US 5,231,178 AGranted

Method for the purification of intact, correctly-folded insulin-like growth factor-1

AssigneeThe Salk Institute Biotechnology/Industrial Associates, Inc.

InventorsGregory C. Holtz, Russell A. Brierley

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Abstract

Method for the recovery and purification of intact, correctly-folded, monomeric insulin-like growth factor-1 peptide from large volumes of IGF-1-containing medium are described, comprising a series of adsorption-desorption steps employing a combination of cation exchange and hydrophobic interaction adsorbents. Product IGF-1 peptide is highly purified and suitable for use in a variety of clinical applications.

Claims56

Dependent claims

Method for the purification of monomeric, intact, correctly-folded insulin-like growth factor-1 peptide (IGF-1) from medium containing IGF-1 peptides, said method comprising:

(a) contacting said medium with a sufficient quantity of first cation exchange material under conditions suitable to adsorb at least about 95% of total IGF-1 from said medium,
(b) eluting the adsorbed IGF-1 from the IGF-1-containing cation exchange material of step (a) by contacting said cation exchange material with a sufficient quantity of a solvent system which has a sufficiently high pH or ionic strength so as to displace aberrant substantially all of said IGF-1 from said cation exchange material,
(c) contacting the IGF-1-containing fractions of the eluate of step (b), in a suitable solvent system, with a sufficient quantity of a first hydrophobic interaction chromatography matrix under conditions suitable to adsorb in the range of about 95 up to 100% of said IGF-1 from said eluate,
(d) eluting the adsorbed IGF-1 from said first hydrophobic interaction chromatography matrix by contacting said matrix first with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having a sufficiently low conductivity so as to displace aberrant IGF-1 peptides from said matrix, without displacing significant quantities of the intact, monomeric, correctly-folded form of adsorbed IGF-1 from said first hydrophobic interaction chromatography matrix, followed by contacting said matrix with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having an elevated pH, wherein said elevated pH is sufficiently high so as to displace substantially all of the remaining adsorbed forms of IGF-1 from said matrix,
(e) contacting the fractions eluted according to step (d) employing said buffer system having an elevated pH which contain, as the predominant form of IGF-1, intact, monomeric, correctly-folded IGF-1, wherein said contacting is carried out with a sufficient quantity of a second cation exchange matrix and under conditions suitable to adsorb in the range of about 95 up to 100% of total IGF-1 from said eluate,
(f) eluting the adsorbed IGF-1 from said second cation exchange matrix by contacting said matrix with at least 1 volume, relative to the volume of matrix, of a buffer system having a sufficient ionic strength so as to differentially displace substantially all of the IGF-1 peptides from said matrix,
(g) contacting the intact, monomeric, correctly-folded IGF-1-containing fractions of the eluate from step (f), in a suitable solvent system, with either,
(1) a sufficient quantity of a second hydrophobic interaction chromatography matrix under conditions suitable to adsorb in the range of about 95 up to 100% of all forms of IGF-1 from said eluate, or,
(2) a sufficient quantity of a gel filtration chromatography matrix having suitable pore size to effect resolution of the intact, monomeric correctly-folded form of IGF-1 from substantially all multimeric forms of IGF-1, and
(h) (1) after step (g) (1) eluting the adsorbed IGF-1 from said second hydrophobic interaction chromatography matrix by contacting said matrix with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having a sufficiently low conductivity so as to displace substantially all forms of IGF-1 other than the intact, monomeric, correctly-folded form of IGF-1 from said matrix, without displacing significant quantities of the intact, monomeric, correctly-folded form of adsorbed IGF-1 from said second hydrophobic interaction chromatography matrix, followed by contacting said matrix with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having a sufficiently high pH so as to displace substantially all of the remaining absorbed IGF-1 from said matrix or,
(2) after step (g) (2) eluting said gel filtration chromatography matrix with a sufficient quantity of elution buffer so as to cause the intact, monomeric correctly-folded form of IGF-1 to be resolved from said multimeric forms of IGF-1.

8 more elements

Method for the purification of monomeric, intact, correctly-folded insulin-like growth factor-1 (IGF-1) peptide from medium containing IGF-1 peptides, wherein said medium containing IGF-1 is the substantially cell-free fermentation broth from a high cell density yeast fermentation operation, and wherein said yeast are transformed with at least one DNA fragment comprising, in the direction of transcription, the following DNA sequences:

(i) a promoter region of a methanol-responsive gene of P. pastoris,
(ii) a DNA sequence encoding a polypeptide consisting of:
(a) The S. cerevisiae AMF pre-pro sequence, including the processing site:
lys-art and
(b) an insulin-like growth factor-1 (IGF-1) peptide;
and
(iii) a transcription terminator functional in P. pastoris,
wherein said DNA sequences are operationally associated with one another for transcription of the sequences encoding said polypeptide, said method comprising:
(a) diluting said IGF-1-containing medium with a low conductivity buffered medium having the same pH as the media used to equilibrate the cation exchange matrix employed in step (b) below,
(b) contacting said medium with a sufficient quantity of a sulfylpropylate cation exchange media under conditions suitable to adsorb at least about 95% of said IGF-1 from said medium;
wherein at least 0.05 liters, per gram of IGF-1 in said medium, of said cation exchange material are employed;
and wherein said contacting is carried out at a temperature in the range of about 2.degree. up to 30.degree. C.,
(c) contacting the IGF-1-containing cation exchange material with at least about 2 volumes, per volume of said cation exchange material, of a 0.02M acetic acid solution, followed by about four volumes of a 0.02M sodium acetate solution having a pH of 5 and containing 0.2M sodium chloride,
(d) eluting the adsorbed IGF-1 from said IGF-1-containing cation exchange matrix material of step (c) by contacting said matrix material with a sufficient quantity of a solvent system comprising a 0.02M sodium acetate solution having a pH of 5.5 and containing 1.0M sodium chloride,
(e) contacting the IGF-1-containing eluate of step (d) with a sufficient volume of a buffered ammonium sulfate-containing solution, pH 4.5, so as to render the ammonium sulfate concentration of the eluate in the range of about 0.4 up to 0.8M, and the pH of the diluted eluate about 4.5,
(f) contacting the product of step (e) with a sufficient quantity of a first hydrophobic interaction chromatography matrix under conditions suitable to adsorb in the range of about 95 up to 100% of said IGF-1 from said eluate, wherein said first hydrophobic interaction chromatography matrix is a butyl-substituted, poly(methacrylate)-supported hydrophobic interaction chromatography matrix;
wherein at least about 0.05 liters, per gram of IGF-1 in said medium, if said first hydrophobic interaction chromatography matrix are employed;
and wherein said contacting is carried out at a temperature in the range of about 20.degree. up to 25.degree. C.,
(g) eluting the adsorbed IGF-1 from said first hydrophobic interaction chromatography matrix by contacting said matrix:
(1) first with a quantity of a linear gradient of a buffered solution having a pH of about 4.5 sufficient to produce a substantially ammonium sulfate-free eluate, then
(2) with a quantity of a linear gradient of a substantially ammonium sulfate-free buffered solution having an initial pH of about 4.5 sufficient to raise the pH of said eluate up to about 6.5,
(h) contacting at least a portion of the eluate fractions obtained in step (g) (2) with a sufficient additional quantity of said first hydrophobic interaction chromatography matrix under conditions suitable to adsorb in the range of about 95 up to 100% of residual quantities of IGF-1 from said eluate;
wherein at least about 0.05 liters, per gram of IGF-1 in said medium, of said first hydrophobic interaction chromatography resin are employed;
and wherein said contacting is carried out at a temperature in the range of about 20.degree. up to 25.degree. C.
(i) eluting the adsorbed IGF-1 from said first hydrophobic interaction chromatography matrix by contacting said matrix:
(1) first with a quantity of a linear gradient of a buffered solution having a pH of about 4.5 sufficient to produce a substantially ammonium sulfate-free eluate, then
(2) with a quantity of a linear gradient of a substantially ammonium sulfate-free buffered solution having an initial pH of about 4.5 sufficient to raise the pH of said eluate up to about 6.5,
(j) contacting those portions of the combined eluate from steps (g) (2) and (i) (2) which contain, as the predominant form of IGF-1, intact, monomeric, correctly-folded IGF-1, wherein said contacting is carried out with a sufficient quantity of a second cation exchange matrix material and under conditions suitable to adsorb in the range of about 95 up to 100% of said IGF-1 from said eluate;
wherein said matrix comprises a sulfylmethylated agarose matrix;
wherein at least 0.05 liters, per gram of IGF-1 in said medium, of said cation exchange material are employed;
and wherein said contacting is carried out at a temperature in the range of about 2.degree. up to 30.degree. C.,
(k) contacting the IGF-1-containing second cation exchange matrix material with at least one, up to about five volumes, per volume of said cation exchange material, of a 0.05M sodium acetate solution, pH 4.5,
(1) eluting the IGF-1 from said second cation exchange matrix material by contacting said matrix material with at least five volumes, relative to the volume of matrix, of a sodium chloride gradient, which is provided by combining, as a linear gradient, a first solvent system and a second solvent system;
wherein said first solvent system comprises a 0.05M sodium acetate solution, pH 5.5, and
wherein said second solvent system comprises a 0.05M sodium acetate/0.3M sodium chloride solution, pH 5.5,
(m) either
(1) diluting the intact, monomeric, correctly-folded IGF-1 containing fractions of the eluate of step (1) with at least one volume of a buffered ammonium sulfate-containing solution, pH 4.5, so as to render the ammonium sulfate concentration of the eluate in the range of about 0.2 up to 2.0M and the pH of the diluted eluate about 4.5, and contacting the diluted eluate with a sufficient quantity of a second hydrophobic interaction chromatography matrix under conditions suitable to adsorb in the range of about 95 up to 100% of said IGF-1 from said eluate;
wherein said second hydrophobic interaction chromatography matrix is a butyl-substituted hydrophobic interaction chromatography matrix;
wherein at least about 0.05 liters, per gram of IGF-1 in said medium, of said second hydrophobic interaction chromatography matrix are employed;
and wherein said contacting is carried out at a temperature in the range of about 20.degree. to 25.degree. C. or,
(2) contacting the intact, monomeric, correctly-folded IGF-1-containing fractions of the eluate from step (1), with a sufficient quantity of a gel filtration chromatography matrix having suitable pore size to effect resolution of the intact, monomeric correctly-folded form of IGF-1 from substantially all multimeric forms of IGF-1, and
(n)
(1) after step (m) (1) eluting the adsorbed IGF-1 from said second hydrophobic interaction chromatography matrix by contacting said matrix:
(i) first with a quantity of a linear gradient of a buffered solution having a pH of about 4.5 sufficient to produce a substantially ammonium sulfate-free eluate, then
(ii) with a quantity of a linear gradient of a substantially ammonium sulfate-free buffered solution having an initial pH of about 4.5 sufficient to raise the pH of said eluate up to about 6.5, or
(2) after step (m) (2) eluting said gel filtration chromatography matrix with a sufficient quantity of elution buffer so as to cause the intact, monomeric correctly-folded form of IGF-1 to be resolved said multimeric forms of IGF-1.

43 more elements

Method for the purification of monomeric, intact, correctly-folded insulin-like growth factor-1 peptide (IGF-1) from medium containing IGF-1 peptides, said method comprising:

(a) contacting said medium with a sufficient quantity of first cation exchange material under conditions suitable to adsorb at least about 95% of total IGF-1 from said medium,
(b) eluting the adsorbed IGF-1 from the IGF-1-containing cation exchange material of step (a) by contacting said cation exchange material with a sufficient quantity of a solvent system which has a sufficiently high pH or ionic strength so as to displace substantially all of said IGF-1 from said cation exchange material,
(c) contacting the IGF-1-containing fractions of the eluate of step (b), in a suitable solvent system, with a sufficient quantity of a first hydrophobic interaction chromatography matrix under conditions suitable to adsorb in the range of about 95 up to 100% of said IGF-1 from said eluate,
(d) eluting the adsorbed IGF-1from said first hydrophobic interaction chromatography matrix by contacting said matrix first with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having a sufficiently low conductivity so as to displace aberrant IGF-1 peptides from said matrix, without displacing significant quantities of the intact, monomeric, correctly-folded form of adsorbed IGF-1 from said firs hydrophobic interaction chromatography matrix, followed by contacting said matrix with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having an elevated pH, wherein said elevated pH is sufficiently high so as to displace substantially all of the remaining adsorbed forms of IGF-1 from said matrix.

1 more element

Dependencies are inferred from the claim text.

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Abstract

Method for the purification of monomeric, intact, correctly-folded insulin-like growth factor-1 peptide (IGF-1) from medium containing IGF-1 peptides, said method comprising:

(a) contacting said medium with a sufficient quantity of first cation exchange material under conditions suitable to adsorb at least about 95% of total IGF-1 from said medium,
(b) eluting the adsorbed IGF-1 from the IGF-1-containing cation exchange material of step (a) by contacting said cation exchange material with a sufficient quantity of a solvent system which has a sufficiently high pH or ionic strength so as to displace aberrant substantially all of said IGF-1 from said cation exchange material,
(c) contacting the IGF-1-containing fractions of the eluate of step (b), in a suitable solvent system, with a sufficient quantity of a first hydrophobic interaction chromatography matrix under conditions suitable to adsorb in the range of about 95 up to 100% of said IGF-1 from said eluate,
(d) eluting the adsorbed IGF-1 from said first hydrophobic interaction chromatography matrix by contacting said matrix first with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having a sufficiently low conductivity so as to displace aberrant IGF-1 peptides from said matrix, without displacing significant quantities of the intact, monomeric, correctly-folded form of adsorbed IGF-1 from said first hydrophobic interaction chromatography matrix, followed by contacting said matrix with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having an elevated pH, wherein said elevated pH is sufficiently high so as to displace substantially all of the remaining adsorbed forms of IGF-1 from said matrix,
(e) contacting the fractions eluted according to step (d) employing said buffer system having an elevated pH which contain, as the predominant form of IGF-1, intact, monomeric, correctly-folded IGF-1, wherein said contacting is carried out with a sufficient quantity of a second cation exchange matrix and under conditions suitable to adsorb in the range of about 95 up to 100% of total IGF-1 from said eluate,
(f) eluting the adsorbed IGF-1 from said second cation exchange matrix by contacting said matrix with at least 1 volume, relative to the volume of matrix, of a buffer system having a sufficient ionic strength so as to differentially displace substantially all of the IGF-1 peptides from said matrix,
(g) contacting the intact, monomeric, correctly-folded IGF-1-containing fractions of the eluate from step (f), in a suitable solvent system, with either,
(1) a sufficient quantity of a second hydrophobic interaction chromatography matrix under conditions suitable to adsorb in the range of about 95 up to 100% of all forms of IGF-1 from said eluate, or,
(2) a sufficient quantity of a gel filtration chromatography matrix having suitable pore size to effect resolution of the intact, monomeric correctly-folded form of IGF-1 from substantially all multimeric forms of IGF-1, and
(h) (1) after step (g) (1) eluting the adsorbed IGF-1 from said second hydrophobic interaction chromatography matrix by contacting said matrix with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having a sufficiently low conductivity so as to displace substantially all forms of IGF-1 other than the intact, monomeric, correctly-folded form of IGF-1 from said matrix, without displacing significant quantities of the intact, monomeric, correctly-folded form of adsorbed IGF-1 from said second hydrophobic interaction chromatography matrix, followed by contacting said matrix with in the range of about 1 up to 10 volumes, relative to the volume of matrix, of a buffer system having a sufficiently high pH so as to displace substantially all of the remaining absorbed IGF-1 from said matrix or,
(2) after step (g) (2) eluting said gel filtration chromatography matrix with a sufficient quantity of elution buffer so as to cause the intact, monomeric correctly-folded form of IGF-1 to be resolved from said multimeric forms of IGF-1.

8 more elements