US 4,503,035 A · Protein purification process and product

Abstract

An improved process for purifying proteins, particularly proteins having molecular weights in excess of 12,000 involves novel applications of high performance liquid chromatography on a preparative scale to provide homogeneous end product in excellent yield. In an embodiment of the process, human interferon is produced as a homogeneous protein.

Claims14

Dependent claims

Human leukocyte interferon as a homogenous protein species having a specific activity of from 0.9.times.10.sup.8 -4.times.10.sup.8 Units/mg. of protein when assayed on the MDBK bovine cell line and from 2.times.10.sup.6 -7.6.times.10.sup.8 Units/mg. of protein when assayed on the AG 1732 human cell line.

A species of human leukocyte interferon designated .alpha..sub.1 being a homogeneous protein characterized by the following:

(a) an amino terminal sequence as follows:
Ser-ASp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly-Asn-Arg-Arg-Thr-Leu-Met-Leu-Leu-Al a-Glu-Met-Arg- . . .
(b) a specific activity on MDBK (bovine cells) of about 2.6.times.10.sup.8 Units/mg;
(c) a specific activity on Ag 1732 (human line) cells of about 2.6.times.10.sup.8 units/mg;
(d) a molecular weight of about 16,500.+-.1000 by polyacrylamide gel electrophoresis
(e) an amino sugar content of less than 1 residue per molecule;
(f) positive growth inhibition activity;
(g) eluted as a single peak at a concentration of 31% n-propanol (0-40% gradient) in a 1M pyridine--2M formic acid aqueous buffer from an octyl bonded silica matrix HPLC 4.6.times.250 mm column at room temperature and a flow rate of 0.2 ml/min;
(h) an amino acid composition (.+-.15%) as based on a molecular weight of 16,500 as contained in the following table:
Asx:
13.8
Thr:
7.7
Ser:
9.2
Glx:
20.3
Pro:
6.1
Gly:
5.1
Ala:
8.4
Val:
7.4
Met:
3.7
ILeu:
7.4
Leu:
18.0
Tyr:
4.0
Phe:
6.9
His:
3.0
Lys:
10.5
Arg:
6.2
Cys:
3.9
(i) providing peptide fragments upon treatment with trypsin, and fragments eluting as peaks at 3(L), 4(L), 4.2(M), 11.5(M), 12.5(S), 14.5(M), 16(S), 18(M), 20(S), 21(S), 22.5(S) and 29(M)% n-propanol when the reaction media is passed through an octyl bonded silica matrix 10.mu. particle size HPLC 4.6.times.250 mm column at room temperature using a 0.03M pyridine-0.1M formic acid aqueous buffer (pH 3) at a flow rate of 0.5 ml/min. with a 1 hour 0 to 40% n-propanol gradient;
and
(j) containing the following amino acid sequences:
(I) . . . Ala-Glu-Ile-Met-Arg . . . ,
(II) . . . Lys-Tyr-Phe-Gln-Arg . . . ,
(III) . . . Glx-Asx-Ser-Ile-Leu-Ala-Val-Arg . . . ,
(IV) . . . Ile-Thr-Leu-Tyr-Leu-Lys . . . ,
(V) . . . Thr-Leu-Met-Leu-Leu-Ala-Gln-Met-Arg . . . ,
(VI) . . . (Ser-2, Ile, Leu-2, Phe, Lys, Cys) . . . ,
(VII) . . . (Asx-2, Thr-2, Ser, Glx-3, Pro, Gly, Ala, Val, Leu-3, Tyr, Lys) . . . ,
(VIII) . . . (Asx, Thr-2, Ser, Glx-3, Pro, Ala, Val, Met, Ile-2, Leu-2, Phe-2, His, Lys) . . . .

51 more elements

A species of human leukocyte interferon designated .alpha..sub.2 being a homogeneous protein characterized by the following:

(a) a blocked amino terminus;
(b) a specific activity on MDBK (bovine cells) of about 4.0.times.10.sup.8 units/mg;
(c) a specific activity on Ag 1732 (human line) cells of about 3.times.10.sup.8 units/mg;
(d) a molecular weight of about 16,200.+-.1000 by polyacrylamide gel electrophoresis;
(e) an amino sugar content of less than 1 residue per molecule;
(f) positive growth inhibition activity;
(g) eluted as a single peak at a concentration of 32% n-propanol (0-40% gradient) in a 1M pyridine--2M formic acid aqueous buffer from an octyl bonded silica matrix HPLC 4.6.times.250 mm column at room temperature and a flow rate of 0.2 ml/min;
(h) resistant to deactivation by treatment with leucine aminopeptidase and aminopeptidase M;
(i) an amino acid composition (.+-.15%) as based on a molecular weight of 16,200 as contained in the following table:
Asx:
11.7
Thr:
8.3
Ser:
10.0
Glx:
20.5
Pro:
4.9
Gly:
4.5
Ala:
8.3
Val:
6.2
Met:
3.8
ILeu:
7.0
Leu:
18.0
Tyr:
4.4
Phe:
8.0
His:
2.8
Lys:
9.0
Arg:
7.1
Cys:
4.0
(j) providing peptide fragments upon treatment with trypsin said fragments eluted as peaks at 3(L), 4(L), 4.2(M), 11.5(M), 12.5(S), 14.5(M), 16(S), 18(M), 27(S) and 29(M) % n-propanol when the reaction media is passed through an octyl bonded silica matrix 10.mu. particle size HPLC 4.6.times.250 mm column at room temperature using a 0.03M pyridine-0.1M formic acid aqueous buffer (pH 3) at a flow rate of 0.5 ml/min. with a 1 hour 0 to 40% n-propanol gradient;
(k) eluted as a single peak at a concentration of 68% n-propanol (72.5-50% gradient) in 0.1M sodium acetate aqueous buffer from a glycerol bonded silica matrix HPLC 4.6.times.250 mm column at room temperature and a flow rate of 0.25 ml/min. and
(l) containing the following amino acid sequences:
(I) . . . Ala-Glu-Ile-Met-Arg . . . ,
(II) . . . Lys-Tyr-Phe-Gln-Arg . . . ,
(III) . . . Glx-Asx-Ser-Ile-Leu-Ala-Val-Arg . . . ,
(IV) . . . Ile-Thr-Leu-Tyr-Leu-Lys . . . ,
(V) . . . Thr-Leu-Met-Leu-Leu-Ala-Gln-Met-Arg . . . ,
(VI) . . . (Ser-2, Ile, Leu-2, Phe, Lys, Cys) . . . ,
(VII) . . . (Asx-2, Thr-2, Ser, Glx-3, Pro, Gly, Ala, Val, Leu-3, Tyr, Lys) . . . ,
(VIII) . . . (Asx, Thr-2, Ser, Glx-3, Pro, Ala, Val, Met, Ile-2, Leu-2, Phe-2, His, Lys) . . . .

51 more elements

A species of human leukocyte interferon designated .beta..sub.1 being a homogeneous protein characterized by the following:

(a) a blocked amino terminus;
(b) a specific activity on MDBK (bovine cells) of about 1.7.times.10.sup.8 units/mg;
(c) a specific activity on Ag 1732 (human line) cells of about 2.2.times.10.sup.8 units/mg;
(d) a molecular weight of about 16,000(.+-.1000) by polyacrylamide gel electrophoresis;
(e) an amino sugar content of less than 1 residue per molecule
(f) positive growth inhibition activity;
(g) eluted as a single peak at a concentration of 31% n-propanol (0-40% gradient) in a 1M pyridine--2M formic acid aqueous buffer from an octyl bonded silica matrix HPLC 4.6.times.250 mm column at room temperature and a flow rate of 0.2 ml/min.;
(h) resistant to deactivation by treatment with leucine aminopeptidase and aminopeptidase M;
(i) an amino acid composition (.+-.15%) as based on a molecular weight of 16,000 as contained in the following table:
Asx:
11.3
Thr:
8.7
Ser:
10.3
Glx:
20.6
Pro:
5.2
Gly:
4.9
Ala:
7.2
Val:
6.0
Met:
4.8
Ile:
6.4
Leu:
18.1
Tyr:
4.4
Phe:
8.6
His:
2.8
Lys:
8.7
Arg:
7.9
Cys:
3.0
(j) providing peptide fragments upon treatment with trypsin, said fragments eluting as peaks at 3(L), 4(L), 11.5(M), 12.5(S), 14.5(M), 16(S), 17.5(S), 18(M) and 29(M) % n-propanol when the reaction media is passed through an octyl bonded silica matrix 10.mu. particle size HPLC 4.6.times.250 mm column at room temperature using a 0.03M pyridine-0.1M formic acid aqueous buffer (pH 3) at a flow rate of 0.5 ml/min. with a 1 hour 0 to 40% n-propanol gradient;
and
(k) containing the following amino acid sequences:
(I) . . . Ala-Glu-Ile-Met-Arg . . . ,
(II) . . . Lys-Tyr-Phe-Gln-Arg . . . ,
(III) . . . Glx-Asx-Ser-Ile-Leu-Ala-Val-Arg . . . ,
(IV) . . . Ile-Thr-Leu-Tyr-Leu-Lys . . . ,
(V) . . . Thr-Leu-Met-Leu-Leu-Ala-Gln-Met-Arg . . . ,
(IV) . . . (Ser-2, Ile, Leu-2, Phe, Lys, Cys) . . . ,
(VII) . . . (Asx-2, Thr-2, Ser, Glx-3, Pro, Gly, Ala, Val, Leu-3, Tyr, Lys) . . . ,
(VIII) . . . (Asx, Thr-2, Ser, Glx-3, Pro, Ala, Val, Met, Ile-2, Leu-2, Phe-2, His Lys) . . . .

51 more elements

A species of human leukocyte interferon designated .beta..sub.2 being a homogeneous protein characterized by the following:

(a) an unblocked amino terminus;
(b) a specific activity on MDBK (bovine cells) of about 4.0.times.10.sup.8 units/mg;
(c) a specific activity on Ag 1732 (human line) cells of about 2.times.10.sup.8 units/mg;
(d) a molecular weight of about 16,500 (.+-.1000) by polyacylamide gel electrophoresis;
(e) an amino sugar content of less than 1 residue per molecule;
(f) positive growth inhibition activity;
(g) eluted as a single peak at a concentration of 32% n-propanol (0-40% gradient) in a 1M pyridine-2M formic acid aqueous buffer from an octyl bonded silica matrix HPLC 4.6.times.250 mm column at room temperature and a flow rate of 0.2 ml/min;
(h) an amino acid composition (.+-.15%) as based on molecular weight of 16,500 as contained in the following table:
Asx:
11.5
Thr:
9.0
Ser:
10.0
Glx:
21.9
Pro:
5.0
Gly:
5.0
Ala:
7.9
Val:
6.7
Met:
4.4
ILeu:
7.4
Leu:
18.9
Tyr:
4.6
Phe:
8.7
His:
3.0
Lys:
8.5
Arg:
8.0
Cys:
1.8
(i) providing peptide fragments upon treatment with trypsin, said fragments eluting at peaks at 3(L), 4(L), 4.2(M), 11.5(M), 12.5(S), 14.5(M), 16(S), 17.5(S), 18(M) and 29(M) % n-propanol when the reaction media is passed through an octyl bonded silica matrix 10.mu. particle size HPLC 4.6.times.250 mm column at room temperature using a 0.03M pyridine-0.1M formic acid aqueous buffer (pH 3) at a flow rate of 0.5 ml/min. with a 1 hour 0 to 40% n-propanol gradient;
(j) eluted as a single peak at a concentration of 66.5% n-propanol (72.5-50% gradient) in 0.1M sodium acetate aqueous buffer from a glycerol bonded silica matrix HPLC 4.6.times.250 mm column at room temperature and a flow rate of 0.25 ml/min.;
and
(k) containing the following amino acid sequences:
(I) . . . Ala-Glu-Ile-Met-Arg . . . ,
(II) . . . Lys-Tyr-Phe-Gln-Arg . . . ,
(III) . . . Glx-Asx-Ser-Ile-Leu-Ala-Val-Arg . . . ,
(IV) . . . Ile-Thr-Leu-Tyr-Leu-Lys . . . ,
(V) . . . Thr-Leu-Met-Leu-Leu-Ala-Gln-Met-Arg . . . ,
(VI) . . . (Ser-2, Ile, Leu-2, Phe, Lys, Cys) . . . ,
(VII) . . . (Asx-2, Thr-2, Ser, Glx-3, Pro, Gly, Ala, Val, Leu-3, Tyr, Lys) . . . ,
(VIII) . . . (Asx, Thr-2, Ser, Glx-3, Pro, Ala, Val, Met, Ile-2, Leu-2, Phe-2, His, Lys) . . . .

51 more elements

A species of human leukocyte interferon designated .gamma..sub.1 being a homogeneous protein characterized by the following:

(a) a blocked amino terminus;
(b) a specific activity on MDBK (bovine cells) of about 2.6.times.10.sup.8 units/mg;
(c) a specific activity on Ag 1732 (human line) cells of about 2.times.10.sup.8 units/mg;
(d) a molecular weight of about 17,700.+-.1000 by polyacrylamide gel electrophoresis;
(e) an amino sugar content of less than 1 residue per molecule;
(f) positive growth inhibition activity;
(g) eluted as a single peak at a concentration of 31% n-propanol (0-40% gradient) in a 1M pyridine-2M formic acid aqueous buffer from an octyl bonded silica matrix HPLC 4.6.times.250 mm column at room temperature and a flow rate of 0.2 ml/min;
(h) resistant to deactivation by treatment with leucine aminopeptidase and aminopeptidase M;
(i) an amino acid composition (.+-.15%) as based on a molecular weight of 17,700 contained in the following table:
Asx:
13.1
Thr:
8.4
Ser:
10.2
Glx:
23.9
Pro:
4.5
Gly:
5.7
Ala:
8.7
Val:
7.3
Met:
4.3
ILeu:
7.9
Leu:
20.3
Tyr:
4.8
Phe:
8.6
His:
3.7
Lys:
10.1
Arg:
8.0
Cys:
3.3
(j) providing peptide fragments upon treatment with trypsin, said fragments eluting as peaks at 3(L), 4(L), 4.2(M), 4.5(S), 5(S), 6.5(S), 11.5(S), 12.5(S), 14.5(M), 16(S), 17.5(M), 18(M) and 29(M) % n-propanol when the reaction media is passed through an octyl bonded silica matrix 10.mu. particle size HPLC 4.6.times.250 mm column at room temperature using a 0.03M pyridine-0.1M formic acid aqueous buffer (pH 3) at a flow rate of 0.5 ml/min. with a 1 hour 0 to 40% n-propanol gradient;
and
(k) containing the following amino acid sequences:
(I) . . . Ala-Glu-Ile-Met-Arg . . . ,
(II) . . . Lys-Tyr-Phe-Gln-Arg . . . ,
(III) . . . Glx-Asx-Ser-Ile-Leu-Ala-Val-Arg . . . ,
(IV) . . . Ile-Thr-Leu-Tyr-Leu-Lys . . . ,
(V) . . . Thr-Leu-Met-Leu-Leu-Ala-Gln-Met-Arg . . . ,
(VI) . . . (Ser-2, Ile, Leu-2, Phe, Lys, Cys) . . . ,
(VII) . . . (Asx-2, Thr-2, Ser, Glx-3, Pro, Gly, Ala, Val, Leu-3, Tyr, Lys) . . . ,
(VIII) . . . (Asx, Thr-2, Ser, Glx-3, Pro, Ala, Val, Met, Ile-2, Leu-2, Phe-2, His, Lys) . . . .

51 more elements

A species of human leukocyte interferon designated .gamma..sub.2 being a homogeneous protein characterized by the following:

(a) a blocked amino terminus;
(b) a specific activity on MDBK (bovine cells) of about 4.0.times.10.sup.8 units/mg;
(c) a specific activity on Ag 1732 (human line) cells of about 1.5.times.10.sup.8 units/mg;
(d) a molecular weight of about 17,700 (.+-.1000) on polyacrylamide gel electrophoresis;
(e) an amino sugar content of less than 1 residue per molecule;
(f) positive growth inhibition activity;
(g) eluted as a single peak at a concentration of 32% n-propanol (0-40% gradient) in a 1M pyridine-2M formic acid aqueous buffer from an octyl bonded silica matrix HPLC 4.6.times.250 mm column at room temperature and a flow rate of 0.2 ml/min;
(h) resistant to deactivation by treatment with leucine aminopeptidase and aminopeptidase M;
(i) an amino acid composition (.+-.15%) as based on a molecular weight of about 17,700 as contained in the following table:
Asx:
13.3
Thr:
9.6
Ser:
7.8
Glx:
25.0
Pro:
4.8
Gly:
5.0
Ala:
8.1
Val:
7.0
Met:
3.9
ILeu:
8.0
Leu:
20.1
Tyr:
4.8
Phe:
9.0
His:
3.3
Lys:
10.0
Arg:
8.5
Cys:
2.9
(j) providing peptide fragments upon treatment with trypsin, said fragments eluting as peaks at 3(L), 4(L), 4.2(M), 4.5(S), 5(S), 11.5(S), 12.5(S), 14.5(S), 16(S), 18(L) and 29(M) % n-propanol when the reaction media is passed through an octyl bonded silica matrix 10.mu. particle size HPLC 4.6.times.250 mm column at room temperature using a 0.03M pyridine-0.1M formic acid aqueous buffer (pH 3) at a flow rate of 0.5 ml/min. with a 1 hour 0 to 40% n-propanol gradient;
and
(k) containing the following amino acid sequences:
(I) . . . Ala-Glu-Ile-Met-Arg . . . ,
(II) . . . Lys-Tyr-Phe-Gln-Arg . . . ,
(III) . . . Glx-Asx-Ser-Ile-Leu-Ala-Val-Arg . . . ,
(IV) . . . Ile-Thr-Leu-Tyr-Leu-Lys . . . ,
(V) . . . Thr-Leu-Met-Leu-Leu-Ala-Gln-Met-Arg . . . ,
(VI) . . . (Ser-2, Ile, Leu-2, Phe, Lys, Cys) . . . ,
(VII) . . . (Asx-2, Thr-2, Ser, Glx-3, Pro, Gly, Ala, Val, Leu-3, Tyr, Lys . . . .

50 more elements

Dependencies are inferred from the claim text.

Facts

Application

06/465,979Filed 14 Feb 1983 · continuation

Priority

24 Nov 1978

Granted

5 Mar 1985

Term ends

Not estimatedThe source records don’t support an estimate.

Classification10 codes

C07K 14/56
Interferons [IFN]: iFN-alpha
C07K 1/20
By chromatography: partition-, reverse-phase or hydrophobic interaction chromatography
C07K 14/665
From animals; from humans: derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
Y10S 435/811
Chemistry: molecular biology and microbiology: interferon

Family

Serial963,257Parent of this continuation-in-part · filed 24 Nov 1978

Serial62,374Parent of this continuation-in-part · filed 31 Jul 1979

Serial77,710Parent of this continuation-in-part · filed 21 Sep 1979

Serial106,644Parent of this continuation-in-part · filed 26 Dec 1979

Serial167,165Parent of this division · filed 9 Jul 1980

Serial247,442Parent of this continuation · filed 25 Mar 1981

US 4,289,690 AParent of this division

US 4,503,035 AThis patent

From the US filing history. Foreign family members aren’t listed.

Protein purification process and product

Life of this patent

Abstract

Claims14

Protein purification process and product

Life of this patent

Abstract

Claims14