Methods for purifying antibodies using protein a affinity chromatography

A protein and affinity technology, applied in peptide preparation methods, chemical instruments and methods, immunoglobulin, etc., can solve the problems of difficult, unsuccessful and time-consuming purification steps

Inactive Publication Date: 2011-08-31
SCHERING AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These protocols are time consuming and they are often unsuccessful, requiring additional chromatographic steps to produce mAb formulations suitable for human use
[0006] Yet another previous approach to reducing protein aggregation in mAb formulations has been the use of stabilizers, which can have several disadvantages, including, changes in protein activity, difficulties with additional purification steps, and potentially undesired immune responses in patients

Method used

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  • Methods for purifying antibodies using protein a affinity chromatography
  • Methods for purifying antibodies using protein a affinity chromatography
  • Methods for purifying antibodies using protein a affinity chromatography

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Embodiment approach

[0081] The present invention provides a first method for purifying monomeric monoclonal antibodies from a sample comprising monomeric monoclonal antibodies, host cell impurities, dimers and higher order aggregates, the method comprising: ( a) exposing the sample to a protein A affinity column; (b) eluting the monomeric monoclonal antibody from the protein A affinity column using elution buffer; and (c) collecting the monomeric monoclonal antibody from step (b). Cloning one or more fractions of the antibody to form a protein A product pool, wherein the product pool (i) comprises less than 5% higher order aggregates, and (ii) has a pH of about 3.5 to about 4.5, whereby Purification of monomeric monoclonal antibodies from samples.

[0082] In one embodiment of the above method, the elution buffer is acetate or citrate.

[0083] In another embodiment, the concentration of citrate in the elution buffer is from about 0.030M to about 0.085M. As used herein, "about" means ± 0.015M. ...

Embodiment 1

[0130] Reduce temperature to reduce protein aggregate levels during protein A affinity chromatography elution and subsequent low pH hold

[0131] To characterize the temperature and pH dependence of protein aggregation, the effect of temperature and pH on the eluted pool of PAP containing citrate (100mM, pH 3.5) was evaluated against an anti-DKK-1 monoclonal antibody. The same procedure can be used for other mAbs, such as anti-ADDL monoclonal antibodies.

[0132] Materials and methods

[0133] Small scale: with AKTA EXPLORER 100 TM , for all small-scale experiments. Phosphate, citrate, and sodium hydroxide buffers were purchased from Hyclone (Logan, UT). Tris base for pH adjustment of PAP was purchased from Hyclone (Logan, UT). Mabselect for Protein A Affinity Chromatography Experiments TM Resins were purchased from GE Healthcare. A depth-filtered centrate was obtained and used as starting material for protein A affinity chromatography experiments. A Thermomixer R (...

Embodiment 2

[0145] Raising the pH of the PAP pool to reduce protein aggregate levels during protein A affinity chromatography elution and subsequent low pH hold

[0146] To characterize the effect of pH on protein aggregation, the effect of pH (3.5-5.0) on the QPAP eluted pool was evaluated against an anti-DKK1 monoclonal antibody. The same procedure can be used for other mAbs, such as anti-ADDL monoclonal antibodies.

[0147] Materials and methods

[0148] Small scale: with AKTA EXPLORER 100 TM (GE Healthcare), for all small-scale experiments. Phosphate, citrate, and sodium hydroxide buffers were purchased from Hyclone (Logan, UT). Tris base for pH adjustment of PAP was purchased from Hyclone (Logan, UT). Mabselect for Protein A Affinity Chromatography Experiments TM Resins were purchased from GE Healthcare. Use depth-filtered centrate as starting material for protein A affinity chromatography experiments. Thermomixer R (Eppendorf) and 2 temperature-controlled chambers were u...

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Abstract

This invention provides a method for purifying a monomeric monoclonal antibody which comprises contacting the sample, wherein the sample comprises the monomeric monoclonal antibody, host cell impurities, dimers, and higher order aggregates, with a Protein A affinity chromatography column; eluting the monomeric monoclonal antibody from the Protein A affinity chromatography column with an elution buffer; and collecting one or more fractions of the monomeric monoclonal antibody to form a Protein A product pool, wherein the product pool comprises less than 5% higher order aggregate, and has a pH from about 3.2 to about 4.5, thereby purifying the monomeric monoclonal antibody from the sample. This invention also provides a method for purifying a monomeric monoclonal antibody which comprises eluting with acetate or citrate, optionally in the presence of amino acids. This invention also provides a method for purifying a monomeric monoclonal antibody which comprises conducting the method within certain temperature ranges.

Description

[0001] Throughout the application, various references are referred to with Arabic numerals in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references can be found at the end of the specification (before the appended claims). Background technique [0002] Applications for therapeutic monoclonal antibodies (mAbs) have increased significantly over the past 10 years. MAb stability represents a current challenge in the purification and formulation of these proteins. MAb instability leads to high levels of mAb aggregation in protein formulations, which can have several disadvantages, including altering protein activity and potentially leading to undesired immune responses in patients. [0003] Protein A affinity chromatography is a powerful and widely used tool for the puri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22C07K16/06
CPCC07K16/18C07K1/22A61K39/39591C07K2317/56
Inventor R. 奇米洛夫斯基E. 格林-特雷克斯勒D. 劳什
Owner SCHERING AG
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