Plasma protein fractionation by sequential polyacid precipitation

Patent metadata

Field	Value
Publication number	US-9624262-B2
Application number	US-201214345302-A
Country	US
Kind code	B2
Filing date	Sep 14, 2012
Priority date	Sep 16, 2011
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

There is a recognized need for novel, more simplified, approaches to isolation of plasma from whole blood, as well as a need to isolate cell-free plasma fractions containing different plasma proteins. Methods are divulged for use of aqueous phase systems, formed in blood or blood containing solutions via addition of a single polymer at relatively low concentration, to effect isolation (clarification) of plasma proteins from blood cells. Methods are also divulged to replace widely used Cohn-type plasma protein fractionation which is based on sequential addition of up to 40% (v/v) ethanol and other precipitants, with simple sequential addition of a polyacid. The latter results in isolation of plasma protein fractions (i.e. fibrinogen, immunoglobulin, albumin) in sequence similar to that obtained with Cohn Fractionation and therefore may be suitable for use to reduce solvent use and solvent-related process complications in existing plasma protein purification processes. It may also support use of polymeric film based containers in novel solvent free plasma fractionation processes. The methods disclosed may also be suitable for use in smaller scale plasma protein isolation, in research and diagnostic applications. The general methodologies are robust and can function over a broad range of process variables such as temperature and pH.

First claim

The invention claimed is:

1. A process for separating at least fibrinogen and immunoglobulin G from a sample of blood plasma, comprising the steps of:
a) providing a sample of blood plasma in a container;
b) adding a polyacid and a salt comprising ≦50 mM sodium chloride and at least one additional salt to the blood plasma, whereby said blood plasma comprises 50-100 mmol/L of total salt at pH between 6 and 8, causing formation of a first protein precipitate that comprises a majority of the fibrinogen in said sample and recovering said first protein precipitate and a first supernatant separately; and
c) adding an additional amount of a polyacid to said first supernatant, causing formation of a second protein precipitate that comprises a majority of the immunoglobulin G in said sample and a second supernatant and recovering said second protein precipitate separately, wherein the polyacid is added in an amount to give a total polyacid concentration in said first supernatant which is 5-8%;
wherein said first protein precipitate comprises a concentrate of the first protein and said second protein precipitate comprises a concentrate of the second protein.

2. The process of claim 1 , wherein said polyacid is selected from the group consisting of polyacrylic acid, polymethacrylic acid, polyvinylsulfonic acid, polystyrenesulfonic acid, carboxymethyl dextran and carboxymethyl cellulose.

3. The process of claim 1 , wherein said additional salt is selected from the group consisting of sodium phosphates, potassium phosphates, ammonium phosphates, sodium citrates, potassium citrates, ammonium citrates, sodium sulphates, potassium sulphates, ammonium sulphates, sodium acetate, potassium acetate, ammonium acetate or any combination thereof.

4. The process of claim 1 , wherein the temperature in steps b) and/or step c) is between 0 and 40° C.

5. The process of claim 1 , wherein in step c) the second protein precipitate comprises essentially all the remaining proteins from the blood plasma.

6. The process of claim 1 , wherein step c) further comprises recovering a second supernatant and further comprising a step d) of adding a polyacid and/or a salt to said second supernatant, causing formation of a third protein precipitate comprising a concentrate of a third protein and recovering said third protein precipitate.

7. The process of claim 6 , wherein said third protein is albumin.

8. The process of claim 1 , wherein said blood plasma is human blood plasma or animal blood plasma.

9. The process according to claim 1 , further comprising a step of pathogen inactivation or removal.

10. The process of claim 1 , wherein at least one protein precipitate is redissolved and subjected to a further step such as precipitation, crystallisation, chromatography, and/or filtration for separation of said first, second and/or third protein.

11. The process of claim 1 , wherein step a) is preceded by a step a′) of separating blood cells from blood by aqueous two-phase separation.

12. The process of claim 11 , wherein step a′) comprises the substeps of:
i) adding a self-associating responsive polymer, and optionally a salt, to a sample of blood;
ii) increasing the temperature or adding salt, causing the formation of a polymer rich aqueous phase, a polymer poor aqueous phase and a phase interface comprising blood cells; and
iii) recovering the polymer poor aqueous phase as the blood plasma.

13. The process of claim 12 , wherein the total responsive polymer content in step ii) constitutes about 4-20 wt % of the total system.

14. The process of claim 12 , wherein pH in step ii) is between 6 and 8.

15. The process of claim 12 , wherein the concentration of added salt in step ii) is in the range of 1-500 mmol/L.

16. The process of claim 12 , wherein the salt is selected from the group consisting of sodium chloride, sodium phosphates, potassium phosphates, sodium sulphate, potassium citrates, sodium citrates, ammonium sulphate and sodium acetate; or any combination thereof.

17. The process of claim 12 , wherein in substep i) or ii) 1-10 wt % ethanol is added to the blood.

18. The process of claim 12 , wherein the self-associating responsive polymer exhibits a cloud point between 2 and 100° C. and is optionally selected from the group consisting of ethylene oxide—propylene oxide copolymers and ethylhydroxyethylcellulose.

19. The process of claim 12 , wherein the self-associating responsive polymer has a weight average molecular weight of 0.9-100 kDa.

20. The process of claim 12 , wherein step a′) is run in a continuous mode.

21. The process of claim 12 , wherein step a′) is performed in plastic containers.

22. The process of claim 21 , wherein the self-associating responsive polymer, and optionally the salt, are present in the plastic containers before introduction of the blood in the containers.

23. The process of claim 12 , wherein said polymer poor aqueous phase is freeze dried or stored for at least one day or one week at a temperature below 8° C.

24. The process of claim 12 , wherein said first and second proteins are suitable for use as pharmaceuticals.

25. The process of claim 12 , wherein stabilizers, anti-aggregation agents and/or anti-proteolytic agents are added to the aqueous phases to promote storage and recovery of active target in a native functioning state.

Assignees

• Van Alstine James
• Berg Mikael
• Kjorning Johanna
• Shanagar Jamil
• Ge Healthcare Bioprocess R&D Ab

Inventors

• Van Alstine James
• Berg Mikael
• Kjorning Johanna
• Shanagar Jamil

Classifications

• C07K1/303

  by salting out · CPC title

• C07K1/30 Primary

  by precipitation · CPC title

• A61K35/16

  Blood plasma; Blood serum (umbilical cord blood A61K35/51) · CPC title

• C07K14/76

  Albumins · CPC title

• C07K16/18

  against material from animals or humans · CPC title