Genentech, Inc.

Stuart E Builder

David Reifsnyder

John R Ogez

Philip M Lester

Roger Hart

William F Bennett

Larry A Gatlin

Charles V Olson

Robert D Van Reis

William R Arathoon

David A Vetterlein

Anthony S Lubiniecki

Eric J Patzer

Billie J Marks

Vincent R Anicetti

Refolding of polypeptides like recombinant insulin-like growth factor

A composition is provided comprising about 0.1 to 15 mg/mL of a polypeptide in a buffer having a pH of about 7-12 comprising about 5-40% (v/v) of an alcoholic or polar aprotic solvent, about 0.2 to 3M of an alkaline earth, alkali metal, or ammonium salt, about 0.1 to 9M of a chaotropic agent, and about 0.01 to 15 .mu.M of a copper or manganese salt. The buffer is suitably used in a method for refolding improperly folded polypeptides.

Refolding of polypeptides

A method is described for isolating an exogenous polypeptide in a non-native conformation from cells, such as an aqueous fermentation broth, in which it is prepared comprising contacting the polypeptide with a chaotropic agent and preferably a reducing agent and with phase-forming species to form multiple aqueous phases, with one of the phases being enriched in the polypeptide and depleted in the biomass solids and nucleic acids originating from the cells. Preferably, the method results in two aqueous phases, with the upper phase being enriched in the polypeptide.

Aqueous multiple-phase isolation of polypeptide

Refolding of misfolded insulin-like growth factor-I

A process is provided for selectively separating a polypeptide of interest from components of differing hydrophobicity in a mixture comprising the steps of:

Method for purifying polypeptides

A process for purifying IGF-I from variants of IGF-I is provided. In this process a mixture containing IGF-I and its variants is loaded onto a reversed-phase liquid chromatography column and the IGF-I is eluted from the column with a buffer at a pH of about 6-8. The buffer contains an alcoholic or polar aprotic solvent at a concentration of about 20-30% (v/v). This process can be preceded by a hydrophobic-interaction chromatography step.

Purification of insulin-like growth factor

A method for purifying a recombinant protein, e.g. human tissue plasminogen activator (t-PA) from a corresponding endogenous protein of the host cell, e.g. Chinese hamster t-PA, is disclosed. The method comprises contacting a fluid containing the recombinant protein with antibodies specifically binding the corresponding endogenous protein. Monoclonal antibodies directed against and an assay for Chinese hamster t-PA are also disclosed.

Method of purifying recombinant proteins from corresponding host cell

A method of producing biologically active human tissue plasminogen activator in suspension culture is provided wherein recombinant Chinese hamster ovary cells are cultured and certain components detrimental to recovery and biological activity are removed as a cell-free filtrate by cross-flow filtration. The human tissue plasminogen activator is recovered from the culture medium.

Process for producing biologically active plasminogen activator in

Disclosed are novel, stable pharmaceutically acceptable compositions containing human tissue plasminogen activator, featuring, for example, an argininium ion containing buffer as a component. Also disclosed are associated means and methods for preparing and using such compositions in various forms.

Stabilized human tissue plasminogen activator compositions

Disclosed are novel, stable pharmaceutically acceptable compositions containing human tissue plasminogen activator, featuring, for example, an argininum ion containing buffer as a component. Also disclosed are associated means and methods for preparing and using such compositions in various forms.

A process for isolating and purifying refractile proteins from host cell culture including treating the host cell culture with a buffered solution to solubilize host protein, leaving refractile protein insoluble, treating the insoluble fraction with a strongly denaturing solution containing a reducing agent to solubilize the refractile protein, and renaturing the refractile protein.

Purification and activity assurance of precipitated heterologous proteins

A process for purifying heterologous proteins from higher molecular weight components, including dissolving the heterologous protein in a strong denaturing solution and removing the higher molecular weight components using a molecular sieve or high speed centrifugation.

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Belmont, CA, United States of America

Stuart E Builder