Cetus Corporation

Sidney N Wolfe

Glenn J Dorin

Ze'ev Shaked

Leo S Lin

James W Thomson

Wolfgang H Hanisch

Flint Smith

Robert Weissburg

Amy Lim

Ze've Shaked

John T Davis

A process for recovering highly pure, recombinant IL-2 from transformed microorganisms in which the cells are disrupted; impure recombinant IL-2 is isolated in the form of refractile bodies from the disruptate; the impure IL-2 is dissolved and denatured with at least 6M guanidine hydrochloride containing a reducing agent; the reduced IL-2 is precipitated and resolubilized; the reduced solubilized IL-2 therein is oxidized by a controlled oxidation; the oxidized IL-2 is refolded by reducing the concentration of guanidine hydrochloride in the solution; and the oxidized, refolded IL-2 is further purified by ion exchange chromatography or hydrophobic interaction chromatography and ion exchange chromatography.

Process for recovering purified, oxidized, renatured recombinant

A process is disclosed for the purification of recombinantly produced biologically active proteins in which a solution containing a mixture of materials, including the biologically active protein, is passed through a continuous porous hydrophobic membrane, and the fraction enriched in the biologically active protein is recovered. Hydrophobic proteins such as TNF and recombinant ricin toxin A chain may be purified according to the process. Conditions for enhanced recovery of purified TNF using the process are disclosed. A highly purified TNF comprising 95% or greater TNF as determined by SDS-PAGE analysis, with an endotoxin content of less than 0.1 ng/mg TNF which is substantially free of pyrogens by the USP rabbit pyrogen test at a dosage range of 1.0 to 2.4.times.10.sup.5 U/kg, is obtained.

N-terminal derivatives of tumor necrosis factor purified by microporous

Catalase and pyranose-2-oxidase are stabilized according to the invention by means of chemical treatment which render the enzymes resistant to thermal inactivation or inactivation of glucosone or both. Stabilized catalase crosslinked with diimido esters, such as dimethyl suberimidate and dimethyl adipimidate are claimed. The method for stabilizing catalase against thermal inactivation or glucosone inactivation or both comprising gradually adding a crosslinking agent to a catalase solution and maintaining pH and temperature control is claimed. Stabilized pyranose-2-oxidase amidinated with an amidinating agent such as ethyl acetimidate is claimed. A method for stabilizing catalase against thermal inactivation comprising gradually adding an amidinating agent to a pyranose-2-oxidase solution preferably with pH control is claimed. An improved process for producing glucosone from glucose using stabilized catalase or pyranose-2-oxidase or both according to the invention is also claimed.

Stabilization of enzymes useful in the production of glucosone and other

Method of oxidizing reduced cysteine-containing microbially produced synthetic proteins, such as synthetic IFN-.beta. or synthetic IL-2, in a controlled manner so that the synthetic proteins have the same disulfide bridging as their native counterparts. The oxidation employs o-iodosobenzoate as oxidizing agent and is carried out in an aqueous medium at a pH at least about one-half pH unit less than the pK.sub.a of the cysteines to be oxidized, a synthetic protein concentration of less than about 5 mg/ml, and an oxidizing agent:protein mol ratio that is at least stoichiometric, provided that the oxidizing agent is in excess in the terminal portion of the reaction.

Controlled oxidation of microbially produced cysteine-containing proteins

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El Cerrito, CA, United States of America

Sidney N Wolfe