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Removal of leaked affinity purification ligand

a technology of affinity chromatography and purification ligand, which is applied in the direction of peptides, drug compositions, separation processes, etc., can solve the problems of difficult to remove ligands from final preparations without, and ligand contamination, and achieves the effects of easy scaling, high efficiency, and high efficiency

Inactive Publication Date: 2016-01-28
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides a better way to remove affinity ligands from a sample of recombinant protein without losing the protein. This is possible because the invention uses a special type of matrix chromatography medium that has a unique design. This design works better than other types of matrix chromatography resins in removing affinity ligands, resulting in higher purity of the recombinant protein.

Problems solved by technology

Although affinity chromatography is a highly effective technique for isolating proteins, one drawback is that the affinity ligand may contaminate the resulting sample of recombinant protein by leaching from the affinity chromatography medium.
Because affinity ligands are chosen for their ability to associate with the recombinant protein, it can be challenging to remove them from a final preparation without also losing the recombinant protein.

Method used

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  • Removal of leaked affinity purification ligand
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Examples

Experimental program
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Effect test

example 1

[0054]The goal of these experiments was to effectively remove contaminating Protein A from a recombinant protein preparation while recovering as much recombinant protein as possible. A recombinant protein, etanercept, was expressed in a transformed CHO (Chinese Hamster Ovary) cell culture, and secreted into the medium. After removal of cells from the medium, etanercept was initially purified by running the medium over a MabSelect SuRe™ Protein A resin column (GE Healthcare Life Sciences). Leached protein A was determined using a sandwich ELISA assay. Microtitre plates were coated with a chicken anti-protein A antibody as a capture antibody that was specifically raised against the MabSelect SuRe ligand. After blocking and washing steps, a biotinylated chicken anti-protein A antibody was used as the detection antibody. The amount of leached protein A in the sample after initial purification over the MabSelect SuRe™ Protein A resin column ranged from about 1 ppm to about 20 ppm.

[0055]I...

example 2

[0057]Using the relatively weak anion exchange resin DEAE Sepharose Fast Flow (GE Healthcare Life Sciences), a variety of different buffers and elution conditions was explored as detailed in the below table. Etanercept and protein A concentrations were determined as in Example 1.

Test Resin: DEAE Sepharose FFMode / ElutionLeachedEquilibrationLoadBufferProtein Aand WashCond.LoadElutionConductivityElutionYieldReductionBuffer(mS / cm)pHBuffer(mS / cm)Flow Rate(%)(%)Bind / Elute / 5.2825 mM Tris,17.140.5mL / min8027.925 mM Tris,150 mMpH 8NaCl, pH 7.4Bind / Elute / 5.2825 mM Tris,17.140.5mL / min8526.225 mM Tris,150 mMpH 8NaCl, pH 7.4Bind / Elute / 5.2825 mM Tris,200.5mL / min8515.125 mM Tris,175 mMpH 8NaCl, pH 7.5Bind / Elute / 5.2825 mM Tris,200.5mL / min8446.325 mM Tris,175 mMpH 8NaCl, pH 7.5Bind / Elute / 5.2825 mM Tris,13.70.5mL / min8655.925 mM Tris,125 mMpH 8NaCl, pH 7.5Bind / Elute / 5.2825 mM Tris,13.70.5mL / min8561.325 mM Tris,125 mMpH 8NaCl, pH 7.5Bind / Elute / 5.2825 mM Tris,13.30.5mL / min72.496.325 mM Tris,100 mMpH 8NaC...

example 3

[0059]In this experiment, a different anion exchange resin was tested for its ability to remove protein A while maintaining a high recovery rate. The resin was Eshmuno® Q, which is available from EMD Millipore, a division of Merck KGaA, Darmstadt, Germany. All runs were in a bind and elute mode.

Resin: Eshmuno QLeached ProteinEquilibrationLoad Cond.LoadElution BufferYieldA Reductionand Wash Buffer(mS / cm)pHand Conductivity(%)(%)25 mM Tris, pH 8,5825 mM Tris, pH 7.4,93.855.6cond. 5 mS / cmcond. 21 mS / cm25 mM Tris, pH 8,5825 mM Tris, pH 7.4,94.460.8cond. 5 mS / cmcond. 21 mS / cm25 mM Tris, pH 8,5825 mM Tris, pH 7.4,9264.7cond. 5 mS / cmcond. 21 mS / cm25 mM Tris, pH 8,5825 mM Tris, pH 7.4,92.563.3cond. 5 mS / cmcond. 21 mS / cm

[0060]Although the recovery of etanercept was high using this resin, reduction of Protein A was not sufficient.

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Abstract

The invention provides for the removal of a large fraction of contaminants from protein preparations while maintaining a high level of recovery using tentacle anion exchange matrix chromatography medium. Using the methods of the invention, leached affinity chromatography contaminants can be removed from recombinant protein preparations.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 61 / 785,038, filed Mar. 14, 2013. The above-identified application is incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention is in the field of removal of contaminants from protein preparations. In one aspect, the invention relates to the removal of leached affinity chromatography contaminants from a protein preparation.BACKGROUND OF THE INVENTION[0003]Affinity chromatography is a powerful tool for purification of proteins such as antibodies and FC-fusion proteins. However, if the proteins are manufactured for therapeutic use, the presence of other proteins, including a protein used as part of an affinity adsorbent, which can leach into a sample during affinity chromatography, is of concern. In addition, other protein contaminants may also be present in a sample, such as, for example, proteins derived from host cells that produce the protein be...

Claims

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Application Information

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IPC IPC(8): C07K1/18C07K14/715
CPCC07K1/18C07K2319/30C07K14/7151B01D15/363C07K14/70575C07K1/22C07K16/00B01D15/1871B01D15/3809A61P17/06A61P19/02A61P29/00
Inventor TREJO, SAMUEL RAYBRAKE, ROBERT PERRY
Owner AMGEN INC
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