Virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF and preparation method thereof
View Patent ↗The invention concerns human platelet extracts rich in growth factors (PGF) for wound healing and stem cell expansion. Accordingly the subject invention relates to a virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF, which is preferably enriched in TGF, IGF and EGF-rich. The present invention further concerns a method for obtaining a platelet lysate comprising the steps of contacting a starting platelet concentrate with a solvent and/or a detergent, incubating the starting platelet concentrate with the solvent and/or detergent for a period of at least 5 minutes to 6 hours, at a pH maintained in a range from about 6.0 to about 9.0, and at a temperature within the range of from 2° C. to 50° C., optionally removing the solvent and/or the detergent by oil extraction and obtaining an aqueous protein phase, and incubating the solvent and/or detergent-treated platelet concentrate or the aqueous protein phase with charcoal.
1. A method for preparing a virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF comprising:
a) containing a starting platelet concentrate with a solvent and a detergent;
b) incubating for a period of at least 5 minutes to 6 hours, at a physiological pH or at a pH ranging from pH 6.8 to 8.2, and at a temperature within the range of from 2° C. to 50° C.;
c) removing the solvent and detergent by oil extraction and obtaining an aqueous protein phase;
d) mixing activated charcoal with the aqueous protein phase obtained in c);
e) centrifuging the mixture of d) to pellet the charcoal; and
f) recovering the supernatant from e), wherein the supernatant is virally-inactivated and depleted of PDGF and VEGF.
2. The method of claim 1 wherein the solvent is selected from the group consisting of di- or trialkylphosphates, di- or trialkylphosphates with different alkyl chains.
3. The method according to claim 2 , wherein the solvent is the tri-n-butylphosphate (TnBP).
4. The method of claim 1 wherein the detergent is selected from the group consisting of polyoxyethylene derivatives of fatty acids, partial esters of sorbitol anhydrides, non-ionic detergents, sodium deoxycholate and sulfobetaines.
5. The method according to claim 4 , wherein the detergent is oxyethylated alkylphenol or polysorbate 80.
6. The method of claim 1 wherein the concentration of both the solvent and detergent in step a) ranges from 0.2 to 5% in volume with respect to the volume of the starting platelet concentrate.
7. The method according to claim 1 , wherein oil extraction is performed with a pharmaceutical grade oil, the oil being used in an amount of from 2 to 20 weight % based on the weight of the mixture of the platelet concentrate with the solvent and the detergent.
8. The method according to claim 7 , wherein the oil is in the amount of from 5 to 15 weight %.
9. The method according to claim 8 , wherein the oil is in the amount of from 5 to 10 weight %.
10. The method according to claim 1 , wherein the activated charcoal is in an amount of from 50 to 250 g/l of said aqueous protein phase.
11. The method according to claim 1 , further comprising centrifuging after step c) to pellet and remove any cell debris or insoluble material and recover the supernatant.
12. The method according to claim 1 , wherein the starting platelet concentrate of step a) is prepared by at least one of apheresis and buffy-coat isolation from whole blood.
13. The method according to claim 1 , wherein the starting platelet concentrate of step a) is obtained from pool platelet concentrates.