CA-3190134-C - A PROCESS FOR THE PURIFICATION OF PROTHROMBIN COMPLEX CONCENTRATE (PCC) AND FIX FROM COMPLETE PLASMA OR CRYO-POOR PLASMA

Abstract

A process for the purification of Prothrombin Complex Concentrate (PCC) from complete plasma or cryo-poor plasma, in particular undiluted cryo-poor plasma, by means of chromatography using a monolithic stationary phase, the method comprising performing an initial sample displacement chromatography step of complete plasma or cryo-poor plasma on an anion exchanger to obtain a first fraction enriched in PCC.

Inventors

• Djuro Josic
• Marija BEGIC
• UROS ANDJELKOVIC

Assignees

• SARTORIUS BIA SEPARATIONS D.O.O.

Dates

Publication Date

20260505

Application Date

20210728

Priority Date

20200728

Claims (14)

1. Claims 1. A process for the purification of Prothrombin Complex Concentrate (PCC) from complete plasma or cryo-poor plasma by means of chromatography using a monolithic stationary phase, the process comprising: a) performing an initial sample displacement chromatography step of complete plasma or cryo-poor plasma on an anion exchanger to obtain a first fraction enriched in PCC.
2. 2. The process of claim 1, wherein the cryo-poor plasma is undiluted cryo-poor plasma.
3. 3. The process of claim 1 or claim 2, wherein the initial chromatography step is performed with a weak anion exchanger or a strong anion exchanger.
4. 4. The process of any one of claims 1 to 3, wherein the process further comprises: b) after elution of the first fraction enriched in PCC from the initial sample displacement chromatography, a second chromatography step of the first fraction enriched in PCC, wherein the chromatography step is performed with an anion exchanger to obtain a second fraction enriched in PCC.
5. 5. The process of claim 4, wherein the process further comprises: c) a third chromatography step of the second fraction enriched in PCC, wherein the chromatography step is performed with a cation exchanger to obtain a third fraction comprising enriched FIX, and in which fraction other components of the PCC are depleted or removed.
6. 6. The process of claim 5, wherein the other components of the PCC are FII, FVII, and FX.
7. 7. The process of claim 5 or claim 6, wherein the cation exchanger has -SO3, -SO4, or -PO4 groups. 16
8. 8. The process of any one of claims 4 to 7, wherein the anion and/or cation exchanger of the second and/or third chromatography step are bulk materials or monolithic materials.
9. 9. The process of any one of claims 1 to 8, further comprising a solvent/detergent virus inactivation step.
10. 10. The process of any one of claims 1 to 9, wherein a stabiliser is added to at least one of the first, second and third fractions.
11. 11. The process of claim 10, wherein the stabiliser is a positively charged amino acid.
12. 12. The process of claim 11, wherein the positively charged amino acid is lysine.
13. 13. The process of any one of claims 1 to 12, wherein a virus filtration step is performed with at least one of the first, second and third fraction.
14. 14. The process of claim 10, wherein heparin is added to at least one of the first, second and third fraction.

Description

A Process for The Purification of Prothrombin Complex Concentrate (PCC) And FIX From Complete Plasma or Cryo-Poor Plasma The invention pertains to a process for the purification of Prothrombin Complex Concentrate (PCC) and FIX from complete plasma or cryo-poor plasma using chromatography, a fraction comprising PCC and a fraction comprising FIX obtainable by the process of the invention. Prothrombin complex concentrate (PCC), also known as factor IX complex, is a medication made up of blood clotting factors II, IX, and X.[1] Some versions also contain factor VII.[2] It is used to treat and prevent bleeding in hemophilia B if pure factor IX is not available.[1][3] It may also be used in those with not enough protein K dependent clotting factors and inhibitors due to other reasons such as warfarin therapy.[3] It is given by slow injection into a vein by a slow intravenous injection".[!] Prothrombin complex concentrate came into medical use in the 1960s.[5] It is on the World Health Organization's List of Essential Medicines, the safest and most effective medicines needed in a health system.[6][7] It is made from human plasma.[4] A version that is made by recombinant methods which only contains factor IX is also available.[8] PCC reverses the effects of warfarin and other vitamin K antagonist anti-coagulants and is used in cases of significant bleeding in people with a coagulopathy. It is also used when such a person must undergo an emergency operation treatment [9]. Other uses include a deficiency of one of the included clotting factors, either congenital or due to liver disease, and haemophilia B.[9] Several guidelines, including those from the American College of Chest Physicians, recommend PCC for warfarin reversal in people with serious bleeding.[10][11][12][13]. EP 0549964 A2 discloses a method for producing a virus-inactivated prothrombin complex concentrate (PCC preparation) with vitamin K dependent clotting factors prothrombin (factor II), proconvertin (factor VII), Stuart-Prower factor (factor X) and hemophilia B factor (factor IX), as well as vitamin K dependent clotting inhibitors protein C, protein S and protein Z wherein stabilized cryopoor plasma is chromatographed on ion exchange materials. After cryoprecipitation and removal of cryoglobulins,citrate-stabilized plasma is treated with an anion exchange material based on QAE-modified dextran. After elution, the resulting fraction is subjected to a first ultrafiltration with a suitable buffer, the so obtained crude PPSB 1 extract is chromatographed on DEAE modified dextran or agarose (Sepharose), and then eluted with a suitable buffer. Summary of The Invention The object underlying the present invention is solved by a process utilizing principles of sample displacement chromatography. Such chromatography is employed with a monolithic anion exchanger separation media at an initial stage of the purification process. Subject matter of the invention is a process for the purification of Prothrombin Complex Concentrate (PCC) from complete plasma or cryo-poor plasma, in particular undiluted cryo-poor plasma, by means of chromatography using a monolithic stationary phase, the method comprising a) performing of an initial sample displacement chromatography step of complete plasma or cryo-poor plasma on an anion exchanger to obtain a first fraction enriched in PCC. In some embodiments, additional chromatography steps are used. In an embodiment of the present invention the initial chromatography step may be performed with a weak anion exchanger or a strong anion exchanger. In another embodiment of the invention the process can further comprise b) after elution of the first fraction enriched in PCC from the initial sample displacement chromatography, a second chromatography step of the first fraction enriched in PCC, wherein the chromatography step is performed with an anion exchanger to obtain a second fraction enriched in PCC. In yet another embodiment of the invention the process may further comprise c) a third chromatography step of the second fraction enriched in PCC, wherein the chromatography step can be performed with a cation exchanger to obtain a third fraction comprising enriched FIX, and in which fraction other components of the PCC, i.e. FII, FVII, and FX, can be depleted or removed. Due to the unexpected finding that undiluted cryopoor plasma can be employed by performing the method of the invention the present method is advantageous over prior art as referenced in [15] which demand dilution of plasma at least two times, most frequently even five times. This saves loss of activity of physiologically active proteins. 2 In still another embodiment of the invention the cation exchanger may be provided with -503, -504, or -PO4 groups. Elution from the 504 (sulphate) monolith can be performed either by salt or by pH gradient. In a further embodiment of the invention the anion and/or cation exchanger of the second and/or third chromatography step can b