US4882275A
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Method of purifying endothelial cell growth factors using immobilized heparin
Assignee: Boston Children's Hospital
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Filing: 1986-04-07
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Granted: 1989-11-21
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Expires: 2006-04-07
Abstract
Abstract Endothelial cell growth factor (ECG) from various sources possesses a strong and specific affinity for heparin. This strong affinity of ECG for heaprin enables removal of undesired impurities from a mixture comprising ECG by: (a) contacting immobilized heparin with the mixture to form a heparin-ECG complex; (b) separating uncomplexed mixture from the complex; and (c) contacting the complex with a salt solution of a salt concentration and pH effective to separate the ECG from the heparin. The resulting purified ECG (or fragment thereof) is useful in therapeutics and as an additive for cell culturing. The purified ECG is also useful to raise antibodies that are used in therapeutics and in ECG immunoassays.
Inventors
- 👤 Michael Klagsbrun
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 21:07
CPC Classifications
C; C07; C07K; C07K16/00; C07K16/18; C07K16/22; C07K14/00; C07K14/435; C07K14/475; C07K14/50; C07K14/501; A; A61; A61K; A61K38/00
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
66 words
A method for purifying a crude solution comprising endothelial cell growth factor (ECG) comprising: contacting immobilized heparin with said crude solution to form a complex between said ECG and said heparin; separating uncomplexed solution from said complex; eluting said ECG from said immobilized heparin by contacting said complex with a salt solution of a salt concentration and pH effective to separate said ECG from said heparin.
Claim 2
Independent Claim
66 words
A method for purifying a crude solution comprising endothelial cell growth factor (ECG) comprising: contacting immobilized heparin with said crude solution to form a complex between said ECG and said heparin; separating uncomplexed solution from said complex; eluting said ECG from said immobilized heparin by contacting said complex with a salt solution of a salt concentration and pH effective to separate said ECG from said heparin.
Claim 3
Dependent Claim
22 words
The method of claim 1 wherein said method further comprises obtaining said crude solution by a method comprising digesting tissue comprising ECG.
Claim 4
Dependent Claim
25 words
The method of claim 1 wherein said method further comprises obtaining said crude solution by a method comprising extracting mammalian tissue or cells comprising ECG.
Claim 5
Dependent Claim
33 words
The method of claim 1 wherein said method comprises subjecting a mixture resulting from tissue digestion, or from cell or tissue extraction, to ion exchange chromatography, prior to said contact with immobilized heparin.
Claim 6
Dependent Claim
41 words
The method of claim 1 wherein said contacting, separating, and eluting steps comprise a cycle that is repeated at least once by contacting immobilized heparin with eluate from the previous cycle, separating non-complexed eluate, and eluting ECG from said immoblized heparin.
Claim 7
Dependent Claim
69 words
The method of claim 5 wherein said heparin contacting, separating, and eluting cycle is performed a sufficient number of times to yield an ECG solution purified to at least one of the following standards: (a) substantially free from foreign protein detectable by silver staining; (b) less than 25 ng of foreign protein per gel; or (c) specific cell growth-factor activity at least 10 4 greater than said crude mixture.
Claim 8
Dependent Claim
15 words
The method of claim 6 wherein said method comprises no more than three said cycles.
Claim 9
Dependent Claim
12 words
The method of claim 3 wherein said tissue is mammalian hypothalamus tissue.
Claim 10
Dependent Claim
15 words
The method of claim 3 wherein said tissue is mammalian pituitary, cartilage, or retinal tissue.
Claim 11
Dependent Claim
14 words
The method of claim 3 wherein said tissue is mammalian bone or brain tissue.
Claim 12
Dependent Claim
13 words
The method of claim 3 wherein chondrosarcoma tumor or hepatoma cells are extracted.
Claim 13
Dependent Claim
44 words
The method of claim 1 wherein said crude solution comprises anionic endothelial cell growth factor (AECG) having pI=4.8-5.2, said method comprising eluting said AECG from said immobilized heparin by contacting said complex with a salt solution having a salt concentration of at least 0.8M.
Claim 14
Dependent Claim
44 words
The method of claim 1 wherein said crude solution comprises cationic endothelial cell growth factor (CECG) having pI=9.5-10.0, said method comprising eluting said CECG from said immobilized heparin by contacting said complex with a salt solution having a salt concentration of at least 0.8M.
Claim 15
Dependent Claim
37 words
The method of claim 1 wherein said separating step is performed by contacting said complex with a flowing salt solution, gradually varying the salt concentration of said flowing solution, and collecting salt solution fractions enriched in ECG.
Claim 16
Independent Claim
154 words
A method of obtaining CECG comprising: (a) culturing hepatoma cells; (b) extracting the cells; (c) subjecting said extract to cation exchange chromatography; (d) contacting immobilized heparin with eluant from said ion exchange chromatography to form a complex between said CECG and said heparin; (e) separating uncomplexed cation exchange eluate from said heparin; (f) eluting said CECG from said immobilized heparin by contacting said complex with a salt solution of salt concentration and pH effective to separate said heparin from said CECG; (g) recovering a CECG-containing solution; and (h) contacting immobilized heparin with said CECG-containing solution to form a complex between said CECG and said immobilized heparin; (i) separating uncomplexed solution from said immobilized heparin; and (j) repeating steps (f) and (g) until the CECG-containing solution recovered has substantially no protein detectable by silver stain, other than CECG, and the specific cell-growth activity of said solution is at least 1 unit per ng of protein.
Claim 17
Independent Claim
154 words
A method of obtaining CECG comprising: (a) culturing hepatoma cells; (b) extracting the cells; (c) subjecting said extract to cation exchange chromatography; (d) contacting immobilized heparin with eluant from said ion exchange chromatography to form a complex between said CECG and said heparin; (e) separating uncomplexed cation exchange eluate from said heparin; (f) eluting said CECG from said immobilized heparin by contacting said complex with a salt solution of salt concentration and pH effective to separate said heparin from said CECG; (g) recovering a CECG-containing solution; and (h) contacting immobilized heparin with said CECG-containing solution to form a complex between said CECG and said immobilized heparin; (i) separating uncomplexed solution from said immobilized heparin; and (j) repeating steps (f) and (g) until the CECG-containing solution recovered has substantially no protein detectable by silver stain, other than CECG, and the specific cell-growth activity of said solution is at least 1 unit per ng of protein.
References Cited (Backward Citations)
No citation records indexed.
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