Patent EA-002349-B1 (EA)
Title: Purification of neurotrophins
Inventor: BURTON LOUIS E
IPC: C07K1/20
Filed: 1997-11-14
Abstract: 1. A process to isolate a neurotrophin from a mixture containing variants of that neurotrophin that can include a misfolded variant, an incorrectly proteolytically processed variant, or a glycosylation variant of that neurotrophin, comprising  a). loading the mixture onto a hydrophobic interaction chromatography resin, b). eluting the neurotrophin from the resin with an elution buffer under conditions in which the neurotrophin separates from the variant, and c). collecting neutrophin. 2. The process of claim 1, in which the resin comprises a phenyl functional group. 3. The process of claim 1, wherein the mixture loaded onto the hydrophobic interaction chromatography resin has a pH of 5 to 8. 4. The process of claim 1, wherein the mixture loaded onto the resin has a salt concentration of 0.5M to 3M. 5. The process of claim 4, wherein the mixture loaded onto the resin has a salt concentration of 0.5M to 2.5M. 6. The process of claim 5, wherein the mixture loaded onto the resin has a salt concentration of 0.7M acetate or l.0M to 2.5M NaCl. 7. The process of claim 1, wherein the elution buffer comprises organic solvent. 8. The process of claim 7, wherein the organic solvent is 5% to 20% by volume. 9. The process of claim 1, wherein the elution buffer pH is pH 5 to pH 8. 10. The process of claim 1, wherein eluting comprises a decreasing salt gradient. 11. The process of claim 1, wherein the neurotrophin is in the NGF superfamily. 12. The process of claim 10, wherein the neurotrophin is NGF, NT-4/5 or NT-3. 13. The process of claim 1, wherein the neurotrophin is prepared from bacterial culture and refolded in vitro prior to using hydrophobic interaction resin. 14. . The process of claim 1, wherein the neurotrophin is isolated from mammalian cell culture. 15. The process of claim 1, further comprising separating the neurotrophin from its chemical variants using high performance cation exchange chromatography resin. 16. The process of claim 15, wherein the high performance cation exchange chromatography separating step comprises loading a mixture comprising the neurotrophin and a chemical variant of that neurotrophin onto a high performance cation exchange chromatography resin and eluting the neurotrophin from the resin under conditions in which the neurotrophin separates from the chemical variant, and wherein the neurotrophin has a high pi. 17. The process of claim 16, wherein the high performance cation-exchange resin is an SP-Sepharose HP, poly aspartic acid, polysulfoethyl cation-exchange, or Fractogel EMD S03 resin. 18. The process of claim 16, wherein the neurotrophin-containing eluate from the high performance cation-exchange resin in desalted or subjected to diafiltration and then formulated with a carrier. 19. The process of claim 1, further comprising separating the neurotrophin from other proteins using silica gel resin by eluting the neurotrophin from the resin with an elution buffer, comprising, preferably, TMAS or TEAS buffered at pH from 3.5 to 8.0 20. The process of claim 19, comprising isolating a neurotrophin from other proteins using a silica gel resin being free of alcoholic or polar aprotic solvent. 21. The process of claim 15, wherein the neurotrophin and its variant are essentially pure prior to separating. 22. The process of claim 15, further optionally comprising the step of separating the neurotrophin from a misfolded variant of that neurotrophin using preparative reversed-phase liquid chromatography resin. 23. The process of claim 22, wherein the resin contains a C4 functional group. 24. The process of claim 1, further comprising the step of purifying using high performance chromatography and thus includes the steps of: (a) loading a mixture comprising the neurotrophin and its variant onto a hydrophobic interaction chromatrography resin at pH 5 to 8; (b) washing the resin with a buffer at pH 5 to 8; (c) eluting the neurotrophin with a buffer at pH 5 to 8 containing an alcoholic or polar aprotic solvent at a concentration of about 5 to 25% (v/v); (d) loading the neurotrophin-containing eluant onto a high performance cation exchange chromatography resin at pH of 5 to 6; and (e) eluting the neurotrophin from the high performance cation exchange chromatography resin with a buffer at pH of 5 to 6 containing a cation at a concentration of 0.2 to 0.5 M. 25. A composition comprising neurotrophin prepared by a process of any one of the preceding claims. 26. The composition of claim 25, further comprising an acceptable carrier. 27. The composition of claim 26 that is sterile.