Assays to monitor bleeding disorders

Patent metadata

Field	Value
Publication number	US-11747351-B2
Application number	US-202016848445-A
Country	US
Kind code	B2
Filing date	Apr 14, 2020
Priority date	Jul 25, 2011
Publication date	Sep 5, 2023
Grant date	Sep 5, 2023

Abstract

The present invention provides methods of dosing Factor VIII or Factor IX chimeric and hybrid polypeptides. The present invention further provides high-sensitivity methods of quantifying an amount of activated FIX protein in a test sample.

First claim

What is claimed is:

1. A method of quantifying an amount of protein capable of exhibiting FIX activity which is in its activated form (activated FIX protein) in a test sample, the method comprising:
(i) measuring thrombin generation activity for the test sample in the presence of FIX-deficient plasma or FIX-deficient blood and in the presence of exogenous thrombin, wherein the exogenous thrombin is present at a concentration of about 5 nM, and
(ii) comparing the thrombin generation activity to a standard curve plotting known activated FIX protein levels to thrombin generation activity;
wherein the measuring is performed in the absence of exogenous tissue factor (TF).

2. The method of claim 1 , wherein the standard curve is constructed by
(a) providing at least two reference samples, each containing a different, known concentration of activated FIX reference protein; and
(b) measuring thrombin generation activity for each reference sample in the presence of FIX-deficient plasma or FIX-deficient blood and in the presence of exogenous thrombin, wherein the exogenous thrombin is present at a concentration of about 5 nM,
wherein the measuring is performed in the absence of exogenous tissue factor (TF).

3. The method of claim 2 , wherein the reference samples comprise from about 0 pM to about 200 pM of activated FIX protein.

4. The method of claim 2 , wherein the reference samples comprise plasma derived activated FIX protein.

5. The method of claim 1 , wherein the FIX-deficient plasma is human FIX-deficient plasma.

6. The method of claim 1 , wherein the method is adapted to accurately measure less than about 100 pM.

7. The method of claim 1 , wherein the test sample comprises a total amount of protein capable of exhibiting factor IX activity, wherein a portion of the total amount is present in its activated form.

8. The method of claim 1 , wherein less than about 1% (w/w) of the total amount of the protein capable of exhibiting FIX activity is present in its activated form.

9. The method of claim 1 , wherein the test sample contains less than 2 pM of activated FIX protein.

10. The method of claim 1 , wherein the protein capable of exhibiting FIX activity comprises a heterologous moiety.

11. The method of claim 10 , wherein the heterologous moiety comprises an immunoglobulin constant (Fc) region or a portion thereof, albumin or a fragment thereof, a straight or branched polyethylene glycol (PEG) moiety, a PAS sequence, and a hydroxyethyl starch (HES) moiety or a derivative thereof, or a combination thereof.

12. The method of claim 10 , wherein the heterologous moiety comprises a first Fc region.

13. The method of claim 12 , wherein the heterologous moiety further comprises a second Fc region, wherein the second Fc region is associated with the first Fc region by a covalent bond or a non-covalent bond.

14. The method of claim 10 , wherein the protein capable of exhibiting FIX activity is a recombinant Factor IX-Fc fusion (FIX-Fc) protein.

15. The method of claim 1 , wherein the measuring is performed in the presence of phospholipids.

Assignees

• Bioverativ Therapeutics Inc

Inventors

• Sommer Jurg
• Jiang Haiyan
• Zhang Xin
• Yang Buyue
• Pierce Glenn

Classifications

• G01N33/86 Primary

  involving blood coagulating time {or factors, or their receptors} · CPC title

• C12N9/644

  Coagulation factor IXa (3.4.21.22) · CPC title

• G01N2333/755

  Factors VIII, e.g. factor VIII C [AHF], factor VIII Ag [VWF] · CPC title

• G01N2333/9645

  Factor IX (3.4.21.22) · CPC title

• G01N2333/974

  Thrombin · CPC title