Process of purification of hCG and recombinant hCG purified by that method
View Patent ↗A process for the purification of recombinant human Chorionic Gonadotropin (hCG) from a sample of crude recombinant hCG in the supernatant of CHO cells comprises the combined use of ion-exchange chromatography and reverse phase HPLC. The ion-exchange chromatography is performed twice and the final use of a size exclusion chromatography allows the purification from any residual traces of contaminants. The specific bioactivity of the highly purified hCG obtained form the process is particularly high, amounting to about 25,000 IU/mg.
1. A process for the purification of recombinant human chorionic gonadotropin (hCG) from a sample containing recombinant hCG, comprising:
(a) eluting the sample containing recombinant hCG through a silica chromatography column;
(b) eluting the sample through an ion-exchange chromatography column;
(c) eluting through a second ion-exchange chromatography column;
(d) eluting through a reverse phase high performance liquid chromatography (HPLC) column; and
(e) applying the eluate to a size exclusion chromatography column.
2. A process for the purification of recombinant hCG according to claim 1 , from a sample containing recombinant hCC, wherein:
the ion-exchange chromatography column of step (b) is a DEAE SEPHAROSE (cross-linked agarose matrix with die thylaminoethyl weak anion exchanger) ion-exchange chromat:ography column;
the second ion-exchange chromatograph column of step
(c) is a CM-SEPHAROSE (cross-linked agarose matrix with carboxymethylweak anion exchanger) ion-exchange chromatography column;
the reverse phase HPLC column of step (d) is a Silica C18 reverse phase HPLC column; and the size exclusion chromatography column of step (e) is a SEPHACRYL (spherical allyl dextran and N,N′-methylenebisacrylamide) size exclusion chromatograph column.
3. A process according to claim 2 , wherein elution thrbuqh the DEAE SEPHAROSE ion-exchange chromatography is carried out in sodium phosphate buffer at pH 7.5.
4. A process according to claim 2 or 3 , wherein elution through the CM-SEPHAROSE ion-exchange chromatography is carried out in sodium phosphate buffer at pH 6.
5. A process according to claim 2 or 3 , wherein the reverse phase HPLC step (d) is carried out with 2-propanol-trisphosphate buffer as mobile phase.
6. A process according to claim 2 or 3 , wherein the size-exclusion chromatography step (e) is carried out wirh ammonium hydrogen carbonate buffer as mobile phase.
7. A process according to claim 2 or 3 , wherein the sample containing recombinant hCG is a culture medium from rebombinant CHO cells that produce human chorionic gonadotropin.