Protein purification by ion exchange chromatography

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Describes a method using ion exchange chromatography purification of the polypeptide, which involves changes in the conductivity of the buffer and / or pH to isolate the polypeptide of interest from one or more contaminants.

Application of this invention is the application number for 99805836.X, filed on May 3, 1999, invented the name for a divisional application "by ion exchange chromatography purification of proteins," the application.

Invention relates generally to the field of protein purification of the present invention. In particular, the present invention relates to the use of ion-exchange chromatography from a composition comprising a polypeptide and at least one contaminant polypeptide (e.g., antibody).

For biotechnology industry, the protein of large-scale, economic purification of a day is more than important issue. Typically, the use of genetically engineered to produce the protein of interest mammalian or bacterial cell lines, the protein recombinant plasmid containing the gene by inserting, after the cell culture to produce a protein. Since the cell lines used are living organisms, they must contain sugars, amino acids and growth factors, usually supplied by the composite animal serum growth medium was prepared to feed. A mixture of compounds fed to the cells from the medium and by-product from the cells themselves, the desired protein is separated, to reach a purity enough as a human therapeutic use, is a difficult challenge.

Purification of proteins from cell debris program first determined by the site of protein expression. Some proteins can directly secreted from the cell into the surrounding growth medium; and other proteins are produced intracellularly. For the first step after a protein purification process involves lysis of the cell, which can be accomplished by various methods, including: mechanical shear, osmotic shock, or enzymatic treatment. These methods make the entire contents of the destruction of cells released into the tissue homogenates and subcellular fragments generated extra due to its small size is difficult to remove. Normally required by differential centrifugation or filtration to remove them. In the process of protein production, due to the release of intracellular host cell proteins within cells and natural death directly secreted proteins, also have the same problem, although a smaller amount.

Once a clarified solution containing the protein of interest, usually try a different combination of chromatographic techniques to the other proteins produced by the cells and their separation. These techniques based on charge, degree of hydrophobicity, or size of the protein to separate the protein mixture. Each of these techniques may be employed several different chromatography resins, which makes it possible to develop specific protein involved in the specific purification scheme. Important These various separation methods is that proteins at different speeds along the length of the column a downward movement, to achieve physical separation, this separation is moved downward along with the protein and then the column is increased, or selective adhesive attached to the separation matrix, and then differentially eluted in different solvents. For some cases, when the impurities specifically adhere to the column, while the protein of interest does not adhere, the desired protein can be separated from impurities, i.e., the protein of interest is present in the "flow-through liquid "in.

Ion exchange chromatography is a commonly used chromatographic technique for protein purification. In ion exchange chromatography, if the ionic strength of the surrounding buffer is low enough, the live part of the solute surface was combined with an opposite charge on the chromatographic matrix attracted. Generally increase the ionic strength of the buffer (i.e., conductivity) exchange sites of the medium charged with solute ions compete to achieve elution. PH change thereby changing the charged amount of solute is another way to achieve elution of the solute. Changing the conductivity or pH may be gradual (gradient elution) or stepwise (step elution). In the past, these changes are gradual, ie, pH or conductivity of the way to increase or decrease.

The present invention provides a method of ion exchange chromatography, wherein the polypeptide of interest in a first conductivity or pH of the material in combination with ion exchange, followed by a different conductivity or pH, or both different intermediate buffer wash the ion exchange material. At a particular time after the intermediate washing, standard methods contrast with the ion exchange chromatography, washed with wash buffer ion exchange material, wherein the change from the intermediate buffer to the conductivity of the wash buffer or pH, or both, and the front Step conductivity or pH, or both to change the direction opposite. In the ion-exchange material ready wash buffer after washing, only by using elution buffer (which has a buffer used in the previous step and the conductivity or pH, or both a different conductivity or pH, or both person) eluting the polypeptide molecule of interest.

This novel application of the method of ion exchange chromatography must take full production scale (which also requires the polypeptide product purity and high recovery) is particularly useful product molecules isolated from the relevant pollutant molecules in close proximity.

Accordingly, the present invention provides a method for purifying polypeptides and contaminants from the group consisting of a polypeptide composition, the method comprising the steps of sequentially: (a) using the loading buffer so that the polypeptide binds to the ion exchange material, wherein loading buffer is at a first conductivity and pH; (b) a second conductivity and / or pH of the intermediate buffer wash the ion exchange material, whereby the contaminant material eluted from the ion exchange;

(C) a third conductivity and / or pH of the wash buffer washing the ion exchange material, wherein the conductivity of the intermediate buffer to the wash buffer and / or pH changes, and buffers from loading buffer to intermediate The conductivity and / or pH changes in the opposite direction; and (d) a fourth exchange material conductivity and / or pH of the elution buffer was washed ions, thus eluted polypeptide from the ion exchange material. The first conductivity and / or pH may be the third conductivity and / or pH the same.