# US 5,344,920 A · Method of separating glycosylated and non-glycosylated prolactin by ion exchange chromatography > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,344,920 A - Document ID: US5344920A - Status: Granted - Kind: A - Application number: 08/057,587 - Filed: 1993-05-04 - Granted: 1994-09-06 - Earliest claimed: 1993-05-04 - Assignees: Genzyme Corporation - Inventors: Albert E. Price - Web page: https://patentgrep.com/patent/US5344920A ## CPC - C07K 14/57554: {Prolactin} - 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 This invention relates to a method for separating glycosylated prolactin from non-glycosylated prolactin. The method comprises the following steps: 1) equilibrating the prolactin containing solution with an equilibrating solution containing at least one organic solvent; 2) adding the equilibrated prolactin solution onto a chromatographic column containing a packing for the ion-exchange affinity separation of glycosylated and non-glycosylated prolactin; 3) eluting the glycosylated prolactin from the chromatographic column with a sufficient quantity of a first eluting solution containing at least one organic solvent and with or without at least one salt, wherein the salt concentration and/or organic solvent concentration and/or pH are appropriate for the eluting of glycosylated prolactin being substantially free of non-glycosylated prolactin; and 4) eluting the non-glycosylated prolactin from the chromatographic column with a sufficient quantity of a first eluting solution containing at least one organic solvent and with or without at least one salt, wherein the salt concentration and/or organic solvent concentration and/or pH are appropriate for the eluting of non-glycosylated prolactin being substantially free of glycosylated prolactin. ## Claims ### Claim 1 A method for separating glycosylated prolactin from non-glycosylated prolactin comprising: a. equilibrating a solution containing glycosylated and non-glycosylated prolactin with an equilibrating solution containing at least one lower alcohol and/or lower glycol; b. adding the equilibrated prolactin solution onto a chromatographic column containing a cationic or anionic packing for the ion exchange separation of glycosylated and non-glycosylated prolactin; c. eluting the glycosylated prolactin from the chromatographic column with a sufficient quantity of a first eluting solution comprising: i. at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume; and ii. at least one salt, wherein the salt concentration, lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of glycosylated prolactin being substantially free of non-glycosylated prolactin; and d. eluting the non-glycosylated prolactin from the chromatographic column with a sufficient quantity of a second eluting solution comprising: i. at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume; and ii. at least one salt, wherein the salt concentration, lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of non-glycosylated prolactin being substantially free of glycosylated prolactin. ### Claim 2 (depends on 1) A method according to claim 1, wherein the prolactin is human prolactin. ### Claim 3 (depends on 1) A method according to claim 1, wherein the lower alcohol is n-butanol. ### Claim 4 (depends on 1) A method according to claim 1, wherein the lower glycol is ethylene glycol. ### Claim 5 (depends on 1) A method according to claim 1, wherein the packing is a resin. ### Claim 6 A method for separating glycosylated prolactin from non-glycosylated prolactin comprising: a. equilibrating a solution containing glycosylated and non-glycosylated prolactin with an equilibrating solution containing at least one lower alcohol and/or lower glycol; b. adding the equilibrated prolactin solution onto a chromatographic column containing a cationic or anionic packing for the ion exchange separation of glycosylated and non-glycosylated prolactin; c. eluting the glycosylated prolactin from the chromatographic column with a sufficient quantity of a first eluting solution comprising: i. at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume; and ii. at least one salt, wherein the salt concentration, lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of non-glycosylated prolactin being substantially free of glycosylated prolactin; and d. eluting the glycosylated prolactin from the chromatographic column with a sufficient quantity of a second eluting solution comprising: i. at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume; and ii. at least one salt, wherein the salt concentration, lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of glycosylated prolactin being substantially free of non-glycosylated prolactin. ### Claim 7 (depends on 6) A method according to claim 6, wherein the prolactin is human prolactin. ### Claim 8 (depends on 6) A method according to claim 6, wherein the lower alcohol is n-butanol. ### Claim 9 (depends on 6) A method according to claim 6, wherein the lower glycol is ethylene glycol. ### Claim 10 (depends on 6) A method according to claim 6, wherein the packing is a resin. ### Claim 11 A method for separating glycosylated prolactin from non-glycosylated prolactin comprising: a. equilibrating a solution containing glycosylated and non-glycosylated prolactin with an equilibrating solution containing at least one lower alcohol and/or lower glycol; b. adding the equilibrated prolactin solution onto a chromatographic column containing a cationic or anionic packing for the ion exchange separation of glycosylated and non-glycosylated prolactin; c. eluting the glycosylated prolactin from the chromatographic column with a sufficient quantity of a first eluting solution containing at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume; wherein the lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of glycosylated prolactin being substantially free of non-glycosylated prolactin; and d. eluting the non-glycosylated prolactin from the chromatographic column with a sufficient quantity of a second eluting solution comprising: i. at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume; and ii. at least one salt, wherein the salt concentration, lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of non-glycosylated prolactin being substantially free of glycosylated prolactin. ### Claim 12 (depends on 11) A method according to claim 11, wherein the prolactin is human prolactin. ### Claim 13 (depends on 11) A method according to claim 11, wherein the lower alcohol is n-butanol. ### Claim 14 (depends on 11) A method according to claim 11, wherein the lower glycol is ethylene glycol. ### Claim 15 (depends on 11) A method according to claim 11, wherein the packing is a resin. ### Claim 16 A method for separating glycosylated prolactin from non-glycosylated prolactin comprising: a. equilibrating a solution containing glycosylated and non-glycosylated prolactin with an equilibrating solution containing at least one lower alcohol or lower glycol; b. adding the equilibrated prolactin solution onto a chromatographic column containing a cationic or anionic packing for the ion exchange separation of glycosylated and non-glycosylated prolactin; c. eluting the glycosylated prolactin from the chromatographic column with a sufficient quantity of a first eluting solution comprising: i. at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume; and ii. at least one salt, wherein the salt concentration, lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of glycosylated prolactin being substantially free of non-glycosylated prolactin; and d. eluting the non-glycosylated prolactin from the chromatographic column with a sufficient quantity of a second eluting solution containing at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume, wherein the lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of non-glycosylated prolactin being substantially free of glycosylated prolactin. ### Claim 17 (depends on 16) A method according to claim 16, wherein the prolactin is human prolactin. ### Claim 18 (depends on 16) A method according to claim 16, wherein the lower alcohol is n-butanol. ### Claim 19 (depends on 16) A method according to claim 16, wherein the lower glycol is ethylene glycol. ### Claim 20 (depends on 16) A method according to claim 16, wherein the packing is a resin. ### Claim 21 A method for separating glycosylated prolactin from non-glycosylated prolactin comprising: a. equilibrating a solution containing glycosylated and non-glycosylated prolactin with an equilibrating solution containing at least one lower alcohol and/or lower glycol; b. adding the equilibrated prolactin solution onto a chromatographic column containing a cationic or anionic packing for the ion exchange separation of glycosylated and non-glycosylated prolactin; c. eluting the glycosylated prolactin from the chromatographic column with a sufficient quantity of a first eluting solution containing at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume, wherein the lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of glycosylated prolactin being substantially free of non-glycosylated prolactin; and d. eluting the non-glycosylated prolactin from the chromatographic column with a sufficient quantity of a second eluting solution containing at least one lower alcohol and/or lower glycol in concentration less than 50% per total volume, wherein the lower alcohol and/or lower glycol concentration and pH are appropriate for the eluting of non-glycosylated prolactin being substantially free of glycosylated prolactin.