Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1999-11-12
2001-08-28
Kemmerer, Elizabeth (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S350000, C530S351000, C530S380000, C530S412000, C530S427000
Reexamination Certificate
active
06281337
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to the isolation and purification of proteins. In particular, the present invention pertains to the isolation of proteins, isolation of isoforms of proteins and conversion of isoforms to the desired proteins.
BACKGROUND
Naturally occurring proteins are widely used for research and clinical purposes. While such proteins may be obtained from their natural source, recombinant techniques can permit the production of these proteins from non-natural sources. For example, fermentation of microorganisms constructed via recombinant technology, such as transformed bacteria, produce large quantities of human interferon at a substantially lower cost than is possible utilizing natural sources. Such recombinant DNA techniques have also been utilized to produce other important proteins, such as insulin and tissue plasminogen activator.
Bacteria altered by recombinant techniques, however, also produce contaminants and structural isoforms of the protein intended to be produced. These contaminants and isoforms include oligomeric proteins and reduced protein isoforms (see U.S. Pat. No. 4,765,903 to D'Andrea et al.), cell debris and viruses (see U.S. Pat. No. 4,732,683 to Georgiadis et al.) and pyruvate-linked isoforms (see Rose et al., J. Biol. Chem. 287:19101 (1992); Prome et al, J. Biol. Chem. 266:13050 (1991); Stevens et al, J. Biol. Chem. 252:2998 (1977); and Shapiro et al, J. Biol. Chem. 255:3120 (1980)). It is desirable to remove these contaminants during purification of the protein.
Clearly, these protein isoforms reduce the purity of the desired protein and the processes for removal of the isoforms reduce the overall yield. If, however, the protein isoforms can be converted to the desired protein, their removal is unnecessary and the overall protein yields would be significantly increased. What is needed is a way to identify undesired protein isoforms and convert them to the desired protein. The present invention addresses such needs.
SUMMARY OF THE INVENTION
The present invention provides methods for preparing highly purified proteins in high yields by isolating adjunct isoforms and converting them to a desired, functional protein. In one embodiment, the present invention provides a method for increasing the yield of an interferon alpha composition, comprising converting an adjunct isoform into interferon alpha. While the present invention is not limited to a particular interferon alpha, in a preferred embodiment the interferon alpha is interferon alpha2b.
In another embodiment, the present invention provides methods for converting a recombinantly produced adjunct isoform to the desired protein comprising chemically removing a cleavable group from the adjunct isoform.
The present invention is not limited by the cleavable group removed. In one embodiment the cleavable group comprises pyruvate.
The present invention is also not limited by the method of chemically removing the cleavable group. In one embodiment, the method comprises exposing the adjunct isoform to acid solution. When the adjunct isoform is a pyruvate adjunct isoform of interferon alpha, it is preferred that the acid solution be at about pH 5.5. In such an embodiment, it is further preferred that the acid solution be at 34-40° C. In another embodiment, however, the adjunct isoform is exposed to a zinc solution. In a preferred embodiment, the zinc solution is at pH 7.8 to pH 8.6. In a further preferred embodiment, the zinc solution is at 30-38° C.
The present invention is also not limited by the type of acid or zinc solution utilized. In preferred embodiments, the acid solution or zinc solution comprise an antioxidant. In particularly preferred embodiments, the antioxidant comprises methionine. In such an embodiment, the preferred concentration of methionine is 5-40 mM.
Definitions
As used herein, the term “desired protein” means a protein of interest that is intended to be purified. The identification of the desired protein is, of course, subject to the ultimate goal of the purification procedure. For example, during a purification procedure it may be desirable to obtain a protein group or groups, including contaminants, in an intermediate step of the purification process. Regardless of the interest in obtaining an intermediate protein group, the protein group that is the ultimate goal of the purification procedure is considered the desired protein.
As used herein, the term “adjunct isoform” means a protein isoform having structural and/or functional characteristics similar to a desired protein, wherein a cleavable group can be removed from the protein to produce the desired protein. A “cleavable group” is understood to mean a chemical group attached to a desired protein that can be chemically removed. “Chemical removal” or “chemically removed” as used herein is understood to designate that a chemical group has been separated from a protein by chemical means, including, but not limited to, acidic solution, basic solutions, metal ion catalysis, etc.
While not being necessary to practice the present invention, if the cleavable group can be chemically identified, the adjunct isoform can be referred to as a specific type of adjunct isoform. For example, a “pyruvate adjunct isoform” is a desired protein having a cleavable group attached that is identifiable as pyruvate.
As used herein, the term “oxidation reaction” means a reaction intended to cause the sulfhydryl groups of two cysteine amino acids to form disulfide bonds.
As used herein, the term “interferon alpha” refers to a family of inducible secreted proteins that confer resistance to viruses on target cells, inhibit cell proliferation and regulate expression of MHC class I antigens. This family includes, but is not limited to interferon alpha-2a (Roferon, Hoffman La-Roche, Nutley, N.J.), interferon alpha 2b (Intron, Schering-Plough, Madison, N.J.), interferon alpha-2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany) or consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen, Amgen, Thousand Oaks, Calif.).
DETAILED DESCRIPTION OF THE INVENTION
The present invention pertains to the isolation and purification of proteins. In one embodiment, the present invention provides for the identification and purification of adjunct isoforms. In another embodiment, the present invention provides methods for producing a desired protein from adjunct isoforms. In yet another embodiment, the present invention provides a highly purified desired protein by the co-purification of the desired protein together with adjunct isoforms and the subsequent conversion of the adjunct isoforms to the desired protein. In this manner, the amount of the adjunct isoform is reduced and the overall yield of the desired protein is increased and/or is more highly purified than previously achievable. The yield of the desired protein can be increased by as much as ten times the yield as obtained without converting adjunct isoforms.
While the present invention is not limited by the source of the desired protein or the adjunct isoforms, in one embodiment, the source is microorganisms constructed via recombinant techniques. There are many such techniques known to those skilled in the art. Such transformed microorganisms may be eukaryotic or prokaryotic cells, bacteria, mammalian cells, etc. For example, interferon alpha may be produced in bacteria by following the teachings of U.S. Pat. No. 4,530,901 to Weissman or by the techniques described in European Patent Application publication number EP032,134.
Likewise, the present invention is not limited to any particular method of extracting the adjunct isoform from the producing cell. When the desired protein is interferon alpha, for example, the methods described in U.S. Pat. Nos. 4,315,852 and 4,364,863 to Leibowitz et al, are suitable.
Likewise, the present invention is not limited by the particular purification techniques employed to isolate the adjunct isoform or the desired protein. Many chromatography and other separation techniq
Cannon-Carlson Susan
Frei Andres
Lee Seoju
Mengisen Roland
Voloch Marcio
Kemmerer Elizabeth
Landsman Robert S.
Schering Corporation
Wyatt Donald W.
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