US4675383A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Purification of T-cell growth factor
Assignee: Salk Institute for Biological Studies
•
Filing: 1984-11-09
•
Granted: 1987-06-23
•
Expires: 2004-11-09
Abstract
Abstract Substantially homogenous T-cell growth factor is isolated. A multiple-step purification procedure of T-cell growth factor-containing biological sample includes at least one reverse-phase HPLC fractionation using a C3 to C5 silica gel stationary phase. A preferred pre-purification protocol includes concentration of the biological sample on a semipermeable medium followed by gel filtration and semi-preparative reverse-phase HPLC on a hydrophobic stationary phase. Fractionating the pre-purified biological sample with two or three reverse-phase HPLC fractionations using C3 to C5 silica gel stationary phase yields T-cells growth factor of over 90% purity.
Inventors
- 👤 Peter Bohlen
- 👤 Gunther Dennert
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 19:44
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/55
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
58 words
A method of obtaining human T-cell growth factor purified to at least about 80% by weight of total protein comprising obtaining a biological sample containing significant levels of human TCGF, pre-purifying said biological sample, and purifying said pre-purified biological sample with at least two reverse-phase HPLC chromatographic fractionations each using a C3 to C5 silica gel stationary phase.
Claim 2
Independent Claim
58 words
A method of obtaining human T-cell growth factor purified to at least about 80% by weight of total protein comprising obtaining a biological sample containing significant levels of human TCGF, pre-purifying said biological sample, and purifying said pre-purified biological sample with at least two reverse-phase HPLC chromatographic fractionations each using a C3 to C5 silica gel stationary phase.
Claim 3
Dependent Claim
32 words
A method according to claim 1 wherein said pre-purification increases the purity of TCGF in said biological sample to at least five percent as expressed as a weight percent of total protein.
Claim 4
Dependent Claim
40 words
A method according to claim 1 wherein said pre-purification comprises concentrating said biological sample to substantially reduce its volume, gel filtrating said concentrated biological sample and collecting biologically active fractions therefrom, and fractionating said biologically active fractions with semi-preparative chromatography.
Claim 5
Dependent Claim
16 words
A method according to claim 3 wherein said semi-preparative fractionation step is a reverse-phase HPLC chromatography.
Claim 6
Dependent Claim
16 words
A method according to claim 3 wherein said biological sample is concentrated with a semipermeable medium.
Claim 7
Dependent Claim
18 words
A method according to claim 5 wherein said biological sample is concentrated with a hollow fiber semipermeable medium.
Claim 8
Dependent Claim
26 words
A method according to claim 3 wherein said concentrating step reduces the volume of said biological sample to at least about 5% of its initial volume.
Claim 9
Dependent Claim
26 words
A method according to claim 1 wherein at least three reverse-phase HPLC fractionations are performed with said C3 to C5 silica gel as the stationary phase.
Claim 10
Dependent Claim
24 words
A method according to claim 1 wherein said biological sample is the culture medium of a TCGF-producing cell line subjected to a lymphokine-inducing agent.
Claim 11
Dependent Claim
36 words
A method according to claim 1 wherein said biological sample is purified on reverse-phase HPLC using an aqueous mobile phase having a pH of below about 5 and an increasing concentration of a water-miscible organic solvent.
Claim 12
Dependent Claim
22 words
A method according to claim 1 wherein C3 to C4 silica gel is the stationary phase for each of said two fractionations.
Claim 13
Independent Claim
70 words
A method of obtaining human T-cell growth factor (TCGF) purified to at least about 80% by weight of total protein comprising obtaining a biological sample containing human TCGF, pre-purifying said biological sample to at least five percent as expressed as a weight percent of total protein, and purifying said pre-purified biological sample with at least two reverse-phase HPLC chromatography fractionations each using a C3 to C5 silica gel stationary phase.
Claim 14
Independent Claim
70 words
A method of obtaining human T-cell growth factor (TCGF) purified to at least about 80% by weight of total protein comprising obtaining a biological sample containing human TCGF, pre-purifying said biological sample to at least five percent as expressed as a weight percent of total protein, and purifying said pre-purified biological sample with at least two reverse-phase HPLC chromatography fractionations each using a C3 to C5 silica gel stationary phase.
Claim 15
Dependent Claim
40 words
A method according to claim 12 wherein said pre-purification comprises concentrating said biological sample to substantially reduce its volume, gel filtrating said concentrated biological sample and collecting biologically active fractions therefrom, and fractionating said biologically active fractions with semi-preparative chromatography.
Claim 16
Dependent Claim
42 words
A method according to claim 13 wherein said semi-preparative chromatography uses a C3-C8 silica gel stationary phase, said first reverse-phase HPLC chromatography uses a C3 silica gel stationary phase and said second reverse-phase HPLC chromatography uses a C4 silica gel stationary phase.
Claim 17
Dependent Claim
20 words
A method according to claim 12 wherein said biological sample is obtained from a cultured cell line of lymphocyte derivation.
Claim 18
Independent Claim
123 words
A method of obtaining a human T-cell growth factor (TCGF) purified to at least about 80% by weight of total protein comprising obtaining culture medium of a TCGF-producing cell line subjected to lymphokine-inducing agent; concentrating said culture medium to reduce its volume to at least about 5% of its initial volume; gel filtrating said concentrated culture medium, fractionating said biologically active fractions with semi-preparative reverse-phase HPLC chromatography conducted on a hydrophobic stationary phase, and collecting biologically active fractions therefrom to increase the TCGF concentration to at least five percent as expressed as a weight percent of total protein; and then purifying said pre-purified biological sample with at least two reverse-phase HPLC chromatography fractionations each using a C3 to C5 silica gel stationary phase.
Claim 19
Independent Claim
123 words
A method of obtaining a human T-cell growth factor (TCGF) purified to at least about 80% by weight of total protein comprising obtaining culture medium of a TCGF-producing cell line subjected to lymphokine-inducing agent; concentrating said culture medium to reduce its volume to at least about 5% of its initial volume; gel filtrating said concentrated culture medium, fractionating said biologically active fractions with semi-preparative reverse-phase HPLC chromatography conducted on a hydrophobic stationary phase, and collecting biologically active fractions therefrom to increase the TCGF concentration to at least five percent as expressed as a weight percent of total protein; and then purifying said pre-purified biological sample with at least two reverse-phase HPLC chromatography fractionations each using a C3 to C5 silica gel stationary phase.
Claim 20
Dependent Claim
43 words
A method according to claim 16 wherein said semi-preparative chromatography uses a C3-C8 silica gel stationary phase, said first reverse-phase HPLC chromatography uses a C3 silica gel stationary phase and said second and third reverse-phase HPLC chromatographies use C4 silica gel stationary phases.
Claim 21
Dependent Claim
20 words
A method according to claim 16 wherein said biological sample is obtained from a cultured cell line of tonsil origin.
Claim 22
Dependent Claim
18 words
A method according to claim 16 wherein said biological sample is concentrated with a hollow fiber semipermeable medium.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-06-22 | 1990-12-22 | 1991-06-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-06-22 | 1994-12-22 | 1995-06-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-06-22 | 1998-12-22 | 1999-06-23 | $7700.00 | Window Closed / Lapsed |