Claims for Patent: 8,617,840

Summary for Patent: 8,617,840

Title:	Lysis/resealing process and device for incorporating an active ingredient, in particular asparaginase or inositol hexaphosphate, in erythrocytes
Abstract:	Lysis/resealing process for preparing erythrocytes which contain an active ingredient (e.g. aspariginase or inositol hexaphosphate), the process comprising the following steps: (1) placing a globular concentrate in suspension in an isotonic solution having a haematocrit level which is equal to or greater than 65%, with refrigeration at from +1 to +8.degree. C., (2) measuring the osmotic fragility based on a sample of erythrocytes from that same globular concentrate, preferably on a sample of the suspension obtained in step (1), (3) lysis and internalization procedure of the active ingredient, inside the same chamber, at a temperature which is constantly maintained at from +1 to +8.degree. C., comprising allowing the erythrocyte suspension having a haematocrit level which is equal to or greater than 65% and a hypotonic lysis solution which is refrigerated at from +1 to +8.degree. C. to circulate in a dialysis cartridge; the lysis parameters being adjusted in accordance with the osmotic fragility previously measured; and (4) resealing procedure carried out in a second chamber at a temperature of from +30 to +40.degree. C. by means of a hypertonic solution.
Inventor(s):	Godfrin; Yann (Lyons, FR)
Assignee:	Erytech Pharma (Lyons, FR)
Application Number:	11/573,093
Patent Claims:	1. A lysis/resealing process for preparing erythrocytes which contain an active ingredient, the process comprising the following steps: (1) placing a globular erythrocyte concentrate in suspension in an isotonic solution at a haematocrit level which is equal to or greater than 65%, with refrigeration at from +1 to +8.degree. C., thereby obtaining an erythrocyte suspension; (2) measuring the osmotic fragility based on a sample of erythrocytes from the globular erythrocyte concentrate, the steps 1 and 2 being able to be carried out in any order; (3) lysing the erythrocytes in the erythrocyte suspension inside a first chamber, at a temperature which is constantly maintained at from +1 to +8.degree. C., by allowing the erythrocyte suspension obtained in step (1) and a hypotonic lysis solution which is refrigerated at from +1 to +8.degree. C., to circulate in a dialysis cartridge, thus obtaining a suspension of lysed erythrocytes, wherein, based on the measurement of osmotic fragility, the flow rate of the erythrocyte suspension which passes into the dialysis cartridge is adjusted, or the osmolarity of the lysis solution is selected; (4) internalizing an active ingredient in the erythrocytes by adding the active ingredient to the erythrocyte suspension obtained in step (1) or in step (3) before or after passing the erythrocyte suspension into the dialysis cartridge, thereby obtaining internalization of the active ingredient in the erythrocytes; and (5) resealing the erythrocytes obtained at step (4) in a second chamber at a temperature of from +30 to +40.degree. C. by means of a hypertonic solution. 2. Process according to claim 1, wherein the osmotic fragility is measured by means of a measuring device which is configured in order to measure the osmotic fragility of a sample of erythrocytes in less than 15 minutes and the result obtained is used within a brief period of time in order to adjust the lysis parameters. 3. Process according to claim 1, wherein the sample of erythrocytes at step (2) is measured for one or more of its haemolysis parameters against a hypotonic solution, having known isotonicity, through a semi-permeable membrane. 4. Process according to claim 3, wherein one or more of the following parameters is/are measured: a. the osmolarity of the medium for which haemolysis appears, b. the velocity of haemolysis, which is established by the gradient of the linear portion of the haemolysis %=f(osmolarity of the medium) curve, c. the percentage of haemolysis for a given osmolarity, d. the osmolarity which allows 50% haemolysis to be obtained, e. the time for obtaining a given percentage of haemolysis. 5. Process according to claim 1, wherein a refrigerated module is provided with temperature control, a pouch of the erythrocyte suspension which is refrigerated at from +1 to +8.degree. C. is placed in the module, and is connected to a sterile, single-use removable assembly which comprises a dialysis cartridge, tubes for connecting the cartridge, at one side, to the pouch and, at the other side, to the flask, the module further comprising means which can bring about the circulation of the erythrocyte suspension and the lysis solution, inside which module the temperature is stabilized at from +1 to +8.degree. C. 6. Process according to claim 5, wherein the lysis procedure is started when the temperature of the suspension in the pouch is from +1 to +8.degree. C. 7. Process according to claim 1, wherein a stable level of haematocrit is maintained in the suspension for the entire time of its passage in the dialysis cartridge. 8. Process according to claim 7, wherein there is used a pouch which is provided with external loop type circulation which can bring about circulation of the suspension to and from the pouch. 9. Process according to claim 1, wherein the active ingredient is present in the suspension pouch and/or is introduced into the suspension circulation before and/or after the passage through the dialysis cartridge. 10. Process according to claim 1, wherein the globular concentrate contains erythrocytes which have previously been processed with a solution which can increase and/or homogenize the osmotic strength of said erythrocytes. 11. Process according to claim 1, wherein the temperature during steps 1 and 3 is maintained at from +2 to +6.degree. C., and is approximately +4.degree. C. 12. Process according to claim 1, wherein the active ingredient is selected from asparaginase and inositol hexaphosphate. 13. Process according to claim 12, to produce erythrocytes incorporating asparaginase intended to treat a patient against acute lymphoblastic leukemias and lymphomas, or to produce erythrocytes incorporating inositol hexaphosphate intended to treat hypoxic tumours in association with a radiotherapy treatment or to treat drepanocytosys or other hypoxic status. 14. Process according to claim 1, wherein the osmotic fragility is measured on a sample of the suspension obtained in step (1). 15. Process according to claim 1, wherein the measurement of the osmotic fragility carried out is rapidly used, that is to say the lysis procedure is carried out a short time after the sample is taken, the time interval between the sample being taken and the start of lysis being less than or equal to 30 minutes. 16. Process according to claim 15, wherein this time interval is less than or equal to 25 minutes. 17. Process according to claim 15, wherein this time interval is less than or equal to 20 minutes. 18. Process according to claim 2, wherein the process is carried out using a device which brings together, at one side and the other of a semi-permeable membrane, the sample of the erythrocyte suspension to be evaluated, and a hypotonic solution, having known isotonicity, in suitable volumes, so as to generate slow haemolysis of the erythrocytes as the NaCl ions diffuse towards the hypotonic solution, for example, distilled water, wherein the progress of haemolysis over time is followed by a measurement of the transmittance by means of a laser beam which has a wavelength of 808 nm and a photoelectric cell measures the variations in the light transmitted through the suspension. 19. Process according to claim 1, wherein the measurement of the osmotic fragility is conducted on erythrocytes or a suspension containing them at a temperature near to or identical to the temperature selected for lysis. 20. Process according to claim 4, wherein the osmotic strength is characterized by means of the parameters b, d, or b and d. 21. Process according to claim 13, wherein the suspension obtained in step (1) contains the active ingredient. 22. Process according to claim 3, wherein the concentration of NaCl in g/L which brings about 50% haemolysis is measured (parameter d.) and the flow rate of the erythrocyte suspension in the dialysis cartridge is adjusted in accordance with the measured concentration values. 23. Process according to claim 1, wherein the lysis procedure is started when the temperature of the erythrocyte suspension is from +1 to +8.degree. C., and the osmotic fragility has been measured and the lysis parameters recorded. 24. Process according to claim 1, wherein the lysis solution flows through the dialysis cartridge at a constant flow rate. 25. Process according to claim 11, wherein the temperature during steps 1 and 3 is maintained at +4.degree. C. 26. Process according to claim 18, wherein the hypotonic solution is distilled water.

Details for Patent 8,617,840

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Recordati Rare Diseases, Inc.	ELSPAR	asparaginase	For Injection	101063	01/10/1978		2024-08-05