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US 7,741,455 B2Granted
Method for purifying FSH
AssigneeAres Trading S.A.
InventorsPascal Valax, Pierre Wenger, Anne Stanley, Lydia Delegrange, Luciano Capponi
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Abstract
The invention relates to a method for purifying recombinant human FSH or an FSH variant starting from crude FSH, comprising the following steps: 1) dye-affinity chromatography; 2) hydrophobic interaction chromatography; and 3) reverse phase chromatography.
Claims5
A method for purifying recombinant follicle stimulating hormone (FSH) or a FSH variant comprising subjecting a liquid containing FSH or a FSH variant to the following steps performed in order:
- (a) subjecting a liquid containing FSH or a FSH variant to anion-exchange chromatography and recovering an eluate containing FSH or FSH variant;
- (b) subjecting the eluate of step (a) to dye affinity chromatography and recovering an eluate containing FSH or a FSH variant;
- (c) subjecting the eluate of step (b) to hydrophobic interaction chromatography and recovering an eluate containing FSH or a FSH variant;
- (d) subjecting the eluate of step (c) to reverse phase chromatography and recovering an eluate containing FSH or a FSH variant;
- and
- (e) subjecting the eluate of step (d) to anion-exchange chromatography and recovering an eluate containing FSH or a FSH variant.
3 more elements
A method for purifying human recombinant FSH comprising subjecting a liquid containing FSH to:
- (a) ultrafiltration of said liquid and recovering a retentate;
- (b) subjecting said retentate to anion exchange chromatography on a resin comprising quaternary ammonium groups and eluting a first eluate with a buffer comprising borate/NaCl and L-methionine at a pH of, or about, 8.5;
- (c) subjecting the first eluate to a step of dye affinity chromatography on a resin comprising cibacron blue and eluting a second eluate with a buffer comprising phosphate, NaCl and L-methionine at a pH of, or about, 7;
- (d) subjecting the second eluate to hydrophobic interaction chromatography on a resin comprising butyl groups and eluting a third eluate with a buffer comprising phosphate, ammonium sulfate and, L-methionine, at a pH of, or about, 7;
- (e) subjecting the third eluate to reverse phase chromatography on a sizing resin with a buffer comprising ammonium acetate, L-methionine and 2-propanol at a pH of, or about, 7.6 and eluting a fourth;
- (f) subjecting the fourth eluate to anion-exchange chromatography on a resin comprising triethylaminoethyl groups with a buffer comprising borate and L-methionine at a pH of, or about, 8.5, and NaCl and eluting a fifth eluate;
- (g) subjecting the fifth eluate to nanofiltration to form a permeate;
- and
- (h) subjecting the permeate of step (g) to ultrafiltration.
6 more elements
Dependencies are inferred from the claim text.