glycoprotein from ascites fluid; and

(m) separating a glycoprotein from clostrum;

(n) separating a glycoprotein from milk;

(o) separating a glycoprotein from plasma or a plasma fraction;

(p) separating a glycoprotein from serum;

(q) separating a glycoprotein from tissue;

(r) separating a glycoprotein from whey;

(s) separating a glycoprotein from whole blood;

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

(u) separating a hormone from ascites fluid;

(v) separating a hormone from clostrum;

(w) separating a hormone from milk;

(x) separating a hormone from plasma or a plasma fraction;

(y) separating a hormone from serum;

(z) separating a hormone from tissue;

(aa) separating a hormone from whey;

(bb) separating a hormone from whole blood;

(cc) separating a liquid to produce a vaccine or vaccine component;

(dd) separating a plasma or a plasma fraction into its constituent parts;

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

(ff) separating a viral culture fluid into its constituent parts;

(gg) separating an antigen from a viral culture containing same;

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

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

(jj) separating an immunoglobulin from ascites fluid;

(kk) separating an immunoglobulin from clostrum;

(ll) separating an immunoglobulin from milk;

(mm) separating an immunoglobulin from plasma or a plasma fraction;

(nn) separating an immunoglobulin from serum;

(oo) separating an immunoglobulin from whey;

(pp) separating an immunoglobulin from whole blood;

(qq) separating an insect cell culture fluid into its constituent parts;

(rr) separating clostrum into its constituent parts;

(ss) separating milk into its constituent parts; and

(tt) separating whey into its constituent parts.

31. The method according to claim 1 or 2 wherein two or more resins are used to purify two or more target substances simultaneously.

33. The method according to any of claims 1-6 wherein the chromatography resin comprises resin particles, wherein at least some of the resin particles comprise multiple ligands thereon, whereby different target substances may be bound by a single resin particle.

34. The method according to any of claims 1-6, wherein said contacting of the liquid and the chromatography resin(s) comprises adding the liquid to a constant volume reservoir comprising chromatographic resin therein.

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

(a) contacting the source liquid with a chromatography resin;

(b) maintaining the contact for a sufficient period of time and under conditions suitable for the chromatography resin to bind a predetermined fraction of target substance(s);

(c) circulating the chromatography resin in a cross-flow filter wherein one or more of the following steps is performed;

(i) separating unbound substances from the chromatography-resin-bound target substance(s) by concentration and/or diafiltration;

(ii) eluting the target substance(s) from the chromatography resin; and

(iii) separating the target substance(s) from the chromatography resin by concentration and/or diafiltration;

(d) recovering the target substance(s); and

(e) optionally concentrating and/or diafiltering the target substance(s).

3. A process for depleting one or more contaminants from a source liquid, said process comprising:

(a) contacting the source liquid with one or more chromatography resins;

(b) maintaining the contact for a sufficient period of time and under conditions suitable for the resins to bind a predetermined fraction of the contaminant(s);

(c) circulating the chromatography resin(s) in a cross-flow filter wherein the unbound substances are separated from the chromatography-resin-bound contaminant(s) by concentration and/or diafiltration.

17. The process of claim 3, 4, 5 or 6, further comprising rejuvenating chromatography resin(s) for reuse by a process comprising eluting the one or more chromatography-resin-bound contaminant(s) and concentrating and/or diafiltering the resin slurry to remove the eluted materials from the resin slurry.

25. The method of any of claims 3-6, wherein the step of contacting the liquid with the chromatography resin(s) to bind the contaminant(s) thereto by adding the liquid to a circulating loop of chromatography resin(s) using a cross-flow filter.

32. The method according to claim 3 or 4 wherein two or more resins are used to capture two or more contaminant(s) simultaneously.

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

(a) contacting the source liquid with a chromatography resin;

(b) maintaining the contact for a sufficient period of time and under conditions suitable for the resins to bind a predetermined fraction of one or more contaminants;

(c) circulating the chromatography resin in a cross-flow filter to separate unbound substances from the chromatography-resin-bound target substance(s) by concentration and/or diafiltration.

5. A process for depleting one or more contaminants from a source liquid, said process comprising:

(a) contacting the source liquid with one or more chromatography resins;

(b) maintaining the contact for a sufficient period of time under conditions suitable for the resins to bind a predetermined fraction of one or more contaminant(s);

(c) circulating the chromatography resin(s) in a cross-flow filter wherein one or more of the following steps is performed;

(i) separating unbound substances from the chromatography-resin-bound contaminant(s) by concentration and/or diafiltration;

(ii) recovering the contaminant(s)-depleted source liquid; and

(iii) optionally concentrating and/or diafiltering of the source liquid.

6. A process for depleting one or more contaminants from a source liquid, said process comprising:

(a) contacting the source liquid with a chromatography resin;

(b) maintaining the contact for a sufficient period of time and under conditions suitable for the resins to bind a predetermined fraction of one or more contaminant(s);

(c) circulating the chromatography resin in a cross-flow filter to separate unbound substances from the chromatography-resin-bound contaminant(s) by concentration and/or diafiltration;

(d) recovering the contaminant(s)-depleted source liquid; and

(e) optionally concentrating and/or diafiltration of the source liquid.

35. A method of purifying a liquid comprising one or more target substances, the method comprising

(a) contacting the liquid with a chromatography resin to bind the target substance(s) thereto;

and

(b) cross-flow filtering the target substance(s)-bound chromatography resin under elution conditions to recover a filtrate comprising the target substance(s).

37. The method of claim 35 or 36, further comprising clarifying the liquid prior to said contacting with the chromatography resin.

38. The method of claim 37, wherein said clarifying step removes contaminant(s) adverse to purification of the liquid.

39. The method of claim 35 or 36, wherein the cross-flow filtering produces a permeate and said permeate is further purified.

36. A method of purifying a liquid purifying comprising one or more target substance(s), the method comprising the steps of contacting the liquid with a chromatography resin to bind one or more non-target substances thereto, and cross-flow filtering the non-target substance(s)-bound chromatography resin under concentration and/or diafiltration conditions to recover a filtrate comprising the target substance(s).

40. A method of purifying one or more target substances from a source liquid comprising one or more bioactive contaminants, the method comprising the steps of:

(a) contacting the source liquid with a chromatography resin;

(b) maintaining the contact for a sufficient period of time and under conditions suitable for the resin to bind a predetermined fraction of the target substance(s);

(c) circulating the chromatography resin in a cross-flow filter wherein one or more of more of the following steps is performed;

(i) concentration and/or diafiltration of the chromatography resin;

(ii) elution of the target substance(s) from the chromatography resin; and

(iii) separation of the target substance(s) from the chromatography resin by concentration and/or diafiltration;

(d) recovering the target substance(s);

(e) optionally concentrating the target substance(s); and

(f) inactivating the bioactive contaminant(s) during said method.

41. The method of claim 40, wherein the bioactive contaminant(s) comprise a bioactive species selected from the group consisting of bacteria, yeasts, mycoplasma, parasites and immunoantigens.

42. The method of claim 40, wherein the bioactive contaminant(s) comprise a virus selected from the group consisting of HIV, hepatitis virus, rabies virus, Epstein-Barr virus, measles virus, mumps virus, chickenpox virus and Parvovirus.

43. The method of claim 40, wherein the inactivating step comprises heating the source liquid.

44. The method of claim 40, wherein the inactivating step comprises heating the permeate containing the target substance(s).

45. The method of claim 40, wherein the inactivating step comprises contacting the bioactive contaminant(s) with one or more inactivating agents.

46. The method of claim 40, wherein the inactivating step comprises contacting the bioactive contaminant(s) with one or more inactivating agents as an initial step of the method.

47. The method of claim 40, wherein the inactivating step comprises contacting the bioactive contaminant(s) with one or more inactivating agents for a sufficient period of time and under conditions suitable, with the source liquid and with the chromatography resin, to inactivate the bioactive contaminant(s).

48. The method of claim 40, wherein the inactivating step comprises contacting the bioactive contaminant(s) with one or more inactivating agent(s) during cross-flow filtration processing.

49. The method of claim 48, wherein the inactivating agent comprises a microbead-bound inactivating agent species.

50. The method of claim 48, wherein the inactivating agent comprises a microbead-bound capture agent for the bioactive contaminant(s).

51. The method of claim 48, wherein the inactivating agent comprises radiation.

52. The method of claim 48, wherein the inactivating agent comprises an agent selected from the group consisting of iodine, chlorine, solvent-detergent compositions, and ultraviolet radiation.

53. A process for purifying one or more target substances from a source liquid comprising the target substance(s) together with one or more viral species, the process comprising:

(a) contacting the source liquid with a viral inactivation agent to inactivate the viral species;

(b) contacting the source liquid with a chromatography resin;

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

(d) circulating the chromatography resin in a cross-flow filter apparatus wherein one or more of the following steps is performed;

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

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

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

(e) recovering the target substance(s); and

(f) optionally concentrating the target substance(s).

54. A process for purifying one or more target substances from a source liquid containing the target substance(s), the process comprising:

(a) contacting the source liquid with a chromatography resin;

(b) circulating the chromatography resin in at least one cross-flow filter apparatus wherein one or more of the following steps is performed;

(i) optionally concentrating the target substance(s) and chromatography resin in the source liquid by tangential flow filtration;

(ii) 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;

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

(iv) separating the target substance(s) from the chromatography resin by concentration and/or diafiltration of the resin recovering the target substance(s); and

(v) optionally concentrating the target substance(s).

55. The process of claim 54, wherein the source liquid comprises one or more pathogenic organisms, the method further comprising contacting the source liquid with a viral inactivation agent for the one or more pathogenic organism(s).

56. The process of claim 54, 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).

57. A method of separating a liquid in a separation system, the method comprising:

(a) providing 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;

(b) clarifying perfusate of the bioreactor in the first cross-flow filtration module to yield a permeate;

(c) flowing the permeate to the chromatographic resin reservoir;

(d) flowing chromatographic resin and permeate to the second cross-flow filtration module for concentration, diafiltration and elution to yield an eluate; and

(e) flowing the eluate to the third cross-flow filtration module for concentration and diafiltration therein.

58. A method of separating a liquid in a separation system, the method comprising:

(a) providing a bioreactor, a chromatographic resin reservoir, a first cross-flow filtration module, and a second cross-flow filtration module;

(b) optionally, clarifying the fluid from the bioreactor;

(c) flowing the fluid to the chromatographic resin reservoir;

(d) flowing chromatographic resin and fluid to the first cross-flow filtration module for concentration and/or diafiltration and elution to yield an eluate; and

(e) flowing the eluate to the second cross-flow filtration module for concentration and/or diafiltration therein.

59. A method for purifying multiple target substances from a source liquid comprising said target substances, utilizing multiple cross-flow filters and multiple chromatography resins the method comprising:

(a) contacting the source liquid with a first chromatography resin;

(b) circulating the first chromatography resin across a first cross-flow filter wherein the following steps are performed;

(i) optionally, concentrating a first target substance and the first chromatography resin in the source liquid by tangential flow filtration;

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

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

(c) separating the first target substance from the first chromatography resin by concentration and/or diafiltration of the first resin recovering the first target substance; and

(d) optionally, concentrating the first target substance;

(e) contacting the permeate of the first cross-flow filter with a second chromatography resin to isolate a second target substance;

(f) circulating the second chromatography resin across a second cross-flow filter wherein one or more of the following steps is performed;

(i) optionally concentrating the second target substance and the second chromatography resin in the permeate of the first cross-flow filter by tangential flow filtration;

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

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

(g) separating the second target substance from the second chromatography resin by concentration and/or diafiltration of the second resin recovering the second target substance; and

(h) optionally concentrating the second target substance; and

(i) sequentially repeating steps sequence of steps (e), (f), (g), and (h) with the permeate generated by the previous cross-flow filter with a subsequent chromatography resin until all the desired target substances are obtained.

60. A method according to claim 59 where the first, second and subsequent cross-flow filters are the same filter.

61. A method according to claim 59 where the source liquid is selected from the group consisting of milk, clostrum, blood, plasma, serum, fruit juice, vegetable juice, and plant extracts.