Antibody purification by cation exchange chromatography

a cation exchange chromatography and antibody technology, applied in antibody medical ingredients, immunological disorders, drug compositions, etc., can solve the problems of difficult removal of subcellular fragments, economic purification of proteins, and difficult use as human therapeutics, and achieve the effect of improving the removal of contaminants of chinese hamster ovary proteins (chop)

Inactive Publication Date: 2009-06-11
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0011]The invention herein concerns an improved method for cation exchange chromatography of antibodies in which a high pH wash step is used to remove contaminants prior to eluting the desired antibody product. The process results, amongst other things, in improved removal of Chinese Hamster Ovary Proteins (CHOP) contaminants.

Problems solved by technology

The large-scale, economic purification of proteins is an increasingly important problem for the biotechnology industry.
Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size.
The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of the protein production run.

Method used

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  • Antibody purification by cation exchange chromatography
  • Antibody purification by cation exchange chromatography
  • Antibody purification by cation exchange chromatography

Examples

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example 1

Purification of a CD20 Antibody

[0116]This example describes an improved cation exchange chromatography process for purifying a CD20 antibody, rituximab. Rituximab is used for therapy of NHL, CLL, RA, MS, etc. The structure of the Rituximab molecule is disclosed in U.S. Pat. No. 5,736,137, Anderson et al., (expressly incorporated herein by reference) as well as FIGS. 1A-1B herein. Rituximab is commercially available from Genentech, Inc.

[0117]Cation-exchange chromatography is used to further reduce the levels of CHOP, DNA, leached protein A, garamycin (GENTAMYCIN®), Rituximab aggregates, and potential viruses. Rituximab binds to the column under the load conditions. The column is then washed, eluted, regenerated / sanitized, and stored until the next use. Multiple cycles may be used to process an entire batch of affinity pool. The cation-exchange pool may be held at room temperature up to 30° C. for up to 3 days or at 5° C. for up to 7 days.

[0118]The cation-exchange resin (POROS 50 HS®,...

example 2

Purification of a VEGF Antibody

[0122]This example describes a cation exchange chromatography process for purifying a recombinant humanized vascular endothelial growth factor antibody (rhuMAb VEGF), bevacizumab. The structure of the bevacizumab molecule is disclosed in U.S. Pat. No. 7,169,901, Presta et al., expressly incorporated herein by reference. See also FIGS. 2A-2B herein. Bevacizumab is commercially available from Genentech, Inc.

[0123]This example summarizes the development studies performed on the cation exchange step for an improved bevacizumab purification process. Three cation exchange resins were evaluated in these studies: CM SEPHAROSE FAST FLOW®, SP SEPHAROSE FAST FLOW® and POROS 50HS®. The cation exchange purification processes using these three resins were evaluated with respect to: process performance (impurities removal, retrovirus removal, and step yield), product quality, process robustness and process fit at all current manufacturing sites. Based on the data gen...

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Abstract

A method for purifying an antibody by cation exchange chromatography is described in which a high pH wash step is used to remove of contaminants prior to eluting the desired antibody using an elution buffer with increased conductivity.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Patent Application No. 60 / 983,825, filed 30 Oct. 2007, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]This invention relates generally to protein purification. In particular, the invention relates to a method for purifying antibody from a composition comprising the antibody and at least one contaminant using cation exchange chromatography, wherein a high pH wash step is used to remove contaminants prior to eluting the desired antibody using an elution buffer with increased conductivity.[0004]2. Description of the Related Art[0005]The large-scale, economic purification of proteins is an increasingly important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either eukaryotic or prokaryotic cell lines engineered to produce the protein of interest by ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K1/00
CPCC07K1/18C07K16/065C07K2317/24C07K16/2887C07K1/36C07K16/22A61P19/02A61P19/08A61P25/00A61P27/02A61P29/00A61P35/00A61P35/02A61P37/02A61P43/00A61P9/08A61P9/14
Inventor LEBRETON, BENEDICTE ANDREEO'CONNOR, DEBORAH ANNSAFTA, AURELIASHARMA, MANDAKINI
Owner GENENTECH INC
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