We claim:
1. A novel method for purification of recombinant human granulocyte colony stimulating factor (rhG-CSF) to pharmaceutical^ acceptable grade, comprising of:
a) Isolation and washing of G-CSF protein inclusion bodies from bacterial cells.
b) Solubilizing the said washed inclusion body pellet.
c) Complete refolding of G-CSF into native form by a one step method.
d) Subjecting to two-step chromatography, where the first step is cation exchange followed by size exclusion.
e) Buffer exchanging by diafiltration to remove salts for formulation of lyophilization.
f) Recovering the pharmaceutical grade human G-CSF.
2. A method as in claim 1, wherein the inclusion bodies are isolated by lysing the cells through high-pressure homogenization or sonication followed by centrifugation.
3. A method as in claim 2, the sonication program is 3.5 min with 5.0 sec ON/OFF mode and amplitude is 40% for complete cell lysis.
4. A method as in claim 1, wherein the inclusion body pellet is washed with nonionic detergents like TRITON-X100 and D.O.C.
5. A method as in claim 4, wherein the concentration of non-ionic detergents are 0.25% only.
6. A method as in claim 4, wherein the inclusion body purity is more than 98% and recovery is about 90%.
7. A method as in claim 1, wherein solubilization of G-CSF inclusion body pellet is effected by urea and cysteine blocking agent at high alkaline pH.
8. A method as in claim 7, wherein concentration of G-CSF in solubilization step is 2 to 8 mg/mL.
9. A method as in claim 7, wherein concentration of urea and cysteine-blocking agent is 2 to 8M and 10 to 600mM.
10. A method as in claim 1, wherein refolding of the solubilized G-CSF is effected by keeping the diluted sample at pH 8.2 to 8.3 for a period of 16 to 18 hrs at 15 to 19 °c or for a period of 18 to 20 hrs at 2 to 8° C.
11. A method as in claim 10, wherein the refolding efficiency is about 99%.
12. A method as in claim 1, wherein purification of refolded protein is effected by two-step chromatography and diafiltration.
13. A method as in claim 12, wherein the first step chromatography is Ion Exchange
chromatography and SP.Sepharose is used as matrix. /
14. A method as in claim 13, wherein the column used for IEC is BPG 140/500, and
the bed volume was 2.0L.
15. A method as in claim 14, wherein the optimum flow rate in the column is 500 mL/min and the protein is eluted by Tris buffer.
16. A method as in claim 15, wherein the purity of the protein is about 99 % and the recovery is about 70%.
17. A method as in claim 1, wherein the IEC pooled protein is acedified to pH 3.0 to 3.5 with acetic acid / HC1.
18. A method as in claim 17, wherein the acedified protein is Buffer exchanged with acetic acid and concentrated by Tangential flow filtration system (pall life science) using 3.0 K.D cassette.
19. A method as in claim 18, wherein the concentration of the protein sample was 1.0 to 3.5mg/mL.
20. A method as in claim 1, wherein the second step chromatography is size . Exclusion chromatography and superdex-75, sephadex-G25 and supharose-12 are used as matrix.
21. A method as claimed in claim 20, where in the column used for SEC is XK 50/1000, and the bed volume is about 1.5 L .
22. A method as in claim 21, wherein the optimum flow rate in the column is 4.0 mL/min and the protein is eluted by 0.2m sodium acetate buffers.
23. A method as in claim 22, wherein the purity of the protein is about 99.9 % and the recovery is about 52%.
24. A method as in claim 1, wherein the SEC pooled protein is Buffer exchanged with lOmM sodium acetate and concentrated by Tangential flow filtration method (pall /milliporore) using 3.0 K.D cassette.
25. A method as in claim 24, wherein the concentration of the protein sample was 0.3 to 0.5 mg/mL.
26. A method as in claim 1, wherein the concentrated protein solution is sterile filtered through 0.2 micron and formulated with 5% sorbitol and 0.004% Tween -80.
27. A method as in claim 1, wherein the second step chromatography is size Exclusion chromatography, and sephadex-G25 is used as matrix.
28. A method as in claim 27, wherein the column used for SEC is XK 50/1000, and the bed height is about 75 % of column length.
29. A method as in claim 28, wherein the optimum flow rate in the column is 1.25-tol.30 cm/min.
30. A method as in claim 29, wherein the column equilibration, washing and protein elution is done by G-CSF formulation buffer.
31. A method as in claim 1, wherein the second step of chromatography is size Exclusion chromatography, and supharose-12 is used as matrix.
32. A method as in claim 31, wherein the column used for SEC is XK 50/1000, and the bed height is about 75 % of column length.
33. A method as claimed in claim 32, where in the optimum flow rate in the column is 25 to 30 mL/min.
34. A method as in claim 33, wherein the column equlibration, washing and protein elution is done by G-CSF formulation buffer.
35. A method as in claim 34, wherein the formulated protein is sterile filtered for directly inj actable usage.
36. A novel method for purification of recombinant human G-CSF to pharmaceutically acceptable grade essentially as described in examples 1-5.