Protein a chromatography

Abstract

A novel method for selectively removing leaked protein A from antibody purified by means of protein A affnitiy chromatography is disclosed.

Description

[0001] The present application is a continuation-in-part application of International Application No. PCT / EP2004 / 002041, filed 1 Mar. 2004, and claims benefit of U.S. Provisional application Ser. No. 60 / 464,973, filed 24 Apr. 2003, U.S. Provisional application Ser. No. 60 / 604,464, filed 26 Ausgust 2004, and EP 0304576.2 filed 28 Feb. 2003, the entire contents of each of which is hereby incorporated by reference.SUMMARY OF THE INVENTION [0002] The present invention relates to the field of antibody purification in biotechnological production. It is an object of the present invention to describe a novel process for purification of such antibody. [0003] Protein A chromatography is widely used in industrial manufacturing of antibodies since allowing for almost complete purification of antibodies, that is usually IgG, in a single step from cell culture supernatants. Protein A affinity columns inevitably are subject to some degree of leakage of ligand from the column upon repeated runs. Pa...

AI Technical Summary

Benefits of technology

[0006] U.S. Pat. No. 4,983,722 teaches selective separation of contaminating protein A from a protein-A purified antibody preparation by absorbing the mixture to an anion exchanger material and to separate both components by sequentially eluting the antibodies and protein A under conditions of increasing ionic strength. This resolution method is highly dependent on the pI of the antibody which is specific and highly variable for a given antibody. Further, throughput is limited by the steepness of the salt gradient required for obtaining separation.

[0007]FIG. 1 shows the result of pretreatment by means of non-reducing 10% SDS-PAGE for a staphylococcal protein A standard (lane 1: native protein A; lane 2: after pretreatment) and pure, uncoupled Streamline™ recombinant protein A (provided by courtesy of Pharmacia, now Amersham-Biosciences; lane 4: native recombinant protein A; lane 5: after pretreatment).

Problems solved by technology

Protein A affinity columns inevitably are subject to some degree of leakage of ligand from the column upon repeated runs.

Unfortunately, this protein A or protein A fragment contaminants retain their affinity for IgG and are difficult to remove from the purified antibody due to ongoing complex formation.

As a concomittant disadvantage, the leakage rate of such recombinant Protein A matrices is often drastically increased in contrast to many traditional, multi-point attached natural Protein A matrices obtained by CNBr coupling.

Low through-put and loss in antibody yield are the disadvantages of this method.

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