Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Purification of proteins using hydrophobic interaction chromatography

a technology of hydrophobic interaction and protein purification, which is applied in the field of protein production and purification, can solve the problems of hic performance deterioration in both product recovery and contaminant reduction, and achieve the effect of greater purification and recovery of proteins

Inactive Publication Date: 2015-10-22
ABBVIE INC
View PDF3 Cites 25 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007]The present invention is directed to methods for purifying a protein of interest, e.g., an antibody, from a sample including the protein of interest and at least one impurity, e.g., an aggregate, by employing a novel hydrophobic interaction chromatography (HIC) method. The present invention is based, at least in part, on the finding that both flow through and bind-elute techniques can be combined to achieve greater purification and recovery of a protein of interest. Moreover, the present invention is predicated, at least in part, on the surprising finding that such methodology can be employed under isocratic wash conditions and at stronger binding conditions than previously appreciated, for example, at a Kp greater than 10, so as to achieve greater purification and recovery.

Problems solved by technology

In addition, U.S. Pat. No. 8,067,182 teaches that Kp values greater than 20 result in decreased product recovery in that the isocratic wash conditions are not effective at washing the bound product off the column in a reasonable number of wash volumes.
Patentees report that HIC performance deteriorates with respect to both contaminant reduction and product recovery at stronger binding conditions.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Purification of proteins using hydrophobic interaction chromatography
  • Purification of proteins using hydrophobic interaction chromatography
  • Purification of proteins using hydrophobic interaction chromatography

Examples

Experimental program
Comparison scheme
Effect test

example 1

Determining Operating Conditions Appropriate for an Mab: Media: Buffer Combination

[0359]The demonstration of the current invention for a specific antibody & media is provided in this example, and consists of: 1) Choosing a salt concentration that allows product and impurities to bind at a given pH; 2) Loading a small amount of protein to the column and then performing a linear gradient elution by decreasing the salt concentration; 3) Determining salt concentration range in which the protein elutes from the HIC media.

[0360]In this example, adalimumab and GE CaptoPhenyl were chosen. The column was equilibrated at 1.1 M AmSO4 pH 7.0 (Tris / Acetate) for 10 CVs. Adalimumab was prepared at 1.1 M AmSO4 and loaded to the column at 20 g-protein / L of resin. The column was washed with 10 CVs of the equilibration buffer. A linear gradient from 1.1M to 0M AmSO4 pH 7.0 (Tris / Acetate) over 20CVs was performed. The process chromatogram is shown in FIG. 4.

[0361]This process can be repeated for any gi...

example 2

Demonstration of Aggregate Reduction with HIC Resins

[0373]This data set is compiled to demonstrate the aggregate reduction achieved with six different HIC adsorbents. Each resin was evaluated with a 500 g / L load of adalimumab at a NaCit concentration near, and slightly higher than, the peak elution concentration determined from the process outlined in Example 1. Table 3 outlines the results from these experiments.

TABLE 3Effect of HIC Resins on Aggregate Reduction of AdalimumabHIC ResinNaCit, pH 5.6ΔAggYieldButyl400 mM1.5%99.8%450 mM1.2%85.7%Hexyl240 mM1.2%93.9%300 mM1.1%100.9%Phenyl400 mM1.5%96.5%450 mM1.2%90.7%Octyl350 mM0.4%98.5%400 mM0.1%103.3%GE Butyl FF550 mM1.2%88.1%600 mM1.7%83.0%PPG450 mM0.2%97.5%600 mM1.0%38.1%

example 3

Demonstration of Aggregate Reduction with Other Antibodies, mAb B and mAb C

[0374]Aggregate reduction technology of the current invention has been demonstrated with multiple antibodies using HIC adsorbents. Antibodies have different hydrophobic properties, leading to interaction behavior on a HIC column that differs from one antibody to another. Therefore the impact of salt type and concentration is different for each antibody.

[0375]Table 4 and Table 5, presented below, provide the data obtained for mAb B and mAB C. The data clearly demonstrates that the aggregate reduction technology is effective for alternatives to adalimumab.

TABLE 4Aggregate reduction for mAb B, pI~9.1HIC ResinAmSO4, pH 5.0ΔAggYieldHexyl370 mM0.8%100%710 mM0.6%93%Phenyl340 mM0.6%95%790 mM0.5%95%Butyl840 mM0.6%99%1000 mM 0.6%96%

TABLE 5Aggregate reduction for mAb C, pI~7.0HIC ResinAmSO4, pH 5.0ΔAggYieldHexyl 80 mM5.0%89.0%330 mM4.5%99.8%Phenyl130 mM3.5%92.8%480 mM2.9%92.8%Butyl690 mM5.2%93.5%880 mM5.4%87.9%

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
concentrationaaaaaaaaaa
concentrationaaaaaaaaaa
Login to View More

Abstract

The present invention is directed to methods for purifying a protein of interest, e.g., an antibody, from a sample comprising the protein of interest and at least one impurity, e.g., an aggregate, by employing a hydrophobic interaction chromatography (HIC) method that allows for binding of both the protein of interest and the at least one impurity under strong binding conditions. The present invention is based, at least in part, on the finding that both flow through and bind-elute techniques can be combined to achieve greater purification and recovery of a protein of interest, e.g., an antibody, under isocratic wash conditions and strong binding conditions.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 14 / 077,574, filed Nov. 12, 2013, which is related and claims priority to U.S. Provisional Application No. 61 / 893,131, filed Oct. 18, 2013, the entire contents of each of which are hereby incorporated herein by reference.FIELD OF INVENTION[0002]The instant invention relates to the field of protein production and purification, and in particular to compositions and processes for reducing the levels of impurities, including process-related impurities (e.g., host cell proteins and media components) and / or product-related substances (e.g., product charge variants, aggregates, and fragments).BACKGROUND OF THE INVENTION[0003]Hydrophobic interaction chromatography (HIC) is a purification technique that exploits the interaction of HIC media with hydrophobic regions present on a protein of interest, such as an antibody, and / or those present on an impurity to separate a protein of interest present i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C07K1/20C07K1/36C07K1/18C07K1/16C07K16/24C07K1/22
CPCC07K1/20C07K16/241C07K1/36C07K1/18C07K1/165C07K1/22C07K1/16
Inventor HERIGSTAD, MATTHEW OMONRICH, LINDA E.LU, STEPHEN MING-TEHRAMASUBRAMANYAN, NATARAJAN
Owner ABBVIE INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com