Analysis of sulfated polysaccharides

• US 7,575,886 B2
• Filed: 03/11/2003
• Issued: 08/18/2009
• Est. Priority Date: 03/11/2002
• Status: Active Grant

• Current Assignee: Momenta Pharmaceuticals Incorporated (Johnson & Johnson)
• Original Assignee: Momenta Pharmaceuticals Incorporated (Johnson & Johnson)
• Inventors: Qi, Yi-Wei, Sundaram, Malikarjun, Sasisekharan, Ram, Shriver, Zachary, Venkataraman, Ganesh
• Primary Examiner(s): Weber; Jon P
• Assistant Examiner(s): Martin; Paul C.
• Application Number:
  US10/386,402
• Publication Number:
  US 20030203385A1
• Time in Patent Office: 2,352 Days
• Field of Search: None
• US Class Current: 435/18
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 11/16 Central respiratory analeptics
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 25/06 Antimigraine agents
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 9/06 Antiarrhythmics
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: G01N 2400/10 : Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• CPC Class Codes: G01N 2400/40 : Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
• CPC Class Codes: G01N 33/66 : involving blood sugars, e.g. galactose
• CPC Class Codes: G01N 33/86 : involving blood coagulating time or factors, or their receptors
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/17 : Nitrogen containing
• CPC Class Codes: Y10T 436/24 : Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

The invention relates to methods and products associated with analyzing and monitoring heterogeneous populations of sulfated polysaccharides. In particular therapeutic heparin products including low molecular weight heparin products and methods of analyzing and monitoring these products are described.

Claims

1. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization;

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin, and

determining the presence of the structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 in a second batch of enoxaparin,

to thereby analyze the enoxaparin sample.

2. The method of claim 1, further comprising selecting a batch as a result of the determination based upon comparison to the reference standard.

54. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 20 or 43, wherein the structural signature is determined using high performance liquid chromatography (HPLC).

55. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the structural signature is determined using NMR.

56. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the structural signature is determined using CE.

57. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the structural signature is determined using MALDI-MS.

58. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the structural signature is determined using ESI-MS.

59. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the structural signature is determined using FPLC.

60. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the structural signature is determined using one or more of fluorometry, ELISA, chromatogenic assays, and colorimetric assays.

61. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the heparin degrading enzymes are selected from the group consisting of heparinase I, heparinase II, heparinase III.

62. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the sample is contacted with heparinase I, heparinase II, and heparinase III.

63. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, further comprising detecting one or more biological activities of the sample.

64. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the sample has one or more of the following properties:

anti-Xa activity of about 100 IU/mg;

a molecular weight distribution of less than or equal to 20% are <

2000 Da species, greater than or equal to 68% are 2000-8000 Da species, and less than or equal to 18% are >

8000 Da species;

an average molecular weight of about 4500 Da; and

a pH of 5.5-7.5.

66. The method of claims 1, 3, 6, 7, 8, 10, 11, 14, 15, 20, 43, 49 or 53, wherein the structural signature is a di- or tetra-saccharide.

3. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization; and

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin, wherein the determination made regarding the comparison to the reference standard is whether to keep or discard the sample,

to thereby analyze the enoxaparin sample.

4. The method of claim 3, wherein the sample is kept.

5. The method of claim 3, wherein the sample is discarded.

6. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization; and

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin, wherein the determination based upon the comparison to the reference standard regards the quality of the sample,

to thereby analyze the enoxaparin sample.

7. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization; and

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin, wherein the determination based upon the comparison to the reference standard regards bioequivalence of the sample,

to thereby analyze the enoxaparin sample.

8. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization;

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin;

detecting one or more of anti-Xa activity, anti-IIa activity, molecular weight distribution and average molecular weight of the sample; and

comparing any of anti-Xa activity, anti-IIa activity, molecular weight distribution and average molecular weight of the sample to a reference standard for enoxaparin,

wherein the reference standard for molecular weight distribution is less than or equal to 20% are <

2000 Da species, greater than or equal to 68% are 2000-8000 Da species, and less than or equal to 18% are >

8000 Da species,

to thereby analyze the enoxaparin sample.

9. The method of claim 8, wherein the reference standard for anti-Xa activity is 100 IU/mg.

10. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization;

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin;

detecting one or more of anti-Xa activity, anti-IIa activity, molecular weight distribution and average molecular weight of the sample; and

comparing any of anti-Xa activity, anti-IIa activity, molecular weight distribution and average molecular weight of the sample to a reference standard for enoxaparin, wherein the reference standard for average molecular weight is 4500 Da,

to thereby analyze the enoxaparin sample.

11. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization; and

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin,

wherein the method comprises determining the presence of the structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from the method used to make enoxaparin and at least one other structural signature,

wherein at least 32 structural signatures are determined,

to thereby analyze the enoxaparin sample.

12. The method of claim 11, wherein the other structural signature is associated with one or more components of peaks 1, 2, 3, 4, 5, 6, 7 and 8 of FIG. 1.

13. The method of claim 11, wherein the presence of one or more structural signatures of components associated with peak 1 or peak 8 of FIG. 1 is determined.

65. The method of claims 11, 15, 20, or 49, wherein two or more structural signatures associated with non-naturally occurring sugars that result from a method that includes β

-eliminative cleavage with a benzyl ester and depolymerization are detected.

67. The method of claim 65, wherein the structural signatures are associated with peaks 9 and 10 of FIG. 1.

68. The method of claim 67, wherein the method comprises determining the presence of the structural signatures associated with the non naturally occurring sugars that result from the method used to make enoxaparin and at least one other structural signature.

69. The method of claim 68, wherein the other structural signature is one or more component associated with peak 1 or peak 8 of FIG. 1.

14. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization;

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin, and

comparing the structural signature determination to a reference database of structural signatures for enoxaparin,

to thereby analyze the enoxaparin sample.

15. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization; and

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin,

wherein the level of one or more structural signatures associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 is determined,

to thereby analyze the enoxaparin sample.

16. The method of claim 15, wherein the level of the structural signature is calculated as the area under the curve or as the percent relative amount of each fraction present in the sample.

17. The method of claim 15, further comprising determining if the level of the structural signature varies less than 2.5% from the reference standard.

18. The method of claim 17, further comprising determining if the level of the structural signature varies less than 2.0% from the reference standard.

19. The method of claim 18, further comprising determining if the quantity of the structural signature varies less than 1.0% from the reference standard.

20. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization;

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin,

wherein the level of two or more structural signatures associated with non-naturally occurring sugars that result from a method that includes β

-eliminative cleavage with a benzyl ester and depolymerization are detected, and wherein the structural signatures of the non naturally occurring sugars are associated with peaks 9 and 10 of FIG. 1;

determining the level of the structural signatures associated with the non naturally occurring sugars that result from the method used to make enoxaparin and at least one other structural signature,

wherein the other structural signature is one or more of;

Δ

U_2SH_NS,6S;

Δ

U_2SH_NS;

Δ

UH_NS,6S;

Δ

U_2SH_NAC,6S;

Δ

UH_NS;

Δ

U_2SH_NAC;

Δ

UH_NAC,6S;

Δ

UH_NAc,6SGH_NS,3S,6S;

Δ

U H_NS,6SGH_NS,3S,6S;

Δ

U H_NS,6SGH_NS,3S and Δ

U H_NAc,6SGH_NS,3S; and

determining if Δ

U_2SH_NS,6S is present at 45-80 mole %,

to thereby analyze the enoxaparin sample.

21. The method of claim 20, wherein the other structural signature is one or more component associated with peak 1 or peak 8 of FIG. 1.

22. The method of claim 20, comprising determining if Δ

U_2SH_NS,6S is present at 50-75 mole %.

23. The method of claim 20, comprising determining if Δ

U_2SH_NS,6S is present at 55-70 mole %.

24. The method of claim 20, comprising determining if Δ

U_2SH_NS,6S is present at 60-65 mole %.

25. The method of claim 20, further comprising determining if Δ

U_2SH_NS is present at 2-15 mole %.

26. The method of claim 20, further comprising determining if Δ

U_2SH_NS is present at 5-10 mole %.

27. The method of claim 20, further comprising determining if Δ

U_2SH_NS is present at 6-9 mole %.

28. The method of claim 20, further comprising determining if Δ

UH_NS,6S is present at 5-18 mole %.

29. The method of claim 20, further comprising determining if Δ

UH_NS,6S is present at 7-15 mole %.

30. The method of claim 20, further comprising determining if Δ

UH_NS,6S is present at 10-12 mole %.

31. The method of claim 20, further comprising determining if Δ

U_2SH_NAC,6S is present at 0.5-7.5 mole %.

32. The method of claim 20, further comprising determining if Δ

U_2SH_NAC,6S is present at 1-5 mole %.

33. The method of claim 20, further comprising determining if Δ

U_2SH_NAC,6S is present at 1.5-3 mole %.

34. The method of claim 20, further comprising determining if Δ

UH_NS is present at 1-7 mole %.

35. The method of claim 20, further comprising determining if Δ

UH_NS is present at 2-5 mole %.

36. The method of claim 20, further comprising determining if Δ

UH_NS is present at 3-4 mole %.

37. The method of claim 20, further comprising determining if Δ

U_2SH_NAC is present at 0.1-5 mole %.

38. The method of claim 20, further comprising determining if Δ

U_2SH_NAC is present at 0.5-3 mole %.

39. The method of claim 20, further comprising determining if Δ

U_2SH_NAC is present at 1-2.5 mole %.

40. The method of claim 20, further comprising determining if Δ

UH_NAC,6S is present at 0.1-12 mole %.

41. The method of claim 20, further comprising determining if Δ

UH_NAC,6S is present at 0.5-10 mole %.

42. The method of claim 20, further comprising determining if Δ

UH_NAC,6S is present at 1-6 mole %.

43. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization;

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin; and

determining if the non naturally occurring sugar associated with peak 9 of FIG. 1 is present in a preselected range,

to thereby analyze the enoxaparin sample.

44. The method of claim 43, wherein the preselected range of the non naturally occurring sugar associated with peak 9 of FIG. 1 is 0.1-2.5 mole %.

45. The method of claim 44, wherein the preselected range is 0.1 to 1 mole %.

46. The method of claim 43, further comprising determining if a non naturally occurring sugar associated with peak 10 of Fig. is present in a preselected range of 0.1-2.5 mole %.

47. The method of claim 46, wherein the preselected range is 0.1 to 1 mole %.

48. The method of claim 46, wherein the preselected range is 0.3 to 0.7 mole %.

49. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization; and

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin,

wherein level of two or more structural signatures associated with non-naturally occurring sugars that result from a method that includes β

-eliminative cleavage with a benzyl ester and depolymerization are detected,

wherein the structural signatures of the non naturally occurring sugars are associated with peaks 9 and 10 of FIG. 1,

wherein the method comprises determining the level of the structural signatures associated with the non naturally occurring sugars that result from the method used to make enoxaparin and at least one other structural signature,

and wherein the other structural signature is one or more of;

Δ

U_2SH_NS,6S;

Δ

U_2SH_NS;

Δ

UH_NS,6S;

Δ

U_2SH_NAC,6S;

Δ

UH_NS;

Δ

U_2SH_NAC;

Δ

UH_NAC,6S;

Δ

UH_NAc,6SGH_NS,3S,6S;

Δ

U H_NS,6SGH_NS,3S,6S;

Δ

U H_NS,6SGH_NS,3S and Δ

U H_NAc,6SGH_NS,3S; and

determining if Δ

UH_NAc,6SGH_NS,3S,6S, Δ

U H_NAc,6SGH_NS,3S, Δ

UH_NS,6SGH_NS,3S,6S, Δ

UH_NS,6SGH_NS,3S, or mixtures thereof are present at 1-15 mole %,

to thereby analyze the enoxaparin sample.

50. The method of claim 49, comprising determining if Δ

UH_NAc,6SGH_NS,3S,6S, Δ

U H_NAc,6SGH_NS,3S, Δ

UH_NS,6SGH_NS,3S,6S, Δ

UH_NS,6SGH_NS,3S, or mixtures thereof are present at 2-10 mole %.

51. The method of claim 49, comprising determining if Δ

UH_NAc,6SGH_NS,3S,6S, Δ

U H_NAc,6SGH_NS,3S, Δ

UH_NS,6SGH_NS,3S,6S, Δ

UH_NS,6SGH_NS,3S, or mixtures thereof are present at 3-8 mole %.

52. The method of claim 49, comprising determining if Δ

UH_NAc,6SGH_NS,3S,6S, Δ

U H_NAc,6SGH_NS,3S, Δ

UH_NS,6SGH_NS,3S,6S, Δ

UH_NS,6SGH_NS,3S, or mixtures thereof are present at 5-7 mole %.

53. A method for analyzing an enoxaparin sample for the presence or amount of a non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that included β

-eliminative cleavage with a benzyl ester and depolymerization, comprising;

providing an enoxaparin sample that has been exhaustively digested with two or more heparin degrading enzymes;

using a separation method to determine, in the enoxaparin sample that has been contacted with two or more heparin degrading enzymes, the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 that results from a method of making enoxaparin that includes β

-eliminative cleavage with a benzyl ester and depolymerization;

making a determination about the enoxaparin sample based upon a comparison of the determination of the presence of a structural signature associated with the non naturally occurring sugar associated with peak 9 to a reference standard for enoxaparin; and

selecting a batch of enoxaparin based upon a comparison of the determination of the presence of the structural signature associated with the non naturally occurring sugar associated with peak 9 of FIG. 1 to a reference standard for enoxaparin,

to thereby analyze the enoxaparin sample.