# US 2014/0155574 A1 · Method for Preparing Insulin Glargine Crystal > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0155574 A1 - Document ID: US20140155574A1 - Status: Published application - Kind: A1 - Application number: 14/116,556 - Filed: 2012-04-19 - Published: 2014-06-05 - Earliest claimed: 2011-05-09 - Assignees: Gan & Lee Pharmaceuticals - Inventors: Damei Wang; Wenjie Li; Jinlei Zhang - Web page: https://patentgrep.com/patent/US20140155574A1 ## CPC - C07K 1/306: {by crystallization} - A61P 3/10: for hyperglycaemia, e.g. antidiabetics - C07K 14/62: Insulins - C30B 7/14: the crystallising materials being formed by chemical reactions in the solution - C30B 29/58: Macromolecular compounds - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C30B: SINGLE-CRYSTAL GROWTH (by using ultra-high pressure, e.g. for the formation of diamonds, B01J3/06); UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL (zone-refining of metals or alloys C22B); PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE (casting of metals, casting of other substances by the same processes or devices B22D; working of plastics B29; modifying the physical structure of metals or alloys C21D, C22F); SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE (for producing semiconductor devices or parts thereof H10); APPARATUS THEREFOR ## Abstract Disclosed is a method for preparing an insulin glargine (GlyA21-ArgB31-AryB32-human insulin) crystal, comprising crystallizing the insulin glargine at pH 7.0-9.0 and in a crystallization solution containing a recombinant insulin glargine, an organic solvent of a 10-30% concentration by volume, a zinc compound, a phenol derivative, a salt and an organic acid. ## Claims ### Claim 1 A method for preparing a recombinant insulin glargine crystal, comprising the steps of: crystallizing the recombinant insulin glargine at pH 7.0-9.0 and in a crystallization solution containing a recombinant insulin glargine, an organic solvent, a zinc compound, a phenol derivative, a salt and an organic acid, wherein the concentration of volume percent of the organic solvent is 10-30%. ### Claim 2 (depends on 1) The method according to claim 1, wherein the organic solvent is selected from acetonitrile, ethanol, n-propanol or isopropanol. ### Claim 3 (depends on 1) The method according to claim 1, wherein the pH of the initial crystallization solution is 8.0-9.0, and during the crystallization process the pH of the crystallization solution is 7.0-8.0. ### Claim 4 (depends on 3) The method according to claim 3, wherein the pH of the initial crystallization solution is 8.0-8.5, and during the crystallization process the pH of the crystallization solution is 7.1-7.8. ### Claim 5 (depends on 1) The method according to claim 1, wherein the temperature of crystallization is 1-30° C. ### Claim 6 (depends on 5) The method according to claim 5, wherein the temperature of the initial crystallization solution is 10-30° C., and during the crystallization process the temperature of the crystallization solution is 2-8° C. ### Claim 7 (depends on 1) The method according to claim 1, wherein the concentration of the recombinant insulin glargine in the crystallization solution is 2-4 g/L. ### Claim 8 (depends on 1) The method according to claim 1, wherein the phenol derivative is selected from phenol, m-cresol and methyl p-hydroxybenzoate or a mixture thereof, and the concentration of phenol derivative in the crystallization solution is 0.05-0.5 g/100 ml. ### Claim 9 (depends on 1) The method according to claim 1, wherein the zinc compound is selected from zinc chloride, zinc oxide, zinc acetate, zinc bromide or zinc sulfate, and the concentration of the zinc compound in the crystallization solution is 20-200 mg/L. ### Claim 10 (depends on 1) The method according to claim 1, wherein the salt is selected from sodium chloride, sodium acetate or sodium citrate, and the concentration of the salt in the crystallization solution is 0.1-0.5M. ### Claim 11 (depends on 1) The method according to claim 1, wherein the organic acid is selected from acetic acid, citric acid or glycine, and the concentration of the salt in the crystallization solution is 0.3-0.8M. ### Claim 12 (depends on 1) The method according to claim 1, comprising the steps of: 1) preparing a crystallization solution containing 2.8-3.2 g/L of recombinant insulin glargine, ethanol, 0.1-0.2 g/100 ml of phenol, 0.4-0.6M of acetic acid, 70-100 mg/L of zinc chloride, 0.3-0.4M of sodium chloride under room temperature, wherein the concentration of volume percent of ethanol is 10-30%; 2) adjusting pH value of the crystallization solution to 8.0-8.5; 3) cooling the crystallization solution to a temperature of 2-8° C., adjusting the pH value to 7.1-7.8, and then crystallizing for at least 3 hours, and thereby obtaining the recombinant insulin glargine crystal. ### Claim 13 (depends on 1) The method according to claim 1, characterizing by further comprising the steps of: suspending the recombinant insulin glargine crystal at a concentration of 20 g/L in 0.02M of ammonium acetate solution, adjusting the pH value to 7.8, and collecting the precipitate by centrifugation, thereby obtaining the purified recombinant insulin glargine crystal. ### Claim 14 (depends on 13) The method according to claim 13, comprising the following steps of: weighing 251.8 g of recombinant insulin glargine, adding thereto 13.1L of 96% ethanol, 2.4 L of pure glacial acetic acid, 148.6 ml of zinc chloride solution at a concentration of 5.08 g/100 ml, 1678 ml of phenol solution at a concentration of 10%, 1472 g of sodium chloride, and then adding water to a final volume of 83.9 L; adjusting the temperature of the above solution to 23±2° C., adjusting the pH value to 8.0-8.5 with strong ammonia, stirring for about 10 min until the solution is slightly turbid, then standing at 2-8° C. for 1 hour and taking it out after cooling, adjusting the pH value to 7.1-7.6 with glacial acetic acid, stirring for about 10 min, and then standing at 2-8° C. for 10 hours; decanting about 77L of the supernatant, adding the remaining 7L of suspension to 1L centrifuge bottle respectively, centrifuging at 4000 rpm for 10 min with a centrifuge, taking the crystal precipitates out of all of the centrifuge bottles, suspending the precipitates in 0.02M of ammonium acetate solution at a concentration of 20 g/L, adjusting the pH value to 7.8, washing, collecting the crystal precipitate by a further centrifugation, washing again, then centrifuging again and removing the supernatant, and freeze-drying for 40 hours.