Fractionation of protein mixtures

• US 4,322,275 A
• Filed: 10/16/1980
• Issued: 03/30/1982
• Est. Priority Date: 01/10/1980
• Status: Expired due to Term

• Current Assignee: Ionics Incorporated
• Original Assignee: Ionics Incorporated
• Inventors: Jain, Surendar M.
• Primary Examiner(s): Prescott, Arthur C.
• Application Number:
  US06/197,394
• Time in Patent Office: 530 Days
• Field of Search: 204/180 P, 204/180 R, 204/301, 260/112 R, 260/112 B, 260/121, 260/122, 424/12, 424/36, 424/177, 424/96, 424/101, 23/902, 23/913, 23/915, 128/214 B, 128/DIG. 22, 210/DIG. 23, 210/257 M, 210/22 R
• US Class Current: 204/527
• CPC Class Codes: A23C 9/144 by electrical means, e.g. electrodialysis
• CPC Class Codes: A23J 1/00 Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
• CPC Class Codes: A23J 1/06 from blood
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61M 1/3482 by filtrating the filtrate using another cross-flow filter, e.g. a membrane filter
• CPC Class Codes: A61M 1/3486 Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
• CPC Class Codes: B01D 2311/18 pH control
• CPC Class Codes: B01D 2311/2642 Aggregation, sedimentation, flocculation, precipitation or coagulation
• CPC Class Codes: B01D 61/422 Electrodialysis
• CPC Class Codes: B01D 61/44 Ion-selective electrodialysis
• CPC Class Codes: C07K 1/303 by salting out
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: C07K 14/745 Blood coagulation or fibrinolysis factors
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 16/065 Purification, fragmentation
• CPC Class Codes: Y10S 128/22 Blood coagulation
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 530/83 Plasma; serum
• CPC Class Codes: Y10S 530/833 Whey; cheese

Abstract

This invention relates to the separation of complex protein mixtures by lowering (desalting) or by raising (salting-out) their electrolytic (ionic) concentration by electrodialysis followed by chilling, pH adjustment, filtration, and/or centrifugation and optionally thereafter restoration of the lost electrolyte or removal of the gained electrolyte and water. This technique is especially adaptable for therapeutic plasma exchange performed in situ for immunepheresis i.e. removal of globulin or immunecomplexes implicated in autoimmune diseases. The techniques are also applicable for antihemophilic factor separation and purification, and separation of proteins from whey.

Claims

1. A process for separating an aqueous protein mixture into fractions having intrinsically distinguishable compositions comprising removing substantially all the turbidity therefrom, subsequently, adding a salting-out agent thereto by passing said mixture at a velocity of between 3-40 cm/sec. into and out of an electrodialysis apparatus containing at least one pair of contiguous membranes defining a liquid flow chamber therebetween, impressing an electric current across said apparatus at about a CD/K=0.1 to 10 (where CD is current denisty in m-amps/cm² and K is the conductivity of the aqueous mixture in milli Siemens/cm) thereby altering the ionic environment of said mixture by increasing its ionic concentration sufficiently to at least partially destabilize one or more proteins in said mixture, allowing said destabilized protein to form turbidity, subsequently removing substantially all of said turbidity and maintaining the temperature of said mixture during the said separation in the range of between about 0°

-40°

C.

2. A process according to claim 1 wherein the ionic environment of said mixture is altered by increasing its ionic concentration by at least about 10 percent.

3. A process according to claim 1 wherein the ionic environment of said mixture is altered by changing its pH substantially toward the isoelectric point of at least one of the said proteins.

4. A process according to claim 1 wherein the membranes of said electrodialysis apparatus are substantially non-ion selective.

5. A process according to claim 1 wherein at least one of the said membranes in every contiguous pair is ion selective.

6. A process according to claim 1 wherein one of the membranes in every contiguous pair is cation selective.

7. A process according to claim 1 wherein one of the membranes in every contiguous pair is cation selective and the other is anion selective.

8. A process according to claim 5 wherein at least a substantial fraction of the electric current is direct current.

9. A process according to claim 1 wherein the ionic environment of said mixture is altered by substantially increasing the concentration of non-monovalent ions.

10. A process according to claim 1 wherein the ionic environment of said mixture is altered by substantially increasing the concentration of both monovalent and non-monovalent ions.

11. A process according to claim 1 wherein said subsequently removed turbidity is resolubilized.

12. A process according to claim 1 wherein said subsequently removed turbidity is at least in part resolubilized and the said resolubilized part is converted to a turbid insoluble fraction and a soluble fraction by again increasing the ionic concentration by electrodialysis and the resulting turbid insoluble and soluble fraction are separated from each other.

13. A process according to claim 1 wherein said aqueous protein mixture comprises plasma proteins and the said subsequently removed turbidity comprises fibrinogen and antihemophilic factor.

14. A process according to claim 1 wherein said aqueous protein mixture comprises substantially undenatured plasma and the said subsequently removed turbidity comprises immunoglobulins.

15. A process according to claim 1 wherein said aqueous protein mixture comprises plasma proteins, the subsequently removed turbidity comprises fibrinogen and antihemophilic factor which turbidity is resolubilized and separated into a fibrinogen rich fraction and an antihemophilic factor rich fraction by contacting with a material selected from the group consisting of ion exchange resin granules, gel permeation granules and mixtures of the same.

16. A process according to claim 1 wherein the aqueous protein mixture comprises plasma proteins and the subsequently removed trubidity comprises albumin.

17. A process according to claim 16 wherein the subsequently removed turbidity is resolubilized for use as a plasma expander.

18. A process according to claim 1 in which the aqueous protein mixture comprises plasma proteins, wherein the ionic concentration of said mixture is increased by adding, substantially non-toxic salts selected from the group consisting of sulfates, citrates, phosphates, chlorides, acetates, perchlorates, nitrates, sulfites, thiosulfates, bromides, iodides, and mixtures of these salts and the resulting turbidity comprising substantially non-albuminous proteins is removed therefrom .

36. The process according to claim 1 wherein said aqueous protein mixture comprises whey.

37. A process according to claim 1 wherein said aqueous protein mixture comprises plasma proteins and the said subsequently removed turbidity comprises antihemophilic factor.

19. A process for separating an aqueous protein solution comprising dispersed euglobulins and dispersed pseudoglobulins into a euglobulin rich fraction and a psuedoglobulin rich fraction comprising removing substantially all the turbidity therefrom, substantially passing said mixture into and out of an electrodialysis apparatus containing at least one pair of contiguous membranes defining a liquid flow chamber therebetween, impressing an electric current across said apparatus thereby increasing the ionic concentration of said mixture sufficiently to result in the formation of a turbid phase relatively rich in euglobulins and relatively poor in pseudoglobulins, subsequently removing substantially all of said thus second formed turbidity, again passing the remaining aqueous mixture into and out of an electrodialysis apparatus, again imposing an electric current across said apparatus thereby further increasing the ionic concentration of the said mixture sufficiently to result in the formation of a turbid phase relatively poor in euglobulins and relatively rich in pseudoglobulins, subsequently removing substantially all of said thus third formed turbidity, again passing the remaining aqueous solution into and out of an electrodialysis apparatus and impressing an electric current across said apparatus thereby substantially decreasing the ionic concentration of the said remaining solution.

20. A process for separating an aqueous protein mixture into fractions having distinguishable compositions comprising removing substantially all the turbidity therefrom, subsequently adding by electrodialysis a salt selected from the group consisting of sulfates, citrates, acetates, phosphates, chlorides, perchlorates, nitrates, thiosulfates, sulfites, bromides, iodides, or mixtures of these salts thereby altering the ionic environment of said mixture by increasing its ionic concentration sufficiently to at least partially destabilize one or more proteins in said mixture, allowing said destabilized proteins to form turbidity, subsequently removing substantially all of said turbidity, subsequently passing the remaining supernatant at a velocity of between about 3-40 cm/sec. into and out of an electrodialysis apparatus containing at least one pair of contiguous membranes defining a liquid flow chamber therebetween, impressing an electric current across said apparatus at about a CD/K=0.1-10 to remove salts therein and maintaining the temperature of said mixture during said salt removl in the range of between about 0°

-40°

C.

21. A process according to claim 20 wherein said subsequently removed turbidity is at least partly resolubilized in electrolyte at least in part recovered by electrodialysis from said protein mixture and said resolubilized protein is then separated into at least two intrinsically distinguishable components.

22. A process according to claim 20 wherein the ionic environment of said mixture is altered by increasing its ionic concentration by at least about 10 percent.

23. A process according to claim 20 wherein the ionic environment of said mixture is further altered by changing its pH to substantially the isoelectric point of at least one of the proteins thereby resulting in the precipitation of at least said protein.

24. A process according to claim 20 wherein the salt addition is accomplished by dialysis.

25. A process according to claim 20 wherein the salt addition is accomplished through a non-ion-selective membrane.

26. A process according to claim 20 wherein the membranes of said electrodialysis apparatus are substantially non-ion-selective.

27. A process according to claim 20 wherein at least one of the said membranes in every contiguous pair is ion selective.

28. A process according to claim 20 wherein one of the membranes in every contiguous pair is cation selective.

29. A process according to claim 20 wherein one of the membranes in every contiguous pair is cation selective and the other is anion selective.

30. A process according to claim 27 wherein at least a substantial fraction of the electric current is direct current.

31. A process according to claim 20 wherein the salts removed by the electrodialysis apparatus from said remaining supernatant is again added to a subsequent protein mixture to cause formation of turbidity.

32. A process according to claim 20 wherein the aqueous protein mixture comprises plasma proteins the the said subsequently removed turbidity comprises fibrinogen and antihemophilic factor.

33. A process according to claim 20 wherein the aqueous protein mixture comprises substantially undenatured plasma proteins and the said subsequently removed turbidity comprises immunoglobulins.

34. A process according to claim 20 wherein the final product of the electrodialysis treatment is a substantially salt free albumin.

35. A process according to claim 34 wherein an outside source of salt is added to the substantially salt free albumin to obtain the required electrolyte balance suitable for intravenous applications.