Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-06-06
2001-12-25
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S387300
Reexamination Certificate
active
06333398
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to protein purification. In particular, the invention relates to a method for purifying C
H
2/C
H
3 region-containing proteins, such as antibodies and immunoadhesins, by Protein A affinity chromatography.
2. Description of Related Art
The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.
Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of the protein production run.
Once a clarified solution containing the protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted by different solvents. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the column, and the protein of interest does not, that is, the protein of interest is present in the “flow-through.”
Affinity chromatography, which exploits a specific interaction between the protein to be purified and an immobilized capture agent, may also be an option for some proteins. Protein A is a useful adsorbent for affinity chromatography of proteins, such as antibodies, which contain an Fc region. Protein A is a 41 kD cell wall protein from
Staphylococcus aureas
which binds with a high affinity (about 10
−8
M to human IgG) to the Fc region of antibodies.
SUMMARY OF THE INVENTION
A problem associated with Protein A chromatography of contaminated protein preparations has been identified herein. In particular, it has been observed that in Protein A chromatography using a glass or silica surface for adsorbing the Protein A (e.g. where the Protein A is immobilized on a controlled pore glass column or a silicic acid column), contaminants in the protein preparation (such as Chinese Hamster Ovary proteins (CHOP), where the protein preparation is derived from a CHO cell) adhere to the glass or silica surface of the solid phase. This was found to occur even when the solid phase is coated with a reagent (such as glycerol) in an attempt to prevent nonspecific adherence thereto. An intermediate wash step has been devised herein which addresses this problem. This wash step serves to remove the contaminants, but not the immobilized Protein A or the protein of interest bound to the Protein A, from the solid phase. In particular, it has been found that hydrophobic electrolytes, e.g., tetramethylammonium chloride (TMAC) and tetraethylammonium chloride (TEAC), can be used in this intermediate wash step.
Accordingly, the invention provides a method for purifying a protein, which comprises a C
H
2/C
H
3 region, from a contaminated solution thereof by Protein A chromatography comprising the following steps performed sequentially: (a) adsorbing the protein to Protein A immobilized on a solid phase comprising silica or glass; (b) removing contaminants bound to the solid phase by washing the solid phase with a hydrophobic electrolyte solvent; and (c) recovering the protein from the solid phase.
In preferred embodiments, the protein is an antibody (e.g. an anti-HER2, anti-IgE or anti-CD20 antibody) or an immunoadhesin (e.g. a TNF receptor immunoadhesin).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
When used herein, the term “Protein A” encompasses Protein A recovered from a native source thereof, Protein A produced synthetically (e.g. by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to bind proteins which have a C
H
2/C
H
3 region. Protein A can be purchased commercially from Repligen, Pharmacia and Fermatech.
The Protein A is immobilized on a solid phase. By “solid phase” is meant a non-aqueous matrix to which the Protein A can adhere. The solid phase of interest herein is one which comprises a glass or silica surface. The solid phase may be a purification column or a discontinuous phase of discrete particles. In preferred embodiments, the solid phase is a controlled pore glass column or a silicic acid column. In certain embodiments, the solid phase is coated with a reagent (such as glycerol) which is intended to prevent nonspecific adherence of contaminants to the solid phase.
The protein of interest herein is one which comprises a C
H
2/C
H
3 region and therefore is amenable to purification by Protein A chromatography. The term “C
H
2/C
H
3 region” when used herein refers to those amino acid residues in the Fc region of an immunoglobulin molecule which interact with Protein A. In preferred embodiments, the C
H
2/C
H
3 region comprises an intact C
H
2 region followed by an intact C
H
3 region, and most preferably comprises a Fc region of an immunoglobulin. Examples of C
H
2/C
H
3 region-containing proteins include antibodies, immunoadhesins and fusion proteins comprising a protein of interest fused to, or conjugated with, a C
H
2/C
H
3 region.
The “intermediate wash step” is a step performed after the protein of interest is loaded on the solid phase and adsorbed to the Protein A, but before the protein is recovered from the column. The intermediate wash step serves to remove contaminants nonspecifically bound to the solid phase, without significantly eluting the protein of interest from the solid phase. In the intermediate wash step, the solid phase is washed with a hydrophobic electrolyte solvent (e.g. the hydrophobic electrolyte solvent is passed through the Protein A column, where the solid phase is a column).
The “hydrophobic electrolyte solvent” in the intermediate wash step is that which is able to elute contaminants bound to the solid phase, without significantly eluting the immobilized Protein A or the protein of interest adsorbed thereto. Preferably the hydrophobic electrolyte solvent is an aqueous carrier (e.g. a buffer) comprising one or more hydrophobic electrolytes. Examples of hydrophobic electrolytes include the alkylamines; tetramethylammonium chloride TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chlor
Genentech Inc.
Lee Wendy M.
Saunders David
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