Assays to monitor bleeding disorders
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.
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.