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Cation exchange chromatography (methods)

Inactive Publication Date: 2012-07-12
MEDAREX LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0003]The present invention provides improved methods for protein purification using cation exchange (CEX) chromatography. These methods generally involve contacting a protein of interest (e.g., an antibody) with a cation exchange resin at a first pH that is less than the isoelectric point (pI) of the most acidic isoform of the protein of interest, such that the protein of interest binds to the resin. The resin is then washed at a second pH that is greater than the first pH, but less than the pI of the most acidic isoform of the protein of interest. The protein is subsequently eluted from the resin at a third pH that is about equal to or less than the first pH. This combination of higher pH wash and lower pH elution results in improved separation of the protein of interest (e.g., an antibody) from contaminants (e.g., HCP), compared to conventional CEX purification methods. The methods of the invention are useful for the commercial purification of recombinant therapeutic proteins, particularly antibodies.

Problems solved by technology

Large-scale, economic purification of proteins is an increasingly important challenge for the biopharmaceutical industry.
However, in therapeutic antibody purification, the current industry-standard, chromatography capture resin, Protein A, is expensive, has a relatively low throughput, and limited life cycles.

Method used

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  • Cation exchange chromatography (methods)
  • Cation exchange chromatography (methods)
  • Cation exchange chromatography (methods)

Examples

Experimental program
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Effect test

example 1

[0042]In this Example a Humab (Humab-1 in table 1) was purified by CEX chromatography using different combinations of load, wash and elution pHs. In all experiments the CEX resin (Poros 50HS (Applied Biosystem)) was packed in a 1 mL column (Φ0.5 cm×H 5 cm).

[0043]In one set of experiments (FIG. 3A), a CEX column was equilibrated with Sodium Phosphate buffer at pH 6.2 and 5.8 mS / cm. A solution of Humab-1 was adjusted to pH 6.2 and 5.8 mS / cm, and loaded onto the column at a concentration of 15 mg / mL. The column was then washed with Sodium Phosphate and Sodium Chloride dual buffer at pH 7.2 or Sodium Phosphate buffer at pH 8.0. After the wash step, the Humab was eluted from the column at pH 8.2, 8.0, 7.5, 7.2, 6.2, or 4.5 with Sodium Phosphate buffer containing sufficient Sodium Chloride to reach the appropriate conductivity for elution.

[0044]In another set of experiments (FIG. 3B), a CEX column was equilibrated with Sodium Citrate and Sodium Phosphate dual buffer at pH 4.5 and 2 mS / cm....

example 2

[0046]In this Example IgG1 (Humab-1 in table 1) and IgG4 (Humab-8 in table 1) Humabs were purified using the methods of the invention (see Table 2). A CEX column was equilibrated to pH 6.2. Unpurified bulk containing either Humab-1 or Humab-8 was buffer-exchanged to the same pH and ionic strength as the column equilibration buffer and loaded onto a cation exchange column. The column was then washed with Sodium Phosphate buffer at a pH just below the pI of each Humab. After the wash step, the Humabs were eluted from the column with a buffer comprising either 35 mM Sodium Phosphate, 75 mM NaCl at pH 6.2 (Humab-1) or 75 mM Sodium Phosphate, pH 6.2 (for Humab-8).

[0047]The data in Table 2 show that the CEX chromatography methods of the invention resulted in about a 1000-fold reduction in the HCP contaminant for both Humab-1 and Humab-8.

TABLE 2IgG1 and IgG4 Humab Purification Using the Methods of the InventionHumab-1 (IgG1)Humab-8 (IgG4)(lowest pI 7.96)(lowest pI 7.33)CHO HCPDNACHO HCPDNA...

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Abstract

The present invention provides improved methods of protein purification using CEX chromatography. Such methods generally comprise the steps of: contacting a protein of interest (e.g., an antibody) with a cation exchange resin at a first pH, that is less than the pI of the most acidic isoform of the protein of interest, such that the protein of interest binds to the resin; washing the cation exchange resin at a second pH that is greater than the first pH, but less than the pI of the most acidic isoform of the protein of interest; and eluting the protein of interest from the resin at a third pH that is about equal to or less than the first pH. The methods of the invention are particularly useful for the commercial purification of recombinant therapeutic proteins (e.g., antibodies).

Description

BACKGROUND OF THE INVENTION[0001]Large-scale, economic purification of proteins is an increasingly important challenge for the biopharmaceutical industry. Therapeutic proteins are typically produced using prokaryotic or eukaryotic cell lines that are engineered to express the protein of interest from a recombinant plasmid containing the gene encoding the protein. Separation of the desired protein from the mixture of components fed to the cells and cellular by-products to an adequate purity, e.g., sufficient for use as a human therapeutic, is a fundamental requirement for biologics manufacturers. However, in therapeutic antibody purification, the current industry-standard, chromatography capture resin, Protein A, is expensive, has a relatively low throughput, and limited life cycles.[0002]Accordingly there is a need in the art for alternative protein purification methods that can be used to expedite the large-scale processing of protein-based therapeutics, such as antibodies, especia...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K14/00C07K1/14
CPCC07K1/18
Inventor ARUNAKUMARI, ALAHARIWANG, JUE
Owner MEDAREX LLC
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