Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-07-12
2004-06-29
Saoud, Christine J. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S399000, C530S303000, C530S412000, C530S414000, C530S418000, C530S417000, C530S422000
Reexamination Certificate
active
06756484
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to protein purification.
BACKGROUND OF THE INVENTION
Insulin-like growth factor-I (“IGF-I;” also known as “somatomedin-C”) is a mammalian growth factor essential for normal growth and development. It has insulin-like effects on muscle and adipose tissue, and it has mitogenic effects on several cell types. IGF-I has a variety of clinical uses. For example, it may be used to enhance the survival of neurons such as non-mitotic cholinergic neurons (Lewis et al, U.S. Pat. No. 5,093,317).
The complete amino acid sequence of the human IGF-I protein is known, and DNA encoding human IGF-I has been cloned and expressed in
E. coil
and yeast (see, e.g., Brierley et al., U.S. Pat. No. 5,324,639). The human a IGF-I protein consists of a single 70-amino acid polypeptide that includes six cysteine residues, all of which participate in the formation of three intrachain disulfide bonds (Axelsson et al.,
Eur. J. Biochem
. 206:987 (1992)). The three disulfide bonds, with all six cysteine residues properly paired, are necessary in order for IGF-I to have its correct (i.e., natural) tertiary structure. Upon reduction and reoxidation of the disulfide bonds, IGF-I can refold in various ways, forming as many as 15 monomeric configurations (Meng et al.,
J. Chrom
. 433:183 (1988)). In addition, IGF-I polypeptides can interact with each other to form multimeric structures. Processes &or obtaining purified, correctly folded IGF-I have been published (see, e.g., Holtz et al., U.S. Pat. No. 5,231,178 (“Holtz”); Chang et al., U.S. Pat. No. 5,288,931; and Hart et al.,
Biotech. Appl. Biochem
. 20:217 (1994)).
SUMMARY OF THE INVENTION
We have discovered an improved process for obtaining purified, monomeric, intact, correctly-folded (i.e., “authentic”) IGF-I. More particularly, we have discovered how to increase, by at least three-fold, the final yield of authentic IGF-I obtained in the IGF-I purification process described in Holtz (supra). The yield increase is obtained primarily by: (1) including an IGF-I protein unfolding/refolding step, carried out after the first cation chromotagraphy step; and (2) substituting a reverse phase chromotagraphy step for the gel filtration step described in Holtz.
Accordingly, the invention features a process for the purification of monomeric, intact, correctly-folded IGF-I polypeptide from a medium containing IGF-I polypeptides, said process comprising the steps of:
(a) contacting:the medium with a sufficient quantity of a first cation exchange matrix under conditions allowing adsorption of at least about 95% of total IGF-I from the medium;
(b) washing the IGF-I-loaded first cation exchange matrix with a first cation exchange wash buffer, which removes a substantial amount of adsorbed non-IGF-I material without removing a substantial amount of authentic or non-authentic IGF-I;
(c) eluting all forms of adsorbed IGF-I from the cation exchange matrix of step (a) by contacting said cation exchange matrix with a sufficient quantity of a first cation exchange elution buffer, which has a sufficiently high pH or ionic strength to displace substantially all of said authentic and non-authentic IGF-I from said cation exchange matrix;
(d) transferring the IGF-I-containing eluate from step (c) into an unfolding/refolding buffer, which: (i) reduces the intrachain disulfide bonds of IGF-I protein and promotes unfolding without permanent denaturation; and (ii) permits refolding of the IGF-I and reoxidation to form properly-paired intrachain disulfide bonds;
(e) contacting the refolded IGF-I from step (d), after transfer into a suitable solvent system, with a sufficient quantity of a hydrophobic interaction chromatography matrix under conditions allowing adsorption of at least about 95% of said IGF-I from said eluate;
(f) washing the IGF-I-loaded hydrophobic interaction chromatography matrix with a hydrophobic interaction wash buffer having an ionic strength sufficiently low to remove most of the non-authentic IGF-I, but not so low as to remove a significant proportion of the authentic IGF-I from the hydrophobic interaction chromatography matrix;
(g) eluting the adsorbed IGF-I from said hydrophobic interaction chromatography matrix by contacting said matrix with a hydrophobic interaction elution buffer, which has a sufficiently elevated pH, or sufficiently low ionic strength, to cause displacement of substantially all of the adsorbed authentic IGF-I from said matrix;
(h) contacting the eluate from step (g) with a sufficient quantity of a second cation exchange matrix under conditions allowing adsorption of at least about 95% of the IGF-I from the eluate;
(i) washing the IGF-I-loaded second cation exchange matrix with a cation exchange wash buffer having a sufficiently high ionic strength, or sufficiently high pH, to remove a significant proportion of non-authentic IGF-I, but not, so high as to remove a significant proportion of authentic IGF-I;
(j) eluting the adsorbed IGF-I from said second cation exchange matrix by contacting said matrix with a second cation exchange elution buffer, which has a sufficiently high ionic strength, or sufficiently high pH, to displace substantially all of the adsorbed authentic IGF-I from said matrix;
(k) contacting the eluate from step (j), in an aqueous buffer, with a suitable quantity of a reverse phase chromatography matrix under conditions allowing adsorption of at least about 95% of the IGF-I from the eluate;
(l) washing the IGF-I-loaded reverse phase chromatography matrix with an aqueous/organic reverse phase wash buffer having an organic solvent concentration sufficiently high to remove a substantial proportion of non-authentic IGF-I, but not so high as to remove a significant proportion of authentic IGF-I;
(m) eluting the adsorbed IGF-I from said reverse phase chromatography matrix with an aqueous/organic buffer having an organic solvent concentration high enough to remove substantially all of the authentic IGF-I without removing a significant proportion of multimeric forms of IGF-I.
Optionally, the non-authentic IGF-I recovered from step (f) is reprocessed at least once through steps (d) to (g), inclusive, before initiation of step (h).
As used herein, the term “authentic” IGF-I means monomeric, intact, correctly folded IGF-I, with three intrachain disulfide bonds involving properly paired cysteine residues, i.e., paired as in naturally-occurring IGF-I.
As used herein, the term “column volume” means the volume occupied by a chromatography matrix including interstitial liquid.
As used herein, the term “degraded” IGF-I means IGF-I in which one or more of the covalent bonds present in the polypeptide backbone or the amino acid side chains of authentic IGF-I have been cleaved.
As used herein, “des-2” IGF-I means IGF-I missing the first two amino acid residues of authentic IGF-I.
As used herein, the term “glycosylated” IGF-I means IGF-I with one or more covalently attached carbohydrate moieties.
As used herein, the term “intact” IGF-I means IGF-I that is not degraded.
As used herein, the term “misfolded” IGF-I means IGF-I whose secondary structure is other than that of authentic IGF-I.
As used herein, the term “multimeric” IGF-I means two or more IGF-I polypeptide chains linked by covalent or non-covalent chemical bonds.
As used herein, the term “oxidized” IGF-I means IGF-I containing at least one oxidized amino acid residue.
As used herein, the term “oxidized” IGF-I means IGF-I in which one or more intrachain disulfide bonds have reformed, following cleavage of those bonds.
As used herein, the term “about” in reference to a numerical value means +/−10% of the value, e.g., “about 50%” means between 45% and 55%.
REFERENCES:
patent: 5093317 (1992-03-01), Lewis et al.
patent: 5231178 (1993-07-01), Holtz et al.
patent: 5288931 (1994-02-01), Chang et al.
patent: 5324639 (1994-06-01), Brierley et al.
patent: 5407810 (1995-04-01), Builder et al.
patent: 5410026 (1995-04-01), Chang et al.
patent: 5446024 (1995-08-01), Builder et al.
patent: 5451660 (1995-09-01), Builder et al.
patent: 5459052 (1995-10
Abrams Joan N.
Brierley Russell A.
Cowgill Cynthia
Dorin Glenn
Hanson John M.
Cephalon Inc.
Saoud Christine J.
Woodcock & Washburn LLP
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