Purified protein having angiogenic activity and methods of preparation
AssigneePresident and Fellows of Harvard College
InventorsBert L. Vallee, James W. Fett
Life of this patent
Estimating the term…
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.
Claims13
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.
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.
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.
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.
Dependencies are inferred from the claim text.