Bio-rad Laboratories, Inc.

Larry J Cummings

Mark A Snyder

Jie He

The present invention discloses methods of neutralizing apatite surfaces, for example during chromatography and before protein elution.

Surface neutralization of apatite

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.

In situ restoration of apatite-based chromatography resins

The present invention discloses methods of regenerating apatite surfaces, for example after purification of a target analyte.

Apatite in-situ restoration

Apatite pretreatment methods are provided. The method is applied to the apatite solid surface prior to first chromatographic use. In one embodiment, the method may be achieved by contacting an apatite solid surface with a phosphate buffered solution at a pH of at least about 6.5 and contacting the apatite solid surface with a solution having a hydroxide.

Apatite pretreatment

Methods of regenerating apatite surfaces, for example after purification of a target analyte. The methods include, for example, regeneration that can involve contacting the apatite solid surface with a buffered calcium solution and a zwitterionic buffer, then contacting the apatite solid surface with a phosphate buffered solution, and then contacting the apatite solid surface with a solution comprising a hydroxide.

The present invention discloses methods of neutralizing apatite surfaces, for example after a flow-through collection of a target and prior to cleaning the chromatography solid support.

Apatite surface neutralization with alkali solutions

Proteins, including monoclonal antibodies, that have been retained on hydroxyapatite resins for purposes of protein separation, purification, or both, are eluted from the resins by a elution buffer that contains controlled amounts of calcium and phosphate ions. The buffer allows elution to be performed in repeated runs at an acidic pH without deterioration of the resin.

Elution of proteins from hydroxyapatite resins without resin deterioration

A chromatography cartridge with a packed bed is manufactured from an inner and outer chamber, the inner chamber being in an insert that is slidable into the outer chamber with openings in each such that a slurry that is originally placed in the interior of the outer chamber flows into the inner chamber as the parts are being pressed into each other. During the assembly, the particles in the slurry are compressed to fill the smaller volume of the inner chamber and excess liquid is discharged through ports in both chambers. The construction allows for a highly precise and reproducible column filling process with a minimum of steps and operator intervention and thereby low cost and reduced error.

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Pleasant Hill, CA, United States of America

Larry J Cummings