Analysis of sulfated polysaccharides
AssigneeMomenta Pharmaceuticals, Inc.
InventorsGanesh Venkataraman, Zachary Shriver, Malikarjun Sundaram, Yi-Wei Qi, Ram Sasisekharan
Life of this patent
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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.
Claims69
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.
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.
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.
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.
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.
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.
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.
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.
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.
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:
- ΔU2SHNS,6S;
- ΔU2SHNS;
- ΔUHNS,6S;
- ΔU2SHNAC,6S;
- ΔUHNS;
- ΔU2SHNAC;
- ΔUHNAC,6S;
- ΔUHNAc,6SGHNS,3S,6S;
- ΔU HNS,6SGHNS,3S,6S;
- ΔU HNS,6SGHNS,3S and ΔU HNAc,6SGHNS,3S;
- and
- determining if ΔU2SHNS,6S is present at 45-80 mole %,
- to thereby analyze the enoxaparin sample.
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.
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:
- ΔU2SHNS,6S;
- ΔU2SHNS;
- ΔUHNS,6S;
- ΔU2SHNAC,6S;
- ΔUHNS;
- ΔU2SHNAC;
- ΔUHNAC,6S;
- ΔUHNAc,6SGHNS,3S,6S;
- ΔU HNS,6SGHNS,3S,6S;
- ΔU HNS,6SGHNS,3S and ΔU HNAc,6SGHNS,3S;
- and
- determining if ΔUHNAc,6SGHNS,3S,6S, ΔU HNAc,6SGHNS,3S, ΔUHNS,6SGHNS,3S,6S, ΔUHNS,6SGHNS,3S, or mixtures thereof are present at 1-15 mole %,
- to thereby analyze the enoxaparin sample.
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.
Dependencies are inferred from the claim text.