Patent EA-000694-B1 (EA)
Title: Process for the purification of erythropoietin
Inventor: ZANETTE DINO
IPC: C07K14/505
Filed: 1996-04-09
Abstract: 1. A process for the purification of erythropoietin which comprises the following steps: a) applying a material containing erythropoietin to a dihydroxyboronyl-containing chromatographic matrix pre-equilibrated with a first equilibrating buffer, b) washing with the equilibrating buffer and c) eluting with a first elution buffer which is an aqueous buffer having a pH between 7.5 and 11 containing a compound having l-hydroxy-2-amino groups, or, additionally a 1,2-cis-diol containing low molecular weight substance and collecting it. 2. A process according to claim 1 wherein at step a) a filtered culture fluid is used as a substance containing erythropoietin and further comprises the following steps: which comprises: d) contacting of the obtained at step b) eluate directly with an anion exchange matrix bearing quaternary ammonium functional groups; e) eluting with a second elution buffer and collecting it. 3. A process according to claim 1, wherein further comprises the steps of: f) ultrafiltrating on a 5,000-30,000 D cutoff membrane g) optionally diafiltering to exchange the buffer to the one suitable for the next step of gel filtering; and h) gel filtering on a separation resin having a separation range between 5,000 and 30,000 D. 4. A process according to claim 2 or 3, wherein a culture fluid used at step a) is adjusted to pH 7.5-9.0 equilibrated with a equilibrating buffer which is an aqueous buffer at a concentration of 25-100 mM, with an ionic strength between  2 20 mS/cm2 and a pH between 7.5 and 9.0; at step b) washing subsequently with the first equilibrating buffer containing from 10 to 100 mM of a 1,2-cis-diol containing low molecular weight substance; at step c) eluting with a first eluting buffer which is an aqueous buffer having a pH between 7.5 and 11.0 containing a compound having 1-hydroxy, 2-amino groups at a concentration of 20-200 mM, possibly in the presence of a chaotropic agent at 2 to 8 mM, a cyanate acceptor at 2-40 mM, and a surfactant from 0.02% to 0.1% (w/w); at stage d) contacting this eluate with an anion exchange matrix bearing quaternary ammonium functional groups equilibrated in equilibrating buffer which is an aqueous buffer having a pH between 7.5 and 11.0 and from 0.01% to 0.1% (w/w) of a surfactant and after applying the eluate on the anion exchange matrix washing subsequently with the second equilibrating buffer and the same buffer containing additionally up to 50 mM salt; at stage f) eluting with a second eluting buffer having a pH between 7.5 and 11.0 in the presence of from 0.01% to 0.1% (w/w) of a surfactant and from 150 to 350 mM salt. 5. A process according to any one of claims 1, 2, 3 or 4 wherein the dihydroxyboronyl bearing chromatographic support is a phenylboronate agarose. 6. A process according to any one of claims 1, 2, 3 or 4 wherein the first equilibrating buffer is selected from glycine, phosphate, trialkylammonium bicarbonate and 4-(2-hydroxyethyl)-l-piperazinoethane sulfonic acid. 7. A process according to claim 6 wherein the first equilibrating buffer is 4-(2-hydroxyethyl)-l-piperazinoethane sulfonic acid at a concentration of about 0.05 M. 8. A process according to any one of claims 1 to 7 wherein the pH of the first equilibrating buffer is about 8.5. 9. A process according to any one of claims 1 or 4 to 8 wherein the 1,2-cis-diol containing low molecular weight substance is selected from small open-chain polyols such as sorbitol, mannitol, adonitol, arabitol, glycerol, erythritol, and cis-inositol, and closed-chain monosaccharides such as ribose and mannose. 10. A process according to claim 9 wherein the 1,2-cis-diol containing low molecular weight substance is present in a concentration of about 0.05 M. 11. A process according to claim 10 wherein the 1,2-cis-diol containing low molecular weight substance is sorbitol. 12. A process according to any one of claims 4 to 11 wherein the second equilibrating buffer contains a compound having 1-hydroxy 2-amino groups at a concentration of 0.02 - 0.2 H. 13. A process according to any one of claims 4 to 11 wherein the second eluting buffer contains a compound having 1-hydroxy 2-amino groups at a concentration of 0.05 H. 14. A process according to any one of claims 4 to 13 wherein the compound having 1-hydroxy, 2-amino groups is selected from 2-amino-2-hydroxymethyl-l,3-propanediol, bis(hydroxymethyl)aminoethane, N-[2-hydroxy-l,l-bis(hydroxymethylethyl)]glycine or N,N-bis(2-hydroxyethyl)glycine. 15. A process according to any one of claims 4 to 14 wherein the chaotropic agent is selected from urea and its derivatives, and guanidine. 16. A process according to claim 15 wherein the chaotropic agent is 6M urea. 17. A process according to any one of claims 4 to 16 wherein the cyanate acceptor is glycine. 18. A process according to any one of claims 2 to 17 wherein the anion exchange matrix bearing quaternary ammonium functional groups is selected from microcrystalline cellulose or crosslinked agarose matrices bearing diethyl aminoethyl, triethyl aminomethyl or trimethyl aminomethyl functional groups. 19. A process according to claim 18 wherein the anion exchange matrix is trimethyl aminomethyl agarose. 20. A process according to any one of claims 3 to 19 wherein the gel filtration is carried out with controlled pore crosslinked dextrane-acrylamide gel matrix.