Process for separating proteins fibrinogen, factor XIII and biological glue from a solubilized plasma fraction and for preparing lyophilised concentrates of said proteins

1. A process for separating proteins fibrinogen, Factor XIII (FXIII) and biological glue from a solubilized plasma fraction, containing fibrinogen and Factor XIII, and for preparing freeze-dried concentrates of said proteins comprising the steps of:

a) chromatographic purification comprising the steps of:

i) equilibrating an anion exchanger of weak base type with a first buffer having a predetermined ionic strength of an alkaline pH,

ii) loading said anion exchanger of weak base type with the solubilized plasma fraction, thus allowing the retention of the biological glue composed of fibrinogen and FXIII,

iii) eluting the biological glue with a second buffer having an alkaline pH and an ionic strength greater than said first buffer,

b) separating FXIII from fibrinogen by addition to at least a part of the biological glue eluate of at least one chemical agent precipitating the FXIII, wherein the chemical agent precipitating the FXIII is present in the form of an aqueous solution comprising 1M sodium citrate, recovering the resulting supernatant solution of purified fibrinogen, and resolubilizing the precipitated FXIII, and

c) diafiltering the fibrinogen, biological glue and resolubilized FXIII solutions, followed by freeze-drying of said solutions.

2. A process according to claim 1 , wherein said first buffer has an ionic strength of less than 0.2.

3. A process according to claim 1 , wherein the pH of said first buffer is in the range of values higher than 7 up to 9.

4. A process according to claim 1 , wherein the process further comprises, prior to the biological glue elution step, a washing step with said first buffer of the anion exchanger until not retained proteins and contaminants are removed.

5. A process according to claim 1 , wherein the biological glue elution is carried out with said second buffer having an ionic strength in a range between 0.5 and 1.3, the pH of which is set to a value of 7.4-7.6.

6. A process according to claim 5 , wherein said second buffer further contains a mixture from 10 to 12 g/l of trisodium citrate, from 1 to 5 g/l of lysine, from 1 to 5 g/l of glycine, from 2 to 5 g/l of Tris, from 25 to 50 g/l of arginine and from 5 to 15 of isoleucine.

7. A process according to claim 1 , wherein the precipitate of FXIII is resolubilized in water or in a buffer containing a mixture from 10 to 12 g/l of trisodium citrate, from 1 to 5 g/l of lysine, from 1 to 5 g/l of glycine, from 2 to 5 g/l of Tris, from 25 to 50 g/l of arginine and from 5 to 15 g/l of isoleucine, at a pH between 6.9 and 7.1.

8. A process according to claim 1 , wherein the diafiltration is carried out against a buffer containing a mixture from 10 to 12 g/l of trisodium citrate, from 1 to 5 g/l of lysine, from 1 to 5 g/l of glycine, from 2 to 5 g/l of Tris, from 25 to 50 g/l of arginine and from 5 to 15 g/l of isoleucine.

9. A process according to claim 1 , further comprising at least one step of viral inactivation and/or viral and contaminants removal treatment, selected from the group consisting of chemical viral inactivation treatment, nanofiltration and dry heat viral inactivation treatment.

10. A process according to claim 9 , wherein the chemical viral inactivation treatment is carried out prior to the step a).

11. A process according to claim 9 , wherein the chemical viral inactivation treatment consists of a solvent-detergent treatment carried out by viral inactivating chemical agents which are a mixture of polysorbate 80-tri(n-butyl) phosphate (TnBP) or octoxynol-9-TnBP.

12. A process according to claim 9 , wherein the nanofiltrafion is carried out with the biological glue eluate obtained in step a) or with the diafiltered fibrinogen, biological glue and resolubilized FXIII solutions, prior to freeze-drying.

13. A process according to claim 9 , wherein the dry heat viral inactivation treatment is carried out on the freeze-dried fibrinogen, biological glue and FXIII.

14. The process according to claim 1 , wherein a concentration step by ultrafiltration is carried out prior to the step of diafiltration or following the said step of diafiltration, prior to freeze-drying.

15. The process according to claim 1 , further comprising, prior to step a), an initial prepurification step of the solubilized plasma fraction by a classical pretreatment with aluminum hydroxide and/or by a precipitation at low temperature.

16. A process for separating proteins fibrinogen and Factor XIII (FXIII) from a solubilized plasma fraction, containing fibrinogen and Factor XIII, and for preparing freeze-dried concentrates of said proteins comprising the steps of:

a) performing a chromatographic step comprising the steps of:

i) equilibrating an anion exchanger of weak base type with a first buffer having a predetermined ionic strength of an alkaline pH,

ii) loading said anion exchanger of weak base type with the solubilized fraction, thus allowing the retention of the biological glue composed of fibrinogen and FXIII,

iii) eluting said biological glue, comprising fibrinogen and Factor XIII, with a second buffer having an alkaline pH and an ionic strength greater than said first buffer,

b) separating FXIII from fibrinogen by addition to the biological glue eluate of step a)iii of a chemical agent precipitating the FXIII, wherein the chemical agent precipitating the FXIII is present in the form of an aqueous solution comprising 1M sodium citrate,

c) recovering the resulting supernatant solution of purified fibrinogen, and resolubilizing the precipitated FXIII, and

d) diafiltering and freeze-drying the fibrinogen and resolubilized FXIII solutions.

17. The process according to claim 16 , wherein said first buffer has an ionic strength of less than 0.2.

18. The process according to claim 16 , wherein the pH of said first buffer is in the range of values higher than 7 up to 9.

19. The process according to claim 16 , wherein the process further comprises, prior to the biological glue elution step, a washing step with said first buffer of the anion exchanger until not retained proteins and contaminants are removed.

20. The process according to claim 16 , wherein the biological glue elution is carried out with said second buffer having an ionic strength in a range between 0.5 and 1.3, the pH of which is set to a value of 7.4-7.6.

21. The process according to claim 20 , wherein said second buffer further contains a mixture from 10 to 12 g/l of trisodium citrate, from 1 to 5 g/l of lysine, from 1 to 5 g/l of glycine, from 2 to 5 g/l of Tris, from 25 to 50 g/l of arginine and from 5 to 15 g/l of isoleucine.

22. The process according to claim 16 , wherein the diafiltration is carried out against a buffer containing a mixture from 10 to 12 g/l of trisodium citrate, from 1 to 5 g/l of lysine, from 1 to 5 g/l of glycine, from 2 to 5 g/l of Tris, from 25 to 50 g/l of arginine and from 5 to 15 g/l of isoleucine.

23. The process according to claim 16 , further comprising at least one step of viral inactivation and/or viral and contaminants removal treatment, selected from the group consisting of chemical viral inactivation treatment, nanofiltration and dry heat viral inactivation treatment.

24. The process according to claim 16 , wherein a concentration step by ultrafiltration is carried out prior to the step of diafiltration or following the said step of diafiltration, prior to freeze-drying.

25. The process according to claim 16 , further comprising, prior to step a), an initial prepurification step of the solubilized plasma fraction by a classical pretreatment with aluminum hydroxide and/or by a precipitation at low temperature.