# US 12,215,139 B2 · Preparation method and application of high-purity plant-derived recombinant human serum albumin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,215,139 B2 - Document ID: US12215139B2 - Status: Granted - Kind: B2 - Application number: 18/518,227 - Filed: 2023-11-22 - Published: 2024-08-08 - Granted: 2025-02-04 - Earliest claimed: 2023-11-22 - Assignees: Wuhan Healthgen Biotechnology Corp - Applicants: Wuhan Healthgen Biotechnology Corp. - Inventors: Daichang Yang; Quan Zhan; Jiquan Ou; Wenhui Yu; Zhijie Qin - Web page: https://patentgrep.com/patent/US12215139B2 ## CPC - C07K 14/765: Serum albumin, e.g. HSA - A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca - A61K 47/643: {Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]} - A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) - C07K 1/18: Ion-exchange chromatography - C07K 1/20: Partition-, reverse-phase or hydrophobic interaction chromatography - 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) - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The present invention provides a method for preparing high-purity recombinant human serum albumin and its applications as a drug and biopharmaceutical excipient. The recombinant human serum albumin from Oryza sativa (OsrHSA) prepared by the present invention can be replaced of plasma-derived human serum albumin for uses of the clinic and pharmaceutical excipients in term of safe and effective. Furthermore, it can effectively maintain the biological activity of biological macromolecules. ## Claims ### Claim 1 A method for preparing high-purity recombinant human serum albumin from Oryza sativa (OsrHSA), comprising the following steps of: (a) preparing a crude or unpurified extract of OsrHSA; (b) subjecting the crude extract of OsrHSA to cation exchange chromatography (A) comprising a cation exchange column, whereby OsrHSA binds to the cation exchange column, (c) providing: a first washing buffer comprising between about 10-20% absolute isopropanol by volume, an equilibration buffer I comprising between about 0-10% absolute isopropanol by volume, and an equilibration buffer II comprising between about 5-15% absolute isopropanol by volume, (d) washing the cation exchange column with the first washing buffer, then washing the column with equilibration buffer I, then washing the column with equilibration buffer II, (e) eluting OsrHSA from the cation exchange chromatography, thereby obtaining a primary product I; wherein the cation exchange chromatography comprises a cationic/hydrophobic composite resin; (f) subjecting the primary product I to anion exchange chromatography B comprising an anion exchange column, to obtain an intermediate product II, wherein the anion exchange chromatography B comprises a composite resin-comprising a matrix having a pore size of about 60 μm and a flow rate of between about 150-750 cm/h; and the parameters of the anion exchange chromatography B comprise: (i) adding or loading onto to the anion exchange column a loading sample under conditions comprising: pH 8.0, conductivity between about 1 to 5 mS/cm; (ii) adding or loading onto to the anion exchange column a washing buffer under conditions comprising: pH 8.0, conductivity between about 9 to 9.5 mS/cm; (iii) adding or loading onto to the anion exchange column an elution buffer comprising: pH 8.0, conductivity between about 30 to 50 mS/cm; thereby eluting off the anion exchange column the intermediate product II, wherein the anion exchange chromatography B comprising an anion exchange column comprises use of the following buffers: an equilibration buffer comprising: between about 10 to 20 mM phosphate buffer (PB), between about 7.9 to 8.1 pH, conductivity between about 1 to 3 mS/cm; a re-equilibration buffer comprising: the same as the equilibration buffer; a second washing buffer comprising: between about 10 to 20 mM PB, between about 150 to 170 mM NaCl, between about 7.9 to 8.1, between about 9 to 9.5 mS/cm conductivity; an elution buffer comprising: between about 10 to 20 mM PB, between about 420 to 460 mM NaCl, between about pH 7.9 to 8.1, between about 41 to 43 mS/cm conductivity; and (g) subjecting the intermediate product II to hydrophobic chromatography (C) comprising loading the intermediate product II in a chromatography buffer onto a hydrophobic column, washing the hydrophobic column by loading the second washing buffer onto the hydrophobic column, and eluting the high-purity OsrHSA by loading an eluting buffer onto the hydrophobic column, thereby generating the high-purity OsrHSA, and the hydrophobic chromatography comprises use of a hydrophobic resin. ### Claim 2 (depends on 1) The method according to claim 1, wherein the OsrHSA is expressed in rice seed. ### Claim 3 (depends on 1) The method according to claim 1, wherein the elution buffer in the step (f) has a conductivity of between about 40 mS/cm and 47 mS/cm, or about 42 mS/cm. ### Claim 4 (depends on 1) The method according to claim 1, wherein the hydrophobic chromatography resin in the step (g) comprises highly crosslinked polymethacrylate beads of uniformed particle size and open pore structure. ### Claim 5 (depends on 3) The method according to claim 3, wherein the elution conductivity of the chromatography in the step (g) is between about 80-92 mS/cm. ### Claim 6 (depends on 4) The method according to claim 4, wherein the elution conductivity of the chromatography in the step (g) is between about 75 to 90 mS/cm or about 84 mS/cm. ### Claim 7 (depends on 1) The method according to claim 1, wherein the chromatography buffer used in step (g) comprises: between about 10 to 20 mM PB, between about 500 to 600 mM (NH4)2SO4, between about 1 and 3 g/L, or about 2 g/L sodium octanoate, between about pH 6.5 to 6.6. ### Claim 8 (depends on 6) The method of claim 6, wherein the elution conductivity of the chromatography in the step (g) is between about 80 to 87 mS/cm. ### Claim 9 (depends on 4) The method of claim 4, wherein the crosslinked polymethacrylate beads have a particle size of about 35 μm and a pore size of about 500 μm. ### Claim 10 (depends on 4) The method of claim 4, wherein the hydrophobic chromatography resin in the step (g) has a dynamic binding capacity of about 30 mg/ml (Lys). ### Claim 11 (depends on 4) The method of claim 4, wherein the hydrophobic chromatography resin in the step (g) has an operating pressure of less than about 0.8M Pa and an operating pH range of between about pH 2 to pH 12. ### Claim 12 (depends on 1) The method of claim 1, wherein the hydrophobic chromatography comprises use of a rigid, hydrophobic resin. ### Claim 13 A method for preparing high-purity recombinant human serum albumin from Oryza sativa (OsrHSA), comprising the following steps of: (a) preparing a crude or unpurified extract of OsrHSA; (b) subjecting the crude extract of OsrHSA to cation exchange chromatography (A) comprising a cation exchange column, whereby OsrHSA binds to the cation exchange column, (c) providing: a first washing buffer comprising between about 10-20% absolute isopropanol by volume, an equilibration buffer I comprising between about 0-10% absolute isopropanol by volume, and an equilibration buffer II comprising between about 5-15% absolute isopropanol by volume, (d) washing the cation exchange column with the first washing buffer, then washing the column with equilibration buffer I, then washing the column with equilibration buffer II, (e) eluting OsrHSA from the cation exchange chromatography, thereby obtaining a primary product I; wherein the cation exchange chromatography comprises a cationic/hydrophobic composite resin; (f) subjecting the primary product I to anion exchange chromatography B comprising an anion exchange column, to obtain an intermediate product II, and the parameters of the anion exchange chromatography B comprise: (i) adding or loading onto to the anion exchange column a loading sample under conditions comprising: pH 8.0, conductivity between about 1 to 5 mS/cm; (ii) adding or loading onto to the anion exchange column a washing buffer under conditions comprising: pH 8.0, conductivity between about 9 to 9.5 mS/cm; (iii) adding or loading onto to the anion exchange column an elution buffer comprising: pH 8.0, conductivity between about 30 to 50 mS/cm; thereby eluting off the anion exchange column the intermediate product II, wherein the anion exchange chromatography B comprising an anion exchange column comprises use of the following buffers: an equilibration buffer comprising: between about 10 to 20 mM phosphate buffer (PB), between about 7.9 to 8.1 pH, conductivity between about 1 to 3 mS/cm; a re-equilibration buffer comprising: the same as the equilibration buffer; a second washing buffer comprising: between about 10 to 20 mM PB, between about 150 to 170 mM NaCl, between about 7.9 to 8.1, between about 9 to 9.5 mS/cm conductivity; an elution buffer comprising: between about 10 to 20 mM PB, between about 420 to 460 mM NaCl, between about pH 7.9 to 8.1, between about 41 to 43 mS/cm conductivity; and (g) subjecting the intermediate product II to hydrophobic chromatography (C) comprising use of a hydrophobic column to obtain the high-purity OsrHSA, comprising: loading the intermediate product II onto the hydrophobic column in a buffer comprising between about 10 to 20 mM PB, between about 500 to 600 mM (NH4)2SO4, between about 1 and 3 g/L, or about 2 g/L sodium octanoate, between about pH 6.5 to 6.6, washing the hydrophobic column by loading the second washing buffer onto the hydrophobic column, and eluting high-purity OsrHSA from the hydrophobic column by loading an eluting buffer onto the hydrophobic column, thereby generating the high-purity OsrHSA, and the hydrophobic chromatography uses a hydrophobic resin. ### Claim 14 (depends on 13) The method of claim 13, wherein the hydrophobic chromatography comprises use of a rigid, hydrophobic resin.