# US 2014/0193357 A1 · Derivatives of recombinant proteins, homo-multimers of granulocyte colony-stimulating factor and method of preparation thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0193357 A1 - Document ID: US20140193357A1 - Status: Published application - Kind: A1 - Application number: 14/237,807 - Filed: 2011-08-09 - Published: 2014-07-10 - Earliest claimed: 2011-08-09 - Assignees: UAB Profarma - Applicants: Uab Profarma - Inventors: Edita Mistiniene; Jonas Henrikas Pesliakas; Milda Pleckaityte; Gintautas Zvirblis - Web page: https://patentgrep.com/patent/US20140193357A1 ## CPC - C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF - C07K 14/56: IFN-alpha - C07K 2319/00: Fusion polypeptide - C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin - 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) ## Abstract The invention relates to derivatives of recombinant proteins, comprising homo-multimers of genetically fused recombinant biologically active protein monomer units, connected via selected peptide linker moiety; and the method of preparation thereof. Derivative of recombinant protein is preferably dimer of human granulocyte colony-stimulating factor, characterised by increased circulation time in vivo. ## Claims ### Claim 1 Derivatives of recombinant proteins, comprising homo-multimers of genetically fused recombinant biologically active protein monomer units, connected via suitable peptide linker moiety, wherein each protein monomer unit comprises an amino acid sequence of native biologically active protein or essentially the same sequence of at least 95% identity, said linker moiety being represented by amino acid sequence, selected from the group, consisting of (S-G4)n-S, wherein n=2-7, and SGLEA-(EAAAK)m-ALEA-(EAAAK)m-ALEGS, wherein m=2-8. ### Claim 2 (depends on 1) Derivatives of recombinant proteins according to claim 1, wherein the suitable linker moiety comprises an amino acid sequence, consisting of (S-G4)n-S, wherein n=2-7, said amino acid sequence being preferably selected from the group, consisting of SEQ ID No:1, SEQ ID No:2, SEQ ID No:3 and SEQ ID No:4. ### Claim 3 (depends on 1) Derivatives of recombinant proteins according to claim 1, wherein the suitable linker moiety comprises an amino acid sequence, consisting of SGLEA-(EAAAK)m-ALEA-(EAAAK)m-ALEGS, wherein m=2-8, preferably represented by SEQ ID No:5. ### Claim 4 (depends on 1) Derivatives of recombinant proteins according to claim 1, wherein said homo-multimer contains single chain of at least two protein monomer units and said biologically active protein monomer unit is selected from the group, consisting of cytokines, growth factors and hormones, wherein cytokines comprise interleukines, colony stimulating factors or interferons, preferably granulocyte colony stimulating factor (G-CSF) or interferon. ### Claim 5 (depends on 1) The derivative of recombinant protein according to claim 1, wherein said biologically active protein monomer unit is recombinant interferon alpha-2a, homo-multimer is dimer, preferably having amino acid sequence SEQ ID No: 6. ### Claim 6 (depends on 1) The derivative of recombinant protein according to claim 1, wherein said biologically active protein monomer unit is recombinant human granulocyte colony-stimulating factor (rhG-CSF). ### Claim 7 (depends on 2) Homo-multimers of granulocyte colony-stimulating factor, comprising of least two genetically fused recombinant granulocyte colony-stimulating factor protein monomers, connected via suitable linker moiety as defined in claim 2. ### Claim 8 (depends on 6) Homo-multimers of granulocyte colony-stimulating factor according to claim 6, which is dimer of granulocyte colony-stimulating factor, comprising amino acid sequence, selected from the group, consisting of SEQ ID No:?, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10 and SEQ ID No: 11. ### Claim 9 (depends on 6) Homo-multimers of granulocyte colony-stimulating factor according to claim 6, characterised by increased circulation time in vivo. ### Claim 10 (depends on 6) Homo-multimers of granulocyte colony-stimulating factor according to claim 6 for use in therapy. ### Claim 11 (depends on 6) Homo-multimers of granulocyte colony-stimulating factor according to claim 6 for the manufacture of the medicament for treating of diseases and conditions in indications where monomeric granulocyte colony-stimulating factor protein is used, such as neutropenia. ### Claim 12 (depends on 6) Pharmaceutical composition comprising an therapeutically effective amount of homo-multimer of granulocyte colony-stimulating factor according to claim 6 in combination with pharmaceutically acceptable carrier, diluent, excipient and/or auxiliary substances. ### Claim 13 (depends on 5) A method for preparation of homo-multimers of granulocyte colony-stimulating factor according to claim 5, comprising the steps of: a) cultivation of microorganism of producer which host cell comprise a nucleotide sequence encoding the target genetically fused protein in a suitable culture medium under conditions, permitting expression of the target protein; b) lysing the microorganism of producer and separating the fraction of insoluble target proteins; c) solubilizing of the fraction of insoluble proteins; d) renaturation of the target protein; e) chromatographic purification of target protein, characterised in that solubilising of the fraction of insoluble proteins is performed in a buffer system containing urea or guanidine hydrochloride as chaotropic agent in the presence of reducing agent; renaturation is oxidizing renaturation of the target protein from inclusion bodies in the presence of reducing agent, such as reduced glutathione or dithiothreitol and oxidizing agent such as oxidized glutathione, or metal ions in solution, or immobilized metal ions on chromatography support, and purification by chromatography consisting of the purification by metal ions affinity chromatography and further additional purification by at least one ion exchange and/or gel-filtration chromatography, or by combination of the ion-exchange chromatography with sequential use of anion-exchange and cation-exchange chromatography. ### Claim 14 (depends on 13) The method according to claim 13, characterised in that said solubilising of the fraction of insoluble proteins is performed in the presence of dithiothreitol (DTT) in a buffer system containing urea as chaotropic agent; and said renaturation of the target protein is performed by any of procedure: a) in the presence of CuSO4 by mixing at ambient temperature for 1-3 hours, preferably 2 hours, and stopping refolding by adding EDTA solution at pH 5.4±0.2 to final concentration of 5-15 mM, preferably 10 mM.; or b) in the presence of a pair reduced (GSH)/oxidized (GSSG) glutathione taken in the concentration ratio 5-10:1 by mixing at least for 12 hours; or c) in the presence of 0.08-0.12 mM GSSG by mixing at least for 12 hours; or d) in the presence of GSSG in such manner that final molar ratio in solution of DTT/GSSG is 1:1.5-10 by mixing at least for 12 hours. ### Claim 15 (depends on 6, 13) Dimer of granulocyte colony-stimulating factor according to claim 6, produced by the method according to claim 13.