Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1998-01-02
1999-11-23
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530350, 530351, 530412, 530415, 530416, 530417, 530419, 530420, 530421, 530427, 530811, 530812, C07K 1700, A23J 100
Patent
active
059902892
DESCRIPTION:
BRIEF SUMMARY
The instant invention relates to a new method for the purification of proteins using copper chelate-affinity chromatography.
Copper chelate-affinity chromatography functions by binding the accessible electron-donating group of a protein, such as histidine, to a copper ion which is held by a chelating group covalently attached on a stationary support. The presence of a single accessible histidine is usually sufficient for the adsorption of a protein on a copper(II)-IDA-chelate (IDA=iminodiacetate). Protein retention in copper chelate-affinity chromatography depends on the number and type of pendant groups on the protein which can interact with the metal. This interaction is affected by a variety of independent variables such as pH, temperature, solvent type, salt type and salt concentration. The proteins that are bound to the copper chelate in a neutral or low alkaline pH region are usually eluted by a decreasing pH gradient (continuous or stepwise) or by an increasing concentration gradient (continuous or stepwise) of a competitive agent, e.g. a Lewis-base such as imidazole, in a buffer. Alternatively, a third possible elution method includes a chelating agent, such as EDTA, in the eluent.
However these methods have several disadvantages: competitive or chelating agents are used desired protein, in order to increase the selectivity. chelating agent the elution contains the protein, the competitive or chelating agent and released copper(II) ions. Therefore, the competitive agent and the copper(II) ions have to be removed by additional purification steps and, furthermore, a lot of proteins are unstable in presence of high concentrations of copper(II) ions in solution (oxidation) or at low pH values (precipitation) and are inactivated.
The current invention now provides a surprising advantageous method for the elution of proteins from a copper chelate-complex. Using the inventive method, it is surprisingly possible to elute the desired protein with deionized water that may optionally contain additional stabilizers for the protein. The use of deionized water has several advantages over the art: purification steps are necessary ions that may cause oxidation problems precipitation or inactivation problems high yield (continuous or stepwise)
DETAILED DESCRIPTION OF THE INVENTION
The current invention concerns a process for the enrichment of proteins comprising the steps: copper(II) ions,
Usually the copper(II) ions are immobilized on a stationary support that is, e.g., an inorganic carrier such as silica or a polymeric matrix.
It is necessary that the stationary support is capable of forming chelats with copper(II) ions. Examples of such stationary supports are those in which chelating groups, such as imidoacetic acid (IDA), N,N,N'-tris(carboxymethyl)-ethylenediamine (TED), carboxymethylated aspartic acid (CM-ASP), tetraethylene pentamine (TEPA) nitriloacetic acid (NTA) or carboxymethylated .alpha., .beta. diamino succinic acid (CM-DASA), are covalently bound to the stationary support. The chelating groups may be attached to the stationary support directly or via spacers. The polymeric carrier may be any polymeric compound conventionally used for the metal ion-affinity-chromatography. Examples of such polymeric materials include agarose, dextrans or other synthetic polymers; preferred are agarose and vinyl polymers. Methods for the preparation of stationary supports having covalently attached chelating groups are described, for example, in Hochuli, CHIMIA (1986), 40, 408-412 (see also Figueroa et al., J. Chromatography (1986), 371, 335-352; Porath, Protein expression and purification (1992), 3, 263-281; L. K.ang.gedal in "Protein purification, principles, high resolution methods and applications" Janson and Ryden Eds. (1989), 227-251, VCH Publishers, New York). In a preferred embodiment of the invention the stationary support is capable of forming chelats with copper(II) ions at a concentration of 1-200, and more preferably 10-100, .mu.mol/ml of the stationary support.
Suitable polymeric carrier for use in the
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Fauquex Pierre Fran.cedilla.ois
Georges Catherine
Kalinchak Stephen G.
Mohamed Abdel A.
Novartis AG
Tsang Cecilia J.
Wissing William K.
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