Evaluating mixtures of low molecular weight heparins by chain profiles or chain mapping

• US 7,790,466 B1
• Filed: 01/25/2008
• Issued: 09/07/2010
• Est. Priority Date: 01/26/2007
• Status: Active Grant

• Current Assignee: Momenta Pharmaceuticals Incorporated (Johnson & Johnson)
• Original Assignee: Momenta Pharmaceuticals Incorporated (Johnson & Johnson)
• Inventors: Gunay, Nur Sibel, Ozug, Jennifer, Wudyka, Steve, Shriver, Zachary, Capila, Ishan, Beccati, Daniela
• Primary Examiner(s): WALLENHORST, MAUREEN
• Application Number:
  US12/011,436
• Time in Patent Office: 956 Days
• Field of Search: 436/94, 436/161, 436/174, 210/656, 705/2
• US Class Current: 436/94
• CPC Class Codes: G01N 2030/8836 involving saccharides
• CPC Class Codes: G01N 30/96 using ion-exchange G01N30/02, G01N30/90 take precedence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

Methods for analyzing mixtures of polysaccharides, for example heparin such as unfractionated heparin and enoxaparin are described. In some instances, the mixtures are analyzed using fast performance liquid chromatography (FPLC) and high liquid performance chromatography (HPLC), e.g., strong anion exchange HPLC.

Claims

1. A method of analyzing an enoxaparin preparation, comprising:

providing an isolated tetrasaccharide fraction from a size fractioned enoxaparin preparation;

analyzing the tetrasaccharide fraction using strong anion exchange high performance liquid chromatography (HPLC) to determine if one or more chains shown in the following table is present or is present in amount identified in the following table;

2. The method of claim 1, wherein the tetrasaccharide fraction is fractionated using fast protein liquid chromatography.

3. The method of claim 2, wherein the method further comprises determining if the enoxaparin preparation has a fraction distribution profile of dodecasaccharides, decasaccharides, octasaccharides, hexasaccharides and tetrasaccharides, as shown in FIG. 2.

4. The method of claim 2, wherein the method further comprises determining if one or more fractions shown in the following tables are present or present in an amount shown in the following tables.

5. The method of claim 1, wherein the presence or amount of one or more of a tetrasaccharide that includes five or six sulfate groups, 0 or 1 galacturonic acids, a mannosamine or glucosamine at the reducing end, a Δ

^4,5 double bond at the non-reducing end, and a 1,6 anhydro structure is determined.

6. The method of claim 1, wherein the tetrasaccharide fraction is evaluated for the presence of one or more of Δ

U_2SH_NS6SI_2SMan_NS6S, Δ

U_2SH_NS6SI_2SMan_NS(1,6 anhydro), and Δ

U_2SH_NS6SI_2SH_NS(1,6 anhydro).

7. A method of analyzing a process of making an enoxaparin preparation comprising:

providing an isolated tetrasaccharide fraction of an enoxaparin preparation;

analyzing the tetrasaccharide fraction using the method of claim 1, and

if said chains shown in the table of claim 1 are present or are present in an amount provided in the table of claim 1, maintaining the process based, at least in part, upon the analysis.

8. A method of processing an enoxaparin preparation comprising:

providing a high performance liquid chromatography (HPLC) determination of whether a tetrasaccharide structure is present in an enoxaparin preparation in an amount provided in the following table;