Massachusetts Institute of Technology

University of Iowa Research Foundation

Rohm and Haas Company

Children's Medical Center Corporation

Charles L Cooney

Robert Samuel Langer

Robert J Linhardt

Ramnath Sasisekharan

Daniel L Lohse

Parrish M Galliher

Ram Sasisekharan

Ganesh Venkataraman

Eugene J Schaefer

Marsha A Moses

Richard C Willson, Iii

Joseph J Zimmermann

Kelley Wilson Moremen

Viswanathan Sasisekharan

Chee Kong Lai

Elizabeth M Robinson

David P Hoult

Matthew A Nugent

Peter Hanson

James C Leung

Ratnaleela Bobba

Arthur Grant

Steffen Ernst

Gerald Fitzgerald

Michael D Klein

Ranganathan Godavarti

Margaret M Flanagan

Eric Bulot

Joseph J Zimmerman

Jean L Romette

G K Raju

Joaquim M Cabral

A single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity is disclosed herein. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. Mono-clonal antibodies to the three heparinases are also described and are useful for detection, isolation and characterization of the heparinases.

Purification, composition and specificity of Heparinase I, II, and III from Flavobacterium heparinum

A purified heparinase I, II and III free of lyase activity and each having a molecular weight of 42,800 84,100, 70,800, respectively, are produced by culturing Flavobacterium heparinum. The kinetic properties of the heparinases have been determined as well as the conditions to optimize their activity and stability.

Purification of Heparinase I, II and III from Flavobacterium heparinum

The invention provides systems and methods for analyzing mixtures and, particularly during processing of the mixtures. The invention provides systems and methods for analyzing mixtures and, particularly, for analyzing mixtures during processing. The methods and systems utilize fluorescence to non-invasively analyze one or more components of the mixture. The analysis may provide a variety of compositional information such as the chemical identity of components, the concentration of components, the uniformity of the mixture and other information. A number of different types of processing equipment can be configured to provide the analysis and, accordingly, the analysis may be performed during a number of processing operations. The system and methods are particularly useful in processing mixtures which are preferably homogeneous blends, such as pharmaceutical products.

Systems and methods for analyzing mixtures using fluorescense

Modified heparinases having altered binding specificity and activity are provided. Isolated nucleic acids encoding the same as as vectors and host cells are provided. Methods for using the modified heparinases are also provided.

Rationally designed polysaccharide lyases derived from heparinase I

The cloning of the heparinase gene from Flavobacterium Heparinum using the polymerase chain reaction is described. The Open Reading Frame (ORF) corresponded to 1152 base pairs encoding a precursor protein of MW 43,800 daltons. The amino acid sequence reveals a 20-residue leader peptide. The gene was expressed in two expression systems in E. coli.

Heparinase gene from flavobacterium heparinum

A computer-implemented process determines candidate three-dimensional shapes of ring-shaped molecules through an exhaustive search of combinations of torsion angles of the bonds of the ring, given known bond lengths and bond angles for the bonds between atoms of the rings, a range of torsion angles and a resolution. Candidate combinations of the torsion angles are selected according to the range and resolution and coordinates of the atoms of the ring are determined for each candidate combination. It is then determined whether each candidate combination defines a physically possible ring. Indications of such candidate combinations are recorded. Candidate combination may then be selected from those recorded so that coordinates of atoms in candidate repetitive structures may be determined.

Computer-implemented process and computer system for estimating the

A single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity is disclosed herein. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. Monoclonal antibodies to the three heparinases are also described and are useful for detection, isolation and characterization of the heparinases.

Purification, composition and specificity of heparinase I, II, and III

Pharmaceutical compositions for delivering an effective dose to a desired site of a heparinase. These compositions are based on the discovery that heparinase alone can inhibit angiogenesis. The effective dosage is dependent not only on the heparinase, but also on the method and means of delivery, which can be localized or systemic. For example, in some applications, as in the treatment of psoriasis or diabetic retinopathy, the inhibitor is delivered in a topical ophthalmic carrier. In other applications, as in the treatment of solid tumors, the inhibitor is delivered by means of a biodegradable, polymeric implant.

Method for inhibiting angiogenesis using heparinase

Purification of heparinase I, II, and III from Flavobacterium heparinum

The present invention is an improved process for purification of active heparinase and heparinase like enzymes from Gram negative organisms, in particular, Flavobacterium heparinum. The primary advantage of the process is the fact that it allows large scale processing and high yield of heparinase. The heparinase is released from the periplasmic space of the organism by osmotic shock treatment, first into an osmotically stabilized medium, secondly into a non-stabilized medium having a pH of approximately pH 6.0 and 8.6 with subsequent release into a second non-stabilized medium containing approximately 0.15 M sodium chloride, followed by fractionation by cation exchange chromatography, and, optionally, electropheresis or gel filtration chromatography. Two proteins having heparinase activity have been isolated, one having a molecular weight of approximately 42,000 Daltons and the other having a molecular weight of 65,000 to 75,000 Daltons. Also described is the construction of a library for screening for the genes encoding the proteins having heparinase activity and two assay for detecting organisms producing heparinase, either F. heparinum or genetically engineered organisms.

Large scale method for purification of high purity heparinase from

In the membrane filtration of a liquid cell culture medium, superior flux rates and product recovery are obtained when a first charged particulate material and optionally a second charged particulate material bearing a charge opposite that of the first material are sequentially added to the medium prior to filtration.

Membrane filtration of cell culture media with charged particles

Apparatus for measuring L-glutamine in a liquid sample includes a membrane on which are immobilized the enzymes glutaminase and glutamate oxidase, whereby any said L-glutamine in the sample is acted upon by the glutaminase to form glutamic acid, the glutamic acid being acted upon by the glutamate oxidase to form an enzymatic reaction product, the membrane being operatively associated with a sensor capable of sensing the enzymatic reaction product or a compound or element consumed or liberated in the formation or degradation thereof.

L-glutamine sensor

A process for forming a purified solute from an aqueous solution is provided whereby a mixture of an extractant, a hydrate former and the aqueous solution is first formed. The hydrate former forms a hydrate with water while the solute does not form a hydrate. The extractant takes up the solute from the aqueous solution. The mixture is subjected to a temperature and pressure sufficient to form the solid hydrate of the hydrate former, an aqueous solution of the solute and a portion comprising the extractant containing the solute. The solute is recovered from the portion comprising the extractant and the solute.

Use of hydrates for aqueous solution treatment

Heparinase is produced by growing the bacterium, Flavobacterium heparinum in an improved defined medium consisting of a carbon source, two or more amino acids and several salts in the absence of protein. The carbon source concentration is specifically kept below a certain level to promote improved heparinase production. The sulfate source concentration is also specifically kept below a certain level to promote improved heparinase synthesis. Heparinase can be produced in this medium with or without the addition of an inducer compound.

Process for producing heparinase

There is disclosed a heparin product obtained by degradation of heparin with heparinase from Flavobacterium heparinum (ATCC 13125) or mutants thereof having activity to reduce the coagulation activity of factor X while not effecting the coagulation activity of thrombin.

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Brookline, MA, United States of America

Charles L Cooney