Method for stabilising suspensions of red blood cells encapsulating an active ingredient
A method for obtaining a stabilized suspension of red blood cells encapsulating an active ingredient, from resealed RBCs incorporating the active ingredient, the method comprising the incubation of the resealed RBCs in an incubation solution at an osmolality of no less than 280 m Osmol/kg, for a time of 30 minutes or more, the incubation solution being a solution that does not contain an agent which is denaturating for the RBC membrane, the liquid medium is then removed from the incubated suspension and the RBCs obtained are placed in suspension in a solution allowing the injection of the suspension in a patient. The suspensions obtained are particularly characterized by an extracellular haemoglobin level maintained at 0.5 or lower, in particular 0.2 g/dl or lower and/or a hemolysis rate maintained at 2 or less, in particular 1% or less, at 72 h after placing in suspension in a preservation solution and at a temperature of between 2 and 8° C.
1. A method for obtaining a stabilised suspension of red blood cells (RBCs) encapsulating an active ingredient, from completely resealed RBCs incorporating the active ingredient, the method comprising the incubation of the resealed RBCs in an incubation solution, at an osmolality of no less than 280 mOsmol/kg, for a time of 30 minutes or more, or a time of 30 minutes to about 3 hours, the incubation solution being a solution that does not contain an agent which is denaturing for the RBC membrane, the liquid medium is then removed from the incubated suspension and the RBCs obtained are placed in suspension in a solution allowing the injection of the suspension in a patient;
wherein the incubation solution does not contain a bridging or cross-linking agent; and
wherein the resealed RBCs were not treated with, nor do they contain, a bridging or cross-linking agent.
2. The method of claim 1 , comprising, before incubation, encapsulating by lysis-resealing the active ingredient into RBCs and obtaining resealed RBCs comprising the active ingredient.
3. The method according to claim 1 , wherein the incubation is performed at an osmolality of between 280 and about 380 mOsmol/kg.
4. The method according to claim 1 , wherein the incubation is performed at an osmolality of between about 290 and about 330 mOsmol/kg.
5. The method of claim 1 , wherein the incubation has a duration that is 1 h or longer.
6. The method of claim 1 , further comprising washing the resealed RBCs before the incubation.
7. The method of claim 6 , wherein washing comprises at least one washing cycle comprising dilution of or placing in suspension the resealed RBCs in a solution, at an osmolality of no less than 280 mOsmol/kg, then separation.
8. The method of claim 7 , wherein washing comprises 2 or 3 washing cycles.
9. The method according to claim 1 , comprising:
(a) providing the resealed RBCs incorporating the active ingredient;
(b) obtaining a suspension or residue comprising the RBCs incorporating the active ingredient and a solution at an osmolality of 280 mOsmol/kg or higher;
(c) incubating the residue or suspension of step (b) as such or after addition of an incubation solution at an osmolality of 280 mOsmol/kg or higher, for a time of 30 minutes or longer;
(d) eliminating the liquid medium from the suspension incubated at step (b),
(e) placing in suspension the RBCs obtained at (d) in a solution allowing injection of the suspension in a patient, preferably a preservation solution allowing injection of the suspension in a patient.
10. The method according to claim 9 , wherein step (b) comprises at least one 1 washing cycle, by dilution of the suspension or residue obtained at (a) in a solution, at an osmolality of 280 mOsmol/kg or higher, then obtaining a residue or suspension of RBCs.
11. The method of claim 9 , wherein at step (d) either a separation is performed, or a dilution before separation.
12. The method of claim 1 , wherein the incubation step is conducted at a temperature of between about 2 and about 39° C.
13. The method of claim 1 , wherein:
the incubation step is conducted at low temperature between about 2 and about 10° C., and lasts about 1 h to about 72 h; or
the incubation step is conducted at a temperature between about 20 and about 39° C., and lasts about 30 min to about 1 h.
14. The method according to claim 1 , comprising the recovering of a stabilised RBC suspension having an extracellular haemoglobin level maintained at 0.5 or lower and/or a haemolysis rate maintained at 2 or less at 72 h after placing in suspension in the preservation solution and at a temperature of between 2 and 8° C.
15. The method according to claim 1 , comprising the recovering of a stabilised RBC suspension having an extracellular haemoglobin level maintained at 0.5 or lower and/or a haemolysis rate maintained at 2 or less for a time of between 24 h and 20 days after placing in suspension in the preservation solution and at a temperature of between 2 and 8° C.
16. The method according to claim 1 , wherein the active ingredient is selected from the group consisting of L-asparaginase, IHP, ADI, Factor VIII, Factor IX, alglucosidase, beta-glucosidase, bisphosphonates, particularly 2 nd and 3 rd generation, uricase, thymidine phosphorylase, and adenosine deaminase.