In situ restoration of apatite-based chromatography resins
AssigneeBio-Rad Laboratories, Inc.
InventorsLarry J. Cummings, Jie He
Life of this patent
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Abstract
Methods and compositions are provided for treatment of an apatite-based resin from which retained solutes have been eluted by an elution buffer that contains an alkali metal salt with solutions of calcium ion, phosphate ion, and hydroxide separately from any sample loading and elution buffers. The treatment solutions restore the resin, reversing the deterioration that is caused by the alkali metal salt in the elution buffer.
Claims16
A method for increasing the resin mass and particle strength of an apatite-based chromatography resin after purification of a target molecule with said apatite-based chromatography resin, wherein said apatite-based chromatography resin is ceramic hydroxyapatite (CHT), said method comprising:
- (a) equilibrating said apatite-based chromatography resin;
- (b) after equilibrating said apatite-based chromatography resin, loading a sample containing a target analyte onto said apatite-based chromatography resin;
- (c) after loading said sample, eluting said target analyte from said apatite-based chromatography resin by passing a solution of elution buffer through said apatite-based chromatography resin;
- and
- (d) after eluting said target molecule, and before subsequent loading of additional target molecule:
- (i) passing a solution of calcium ion having a calcium ion concentration of about 10 ppm to about 2000 ppm through said apatite-based chromatography resin;
- (ii) after (i), passing a solution of phosphate ion having a pH of at least about 6.5 and a phosphate ion concentration of about 50 mM to about 1 M through said apatite-based chromatography resin;
- and
- (iii) after (ii), passing a solution of hydroxide ion having a hydroxide ion concentration of about 0.1 M to about 5.0 M through said apatite-based chromatography resin, wherein the hydroxide ion is an alkali metal hydroxide;
- wherein (i), (ii), and (iii) are performed at volumes of said solution of calcium ion, solution of phosphate ion, and solution of hydroxide ion that are effective to increase the resin mass and particle strength of said apatite-based chromatography resin.
In a method for purifying target molecules from a plurality of samples on an apatite-based chromatography resin by loading said samples in succession onto said resin and eluting said target molecules from said resin after each such loading with an elution buffer comprising an alkali metal salt, wherein said apatite-based chromatography resin is ceramic hydroxyapatite (CHT), the improvement comprising treating said resin after each target molecule elution and before each subsequent sample loading by:
- (a) passing a solution of calcium ion having a calcium ion concentration of about 10 ppm to about 2000 ppm through said resin;
- (b) after (a), passing a solution of phosphate ion having a pH of at least about 6.5 and a phosphate ion concentration of about 50 mM to about 1 M through said resin;
- and
- (c) after (b), passing a solution of hydroxide ion having a hydroxide ion concentration of about 0.1 M to about 5.0 M through said resin, wherein the hydroxide ion is an alkali metal hydroxide;
- wherein steps (a), (b), and (c) are performed at volumes of said solution of calcium ion, solution of phosphate ion, and solution of hydroxide ion that are effective to increase resin mass and particle strength of said apatite-based chromatography resin.
Dependencies are inferred from the claim text.