Claims for Patent: 9,933,432

Summary for Patent: 9,933,432

Title:	Multi-dimensional chromatographic methods for separating N-glycans
Abstract:	A multi-dimensional chromatographic method for the separation of N-glycans. The method comprises providing a glycan preparation that includes at least one negatively charged N-glycan. The glycan preparation is then separated by anion-exchange chromatography and at least one secondary chromatographic technique.
Inventor(s):	Parsons; Ian Christopher (Belmont, MA), Zheng; Ting (Lake Oswego, OR), Gunay; Nur Sibel (Chestnut Hill, MA), Bosques; Carlos J. (Arlington, MA)
Assignee:	Momenta Pharmaceuticals, Inc. (Cambridge, MA)
Application Number:	15/241,717
Patent Claims:	1. A method of producing of a therapeutic product, the method comprising: producing a test preparation of a glycoprotein in a cell culture; removing at least a first sample and a second sample of the test preparation, wherein the first and second samples include at least one charged N-glycan; adding a known quantity of a reference N-glycan to the first and second samples, wherein the reference N-glycan is labeled with a labeling agent; for each of the first and second samples: (i) separating the sample by anion-exchange chromatography to generate a plurality of fractions; (ii) separating a first portion of the plurality of fractions by at least one secondary chromatographic technique to obtain first separated fractions; (iii) separating a second portion of the plurality of fractions by at least one secondary chromatographic technique that differs from the secondary chromatographic technique from (ii) to obtain second separated fractions; (iv) quantifying at least one N-glycan relative to the reference N-glycan in at least a portion of the first separated fractions, or in at least a portion of the second separated fractions, or both; comparing the result of (iv) from the first sample with the result of (iv) from the second sample to obtain a glycosylation pattern for the test preparation; and producing a therapeutic product comprising at least a portion of the test preparation if the glycosylation pattern for the test preparation has at least 90% correlation to a glycosylation pattern of a reference preparation of the glycoprotein. 2. The method of claim 1, wherein the at least one secondary chromatographic technique from (ii) is selected from the group consisting of reversed phase liquid chromatography (RP), normal phase liquid chromatography (NP), ion-pairing reverse phase chromatography (IP-RP), size exclusion chromatography, affinity chromatography (AC), capillary electrophoresis (CE); fluorophore-assisted carbohydrate electrophoresis (FACE); electrochromatography, and micellar electrokinetic chromatography (MEKC). 3. The method of claim 1, wherein the at least one secondary chromatographic technique from (ii) is a reversed phase chromatographic technique. 4. The method of claim 3, wherein the reversed-phase chromatographic technique employs a C18 reverse phase resin. 5. The method of claim 3, wherein the reversed-phase chromatographic technique employs a porous graphitized-carbon (PGC) resin. 6. The method of claim 1, wherein the at least one secondary chromatographic technique from (ii) is a normal phase chromatographic technique. 7. The method of claim 6, wherein the normal phase chromatographic technique employs a modified silica gel. 8. The method of claim 7, wherein the modified silica gel is selected from the group consisting of cyano-modified silica, amine-modified silica, and amide-modified silica. 9. The method of claim 1, wherein the at least one secondary chromatographic technique from (iii) is selected from the group consisting of reversed phase liquid chromatography (RP), normal phase liquid chromatography (NP), ion-pairing reverse phase chromatography (IP-RP), size exclusion chromatography, affinity chromatography (AC), capillary electrophoresis (CE); fluorophore-assisted carbohydrate electrophoresis (FACE); electrochromatography, and micellar electrokinetic chromatography (MEKC). 10. The method of claim 1, wherein the at least one secondary chromatographic technique from (iii) is a reversed phase chromatographic technique. 11. The method of claim 10, wherein the reversed-phase chromatographic technique employs a resin selected from the group consisting of a C18 reverse phase resin and a porous graphitized-carbon (PGC) resin. 12. The method of claim 1, wherein the at least one secondary chromatographic technique from (iii) is a normal phase chromatographic technique. 13. The method of claim 12, wherein the normal phase chromatographic technique employs a modified silica gel. 14. The method of claim 13, wherein the modified silica gel is selected from the group consisting of cyano-modified silica, amine-modified silica, and amide-modified silica. 15. The method of claim 1, wherein the at least one secondary chromatographic technique from (ii) is a normal phase chromatographic technique and the at least one secondary chromatographic technique from (iii) is a reverse phase chromatographic technique. 16. The method of claim 1, wherein the first portion of the plurality of fractions comprises the first half to two-thirds of the fractions from the anion exchange chromatography. 17. The method of claim 1, wherein the second portion of the plurality of fractions comprises the second half to one-third of the fractions from the anion exchange chromatography. 18. The method of claim 1, wherein the glycoprotein is a therapeutic glycoprotein. 19. The method of claim 18, wherein the therapeutic glycoprotein is an antibody or fusion protein. 20. The method of claim 19, wherein the antibody is alemtuzumab, etanercept, adalimumab, abatacept, infliximab, bevacizumab, rituximab, natalizumab, or cetuximab. 21. The method of claim 1, wherein the method comprises producing the therapeutic product comprising at least a portion of the test preparation if the comparison has an at least 95% correlation to a glycosylation pattern of a reference preparation of the glycoprotein. 22. The method of claim 1, wherein the method comprises producing the therapeutic product comprising at least a portion of the test preparation if the comparison has an at least 98% correlation to a glycosylation pattern of a reference preparation of the glycoprotein.

Details for Patent 9,933,432

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	RITUXAN	rituximab	Injection	103705	11/26/1997		2027-04-16
Janssen Biotech, Inc.	REMICADE	infliximab	For Injection	103772	08/24/1998		2027-04-16
Immunex Corporation	ENBREL	etanercept	For Injection	103795	11/02/1998		2027-04-16
Immunex Corporation	ENBREL	etanercept	For Injection	103795	05/27/1999		2027-04-16