# US 5,702,699 A · Process for the recovery of lipophilic proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,702,699 A - Document ID: US5702699A - Status: Granted - Kind: A - Application number: 08/474,768 - Filed: 1995-06-07 - Granted: 1997-12-30 - Earliest claimed: 1982-09-19 - Assignees: Cetus Corporation - Inventors: Wolfgang H. Hanisch; Peter Fernandes - Web page: https://patentgrep.com/patent/US5702699A ## CPC - C07K 14/565: IFN-beta - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract An improved process for recovering and purifying lipophilic recombinant proteins such as human .beta.-interferon from their hosts involves concentrating the bacteria; disrupting the cell wall and solubilizing the protein into an aqueous medium with an appropriate solubilizing agent; extracting the protein from the aqueous medium with 2-butanol, 2-methyl-2-butanol or mixtures thereof; precipitating and isolating the protein from the alcohol phase; purifying the protein by chromatography and diafiltering the protein against distilled water or aqueous solutions of ethanol or glycerol at a pH of about 12. Therapeutic formulations of such purified proteins with stabilizers contain SDS levels reduced to less than 10 p.p.m. ## Claims ### Claim 1 A pharmaceutical composition comprising a therapeutically effective amount of unglycosylated recombinant human interferon-.beta. (IFN-.beta.) dissolved in a non-toxic, therapeutically compatible aqueous-based carrier medium comprising as a stabilizer at pH of 6.8 to 7.8 wherein said stabilizer comprises human serum albumin wherein the composition is produced by a process comprising: 1) recovering the recombinant human IFN-.beta. from a bacterial host transformed to produce it; 2) isolating and purifying the recombinant human IFN-.beta. in a solution containing a solubilizer; 3) adjusting the pH of the solution to about 10.5 to 12.5; 4) diafiltering the solution at a pH of about 10.5 to 12.5 against pure water or water/aliphatic alcohol mixtures adjusted to a pH of about 11 to remove free or IFN-.beta.-bound solubilizer. 5) adding an aqueous-based carrier medium of a pH of about 10.5 to 12.5 to the diafiltered solution containing the recombinant human IFN-.beta.; 6) maintaining the resulting solution at a pH of about 10.5 to 12.5 for 1 to 15 minutes; 7) lowering the pH of the resulting solution to between about 6.8 and 7.8; wherein the pharmaceutical composition contains less than 10 ppm of solubilizer when the solubilizer is sodium dodecyl sulfate, and wherein the stabilizer in the aqueous-based carrier medium is effective to stabilize the recombinant human IFN-.beta. against denaturation and loss of biological activity and to prevent precipitation of the recombinant human IFN-.beta. from aqueous-based carrier medium. ### Claim 2 (depends on 1) The composition of claim 1 wherein the human serum albumin is present in concentration range of about 0.5 to 10% (w/v). ### Claim 3 (depends on 1) The composition of claim 1 wherein the stabilizer is a mixture of human serum albumin and dextrose. ### Claim 4 (depends on 1) The composition of claim 1 wherein the solubilizer is sodium dodecyl sulfate (SDS). ### Claim 5 (depends on 1) The composition of claim 1 wherein the therapeutically effective amount of recombinant human IFN-.beta. is in the range of 0.1 to 1.0 mg recombinant human IFN-.beta./mL of aqueous-based carrier. ### Claim 6 (depends on 1) The composition of claim 1 wherein the level of sodium dodecyl sulfate is in the range of about 2 to 6 ppm. ### Claim 7 (depends on 1) The composition of claim 1 wherein the aqueous-based carrier medium is selected from the group consisting of physiological saline solution, distilled water, Ringer's solution and Hank's solution. ### Claim 8 (depends on 1) The composition of claim 1 wherein the recombinant human IFN-.beta. is ser.sub.17 IFN-.beta.. ### Claim 9 (depends on 1) The composition of claim 1 wherein the specific activity of the recombinant human INF-.beta. is in the range of 1-2.times.10.sup.7 IU/mg recombinant human INF-.beta.. ### Claim 10 (depends on 1) The composition of claim 1 wherein the bacterial host is E. coil bacteria. ### Claim 11 (depends on 3) The composition of claim 3 wherein the human serum albumin and the dextrose are each present in the amount of 1.25% (w/v). ### Claim 12 (depends on 8) The composition of claim 8 wherein the composition is lyophilized. ### Claim 13 (depends on 9) The composition of claim 9 wherein the therapeutically effective amount of recombinant human INF-.beta. is in the range of 0.1 to 1.0 mg recombinant human IFN-.beta./mL of aqueous-based carrier, the stabilizer is human serum albumin, the solubilizer is sodium dodecyl sulfate present at a level of 2 to 6 ppm, and the aqueous-based carrier is selected from the group consisting of physiological saline solution, distilled water, Ringer's solution and Hank's solution. ### Claim 14 (depends on 12) The composition of claim 12 which is reconstituted in an aqueous-based carrier medium at a concentration equivalent to the prelyophilization concentration, wherein the reconstituted concentration of recombinant human IFN-.beta. is therapeutically effective and the recombinant human IFN-.beta. is substantially fully recovered in soluble and biologically active form.