Chiron Corporation

Cetus Corporation

Other

Michael G Kunitani

Kirston E Koths

Kenneth L Wilson

James W Thomson

Hugh Parker

Wolf Hanisch

An D Tran

Gavin D Dollinger

Robert L Cunico

Robert F Halenbeck

Flint Smith

Methods for improving purification and quantification of platelet derived growth factor (PDGF) proteins having structural heterogeneity are provided. Preparation of substantially pure isoforms of these proteins is achieved using TSK sulfopropyl cation exchange chromatography and reverse phase high performance liquid chromatography. A reverse charged capillary zone electrophoresis method enables quantification of substantially pure isoforms of these proteins resulting from endoproteolytic post-translational modifications. Compositions of the invention are substantially purified isoforms of secreted PDGF proteins having structural heterogeneity, more particularly purified intact, single-clipped, and double-clipped isoforms of recombinant PDGF-BB. Pharmaceutical compositions comprising at least one of these substantially purified recombinant PDGF isoforms and methods for their use in promoting wound healing are also provided.

Analysis and separation of platelet-derived growth factor proteins

Therapeutic uses of resolved intact or clipped native-sequence PDGF-BB

A process for recovering microbially produced IL-2 in a highly pure form from the cellular material of the microorganisms that produced it comprising: disrupting the-cell membranes of the microorganisms; extracting the disruptate with a chaotropic agent, such as urea, that selectively extracts microbial proteins from the cellular material; solubilizing the IL-2 in the solid phase of the extraction mixture with an aqueous solution of a solubilizing agent, such as SDS, containing a reducing agent; and separating the IL-2 from the resulting solution by an optional extraction with 2-butanol or 2-methyl-2-butanol followed by gel filtration chromatography, oxidizing the IL-2 and purifying the oxidized IL-2 by RP-HPLC.

Compositions containing reduced, microbially produced interleukin-2

A process for recovering microbially produced IL-2 in a highly pure form from the cellular material of the microorganisms that produced it comprising: disrupting the cell membranes of the microorganisms; extracting the disruptate with a chaotropic agent, such as urea, that selectively extracts microbial proteins from the cellular material; solubilizing the IL-2 in the solid phase of the extraction, mixture with an aqueous solution of a solubilizing agent, such as SDS, containing a reducing agent; and separating the IL-2 from the resulting solution by an optional extraction with 2-butanol or 2-methyl-2-butanol followed by gel filtration chromatography, oxidizing the IL-2 and purifying the oxidized IL-2 by RP-HPLC.

Purified, reduced recombinant interleukin-2 compositions

A high angle light scattering detector using classical Rayleigh scattering. A high intensity arc light source, filtered to leave only one wavelength illuminates a flow cell. Through the flow cell, very small particles such as biological proteins flow in solution after separation by HPLC or some other means. A UV detector generates data regarding the weight concentration of the eluting particles and a scattered light detector collecting scattered light at angles of approximately 90.degree. generates a scattered light signal. The incident light intensity is also measured. The average molecular weight is then computed using the scattered and incident light data the weight concentration data and a simplified mathematical relationship from which the size factor P and the viral coefficients have been eliminated.

HPLC light scattering detector for biopolymers

A high angle light scattering detector using classical Rayleigh scattering. A high intensity arc light source, filtered to leave only one wavelength illuminates a flow cell. Through the flow cell, very small particles such as biological proteins flow in solution after separation by HPLC or some other means. A UV detector generates data regarding the weight concentration of the eluting particles and a scattered light detector collecting scattered light at angles of approximately 90.degree. generates a scattered light signal. The incident light intensity is also measured. The average molecular weight is then computed using the scattered and incident light data, the weight concentration data and a simplified mathematical relationship from which the size factor P and the viral coefficients have been eliminated.

A process for recovering substantially pure rIL-2 from transformed microorganisms in which the cells are disrupted, impure insoluble rIL-2 is separated from the bulk of the cellular components, the separated impure rIL-2 is solubilized and partially purified in a reduced form, the solubilized rIL-2 is oxidized, the oxidized rIL-2 is purified to clinically acceptable levels, and the oxidized, purified IL-2 is denatured by placing it into a solution of a chaotropic agent, solids are removed from the solution and rIL-2 is renatured from the solution.

Process for recovering microbially produced interleukin-2

A process for recovering microbially produced IL-2 in a highly pure form from the cellular material of the microorganisms that produced it comprising: disrupting the cell membranes of the microorganisms; extracting the disruptate with a chaotropic agent, such as urea, that selectively extracts microbial proteins from the cellular material; solubilizing the IL-2 in the solid phase of the extraction mixture with an aqueous solution of a solubilizing agent, such as SDS, containing a reducing agent; and separating the IL-2 from the resulting solution by an optional extraction with 2-butanol or 2-methyl-2-butanol followed by gel filtration chromatography, oxidizing the IL-2 and purifying the oxidized IL-2 by RP-HPLC.

Growing community of inventors

San Rafael, CA, United States of America

Michael G Kunitani