Barofold, Inc.

University of Colorado

Nuron Biotech, Inc.

Pressure Biosciences, Inc.

Matthew B Seefeldt

Theodore W Randolph

John Frank Carpenter

Lyndal K Hesterberg

Jeffrey L Cleland

Stephen P Eisenberg

Mary S Rosendahl

Robert Nelson

Amber Haynes Fradkin

Eliana Gomez

Methods for refolding antibodies, particularly monoclonal antibodies, from aggregated and/or denatured preparations by subjecting the antibody preparation to high hydrostatic pressure are provided. Refolded preparations of antibodies produced by the methods described herein are also provided.

High pressure refolding of monoclonal antibody aggregates

The present invention provides methods for reducing and/or evaluating the immunogenic potential of a therapeutic protein preparation. The present invention further provides pharmaceutical compositions of therapeutic proteins and methods of treatment with the same, the compositions having low immunogenic potential and/or improved efficacy. The invention achieves these goals by evaluating therapeutic protein preparations for subvisible protein particulates, which can contribute significantly to the overall immunogenic potential of the protein preparation. Further, by maintaining the content of such subvisible protein particulates to below an immunogenic threshold level, the resulting pharmaceutical composition is less likely to result in a loss of tolerance (e.g., upon repeated administration), thereby improving both the safety and efficacy profile of the therapeutic.

Method for reducing immunogenicity of therapeutic protein compositions

High pressure to treat aggregated interferons, particularly recombinant human interferon-&#x3b2;, to reduce the aggregate content of interferon material. Highly pure, soluble monomeric recombinant interferon-&#x3b2; is prepared in representative embodiments. Multiple strategies may be used in combination that make nonglycosylated IFN-&#x3b2; more amenable to high pressure treatment. It has been found that refolding yields of high pressure treatment can be significantly improved by use of a combination of strategies, including, or example a pre-treatment of the IFN-&#x3b2; that involves solubilizing and then precipitating the protein. This pre-treatment is particularly effective with respect to recombinant IFN-&#x3b2; inclusion bodies recovered from host cells such ascells. According to another strategy, refolding under high pressure is much more effective when the refolding reagent incorporating the IFN-&#x3b2; incorporates a zwitterionic surfactant and/or a cholate salt. When a solubilization and precipitation pre-treatment is used, the effectiveness of the high pressure treatment is further enhanced when the refolding reagent incorporating the protein incorporates a disulfide shuffling chemistry such as cysteine/cystine. According to still yet another strategy, high pressure treatment is more effective when using atypically high treatment pressures. When coupled with purification techniques, these strategies singly or in combination provide a low aggregate or substantially aggregate free, biologically active solution. Biologically active solutions comprising nonglycosylated interferon, said interferon comprising less than about 5 weight percent of protein aggregation has been found to exhibit improved PK/PD characteristics.

High pressure treatment of aggregated interferons

Described herein are methods for the solubilization and proteolytic cleavage of fusion protein aggregates, including autocatalytic fusion proteins, at pressures greater than atmospheric pressure to yield soluble target polypeptides.

High-pressure inclusion body solubilization and protease clipping of recombinant fusion proteins

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Denver, CO, United States of America

Matthew B Seefeldt