High pressure treatment of aggregated interferons
View Patent ↗High pressure to treat aggregated interferons, particularly recombinant human interferon-β, to reduce the aggregate content of interferon material. Highly pure, soluble monomeric recombinant interferon-β is prepared in representative embodiments. Multiple strategies may be used in combination that make nonglycosylated IFN-β 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-β that involves solubilizing and then precipitating the protein. This pre-treatment is particularly effective with respect to recombinant IFN-β inclusion bodies recovered from host cells such as E. coli cells. According to another strategy, refolding under high pressure is much more effective when the refolding reagent incorporating the IFN-β 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.
1. A method of preparing a composition comprising nonglycosylated interferon, comprising the steps of:
a) solubilizing the aggregated interferon;
b) precipitating the solubilized interferon;
c) incorporating the precipitated interferon into a refolding admixture comprising 0.05 weight percent to 5.0 weight percent of a zwitterionic surfactant and having an absence of a chaotrope or denaturing surfactant; and
d) applying greater than 3000 bars of pressure to the refolding admixture incorporating the interferon effective to refold at least a portion of the interferon to form a composition comprising nonglycosylated interferon,
wherein the resulting composition comprises less than about 5 weight percent of protein aggregation.
2. A method of preparing a composition comprising nonglycosylated interferon, comprising the steps of:
a) solubilizing the aggregated interferon;
b) precipitating the solubilized interferon;
c) incorporating the precipitated interferon into a refolding admixture comprising a zwitterionic surfactant and a disulfide shuffling reagent wherein the zwitterionic surfactant is provided in an amount between 0.05% to 5.0% of the refolding admixture and wherein the refolding admixture has an absence of a chaotrope or denaturing surfactant; and,
d) applying greater than 3000 bars of pressure to the refolding admixture incorporating the interferon effective to refold at least a portion of the interferon;
wherein the resulting composition comprises less than about 5 weight percent of protein aggregation.
3. A method of preparing a composition comprising nonglycosylated interferon to minimize the presence of inclusion bodies and aggregated nonglycosylated interferon, comprising the steps of:
a) solubilizing the aggregated interferon in an aqueous medium;
b) extracting the solubilized interferon into an organic solvent;
c) precipitating the solubilized interferon;
d) incorporating the precipitated interferon into a refolding admixture comprising 0.05 weight percent to 5.0 weight percent of a zwitterionic surfactant and having an absence of a chaotrope or denaturing surfactant; and,
e) applying greater than 3000 bars of pressure to the refolding admixture incorporating the interferon effective to refold at least a portion of the interferon; wherein the resulting composition comprises less than about 5 weight percent of protein aggregation.
4. The method of claim 3 , wherein the solubilized interferon is precipitated from the organic solvent by addition of a solvent selected from methanol, ethanol, isopropanol, and acetonitrile thereto.
5. The method of claim 3 , wherein the refolding admixture further comprises a disulfide shuffling reagent.
6. The method of claim 3 , wherein the yield of nonglycosylated interferon is at least about 30%.
7. The method of claim 3 , wherein the yield of nonglycosylated interferon is at least about 40%.