US4727137A
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Purified protein having angiogenic activity and methods of preparation
Assignee: Harvard University
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Filing: 1985-09-20
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Granted: 1988-02-23
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Expires: 2005-09-20
Abstract
Abstract A substantially pure protein having angiogenic activity is disclosed. A method for preparing proteins having angiogenic activity from cell culture media is also disclosed. Proteins produced according to the invention are useful in the diagnosis of malignancies, for promoting wound healing, and for other diagnostic and therapeutic purposes.
Inventors
- 👤 Bert L. Vallee
- 👤 James W. Fett
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 16:42
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/16; C12N9/22; A; A61; A61P; A61P9/00; C07; C07K; C07K14/00; C07K14/435; C07K14/475; C07K14/515; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/74; A61K; A61K39/00; Y; Y10; Y10S; Y10S436/00; Y10S436/811; Y10S436/815; Y10S530/00; Y10S530/827; Y10S930/00; Y10S930/01; Y10S930/26
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
91 words
A process for obtaining a substantially pure protein having angiogenic activity from a conditioned cell culture medium, comprising the steps of: treating said medium to remove high molecular weight proteins; binding a portion of said treated medium to a cation exchange matrix; eluting said bound portion from said matrix to produce an eluate; factionating said eluate by high performance liquid chromatography; and collecting the fraction containing said protein, said protein having a purity such that 50 ng of said protein produces a positive angiogenic response in the rabbit cornea implantation test.
Claim 2
Dependent Claim
91 words
A process for obtaining a substantially pure protein having angiogenic activity from a conditioned cell culture medium, comprising the steps of: treating said medium to remove high molecular weight proteins; binding a portion of said treated medium to a cation exchange matrix; eluting said bound portion from said matrix to produce an eluate; factionating said eluate by high performance liquid chromatography; and collecting the fraction containing said protein, said protein having a purity such that 50 ng of said protein produces a positive angiogenic response in the rabbit cornea implantation test.
Claim 3
Dependent Claim
27 words
The process of claim 1 wherein said treating step comprises acidification of the medium followed by centrifugation to separate high molecular weight proteins from the treated medium.
Claim 4
Dependent Claim
27 words
The process of claim 1 wherein said fractionating step comprises reversed phase high performance liquid chromatography using a solvent consisting of isopropanol/acetonitrile/water (5:5:4 v/v/v) containing 0.08% TFA.
Claim 5
Dependent Claim
39 words
The process of claim 1 wherein said fractionating step comprises fractionating said eluate by high performance liquid chromatography on an ion exchange column to produce a second eluate and fractionating said second eluate by reversed-phase high performance liquid chromatography.
Claim 6
Dependent Claim
13 words
The process of claim 1 wherein said cation exchange matrix is carboxymethyl cellulose.
Claim 7
Dependent Claim
50 words
The process of claim 5 wherein said cells are human adenocarcinoma HT-29 cells and said protein is characterized by a molecular weight of approximately 14,193 daltons as determined by amino acid sequence analysis, as isoelectric point greater than 9.5, a lack of mitogenic activity toward 3T3 cells, and ribonuclease activity.
Claim 8
Dependent Claim
22 words
The process of claim 1 wherein said conditioned cell culture medium is filtered to remove cell debris prior to the treating step.
Claim 9
Dependent Claim
39 words
The process of claim 1 further comprising the steps of binding a portion of said fraction containing said protein to a cation exchange matrix to produce a second bound portion; and eluting said second bound portion from said matrix.
Claim 10
Dependent Claim
159 words
A process for obtaining a substantially pure protein having angiogenic activity from a conditioned cell culture medium, comprising the steps of: filtering said medium to remove cell debris; acidifying said filtered medium to precipitate high molecular weight proteins; centrifuging said acidified filtered medium to produce a treated medium; binding a portion of said treated medium to a cation exchange matrix; eluting said bound portion from said matrix to produce a first eluate; fractionating said first eluate by high performance liquid chromatography on an ion exchange column to produce a second eluate; fractionating said second eluate by reversed-phase high performance liquid chromatography; collecting the fraction containing said protein; binding a portion of said fraction containing said protein to a cation exchange matrix to produce a second bound portion; and eluting said second bound portion from said matrix, said protein having a purity such that 50 ng of said protein produces a positive angiogenic response in the rabbit cornea implantation test.
Claim 11
Dependent Claim
159 words
A process for obtaining a substantially pure protein having angiogenic activity from a conditioned cell culture medium, comprising the steps of: filtering said medium to remove cell debris; acidifying said filtered medium to precipitate high molecular weight proteins; centrifuging said acidified filtered medium to produce a treated medium; binding a portion of said treated medium to a cation exchange matrix; eluting said bound portion from said matrix to produce a first eluate; fractionating said first eluate by high performance liquid chromatography on an ion exchange column to produce a second eluate; fractionating said second eluate by reversed-phase high performance liquid chromatography; collecting the fraction containing said protein; binding a portion of said fraction containing said protein to a cation exchange matrix to produce a second bound portion; and eluting said second bound portion from said matrix, said protein having a purity such that 50 ng of said protein produces a positive angiogenic response in the rabbit cornea implantation test.
Claim 12
Independent Claim
81 words
A method for promoting the development of angiogenesis in a mammal, comprising the step of administering to said mammal a therapeutic composition comprising (1) a substantially pure human protein having angiogenic activity and characterized by a molecular weight between 12,500 and 17,500 daltons as determined by SDS-PAGE and an isoelectric point greater than 9.5, the purity being such that 50 ng of said protein produces a positive angiogenic response in the rabbit cornea implantation test, and (2) a pharmaceutically acceptable carrier.
Claim 13
Independent Claim
81 words
A method for promoting the development of angiogenesis in a mammal, comprising the step of administering to said mammal a therapeutic composition comprising (1) a substantially pure human protein having angiogenic activity and characterized by a molecular weight between 12,500 and 17,500 daltons as determined by SDS-PAGE and an isoelectric point greater than 9.5, the purity being such that 50 ng of said protein produces a positive angiogenic response in the rabbit cornea implantation test, and (2) a pharmaceutically acceptable carrier.
Claim 14
Independent Claim
96 words
A method for determining the presence, in a biological sample, of antibodies to a protein having angiogenic activity, comprising the steps of: incubating said biological sample with a composition comprising a substantially pure human protein having angiogenic activity and characterized by a molecular weight between 12,500 and 17,500 daltons as determined by SDS-PAGE and an isoelectric point greater than 9.5, the purity being such that 50 ng of said protein produces a positive angiogenic response in the rabbit cornea implantation test and detecting the formation of antigen-antibody complexes, and therefrom determining the presence of said antibody.
Claim 15
Independent Claim
96 words
A method for determining the presence, in a biological sample, of antibodies to a protein having angiogenic activity, comprising the steps of: incubating said biological sample with a composition comprising a substantially pure human protein having angiogenic activity and characterized by a molecular weight between 12,500 and 17,500 daltons as determined by SDS-PAGE and an isoelectric point greater than 9.5, the purity being such that 50 ng of said protein produces a positive angiogenic response in the rabbit cornea implantation test and detecting the formation of antigen-antibody complexes, and therefrom determining the presence of said antibody.
Claim 16
Dependent Claim
15 words
A method according to claim 10 wherein the protein has the amino acid sequence: ##STR1##
Claim 17
Dependent Claim
15 words
A method according to claim 11 wherein the protein has the amino acid sequence: ##STR2##
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) | 1991-02-22 | 1991-08-24 | 1992-02-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-02-22 | 1995-08-24 | 1996-02-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-02-22 | 1999-08-24 | 2000-02-23 | $7700.00 | Window Closed / Lapsed |