Multi-dimensional chromatographic methods for separating N-glycans

Patent metadata

Field	Value
Publication number	US-9933432-B2
Application number	US-201615241717-A
Country	US
Kind code	B2
Filing date	Aug 19, 2016
Priority date	Apr 16, 2007
Publication date	Apr 3, 2018
Grant date	Apr 3, 2018

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.

First claim

We claim:

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.

Assignees

• Momenta Pharmaceuticals Inc

Inventors

• Parsons Ian Christopher
• Zheng Ting
• Gunay Nur Sibel
• Bosques Carlos J

Classifications

• B01D15/363

  Anion-exchange · CPC title

• B01D15/325

  Reversed phase · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title

• B01D15/166

  Fluid composition conditioning, e.g. gradient · CPC title

• B01D15/426

  Specific type of solvent · CPC title