Method for manufacturing a tissue factor-based prothrombin time reagent

• US 20040086953A1
• Filed: 11/05/2002
• Published: 05/06/2004
• Est. Priority Date: 11/05/2002
• Status: Active Grant

• Current Assignee: Cilag Gmbh International (Johnson & Johnson)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Earp, Brian, Haley, Paul E., Jenny, Richard
• Granted Patent: US 7,049,087 B2
• Time in Patent Office: Days
• US Class Current: 435/13
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: G01N 2333/7454 Tissue factor (tissue thromboplastin, Factor III)

Abstract

A method for manufacturing a tissue factor-based prothrombin-time reagent includes combining predetermined quantities of tissue factor (TF), phospholipid and detergent-containing buffer to create a TF/phospholipid mixture. In this combining step, the quantity of TF is predetermined based upon its measured TF activity. Next, the detergent is removed from the TF/phospholipid mixture to produce an essentially detergent-free TF/phospholipid mixture that is useful as a tissue factor-based PT reagent. The method can include the use of hydrophobic resin that has been prescreened based on phospholipid recovery to accomplish the removal of the detergent.

Claims

1. A method for manufacturing a tissue factor-based prothrombin time (PT) reagent, the method comprising:

combining predetermined quantities of tissue factor (TF), phospholipid and detergent-containing buffer to create a TF/phospholipid mixture, wherein the predetermined quantity of TF is based on a measured TF activity thereof; and

removing the detergent from the TF/phospholipid mixture to create an essentially detergent-free TF/phospholipid vesicle mixture, thereby creating a tissue factor-based PT reagent.

2. The method of claim 1, wherein the combining step includes adding the predetermined quantity of TF to a mixture of phospholipid and detergent-containing buffer.

3. The method of claim 1, wherein the combining step includes adding a predetermined quantity of recombinant tissue factor (rTF).

4. The method of claim 1, wherein the removing step is accomplished using a technique that includes:

adding a hydrophobic resin to the TF/phospholipid mixture;

incubating the TF/phospholipid mixture and the added resin until the TF/phospholipid mixture is essentially free of detergent; and

removing said hydrophobic resin from the TF/phospholipid mixture.

5. The method of claim 4, wherein the removing step is accomplished using a technique that further includes the step of prescreening the hydrophobic resin prior to the adding step.

6. The method of claim 5, wherein the prescreening includes prescreening the hydrophobic resin based on solubilized phospholipid recovery.

7. The method of claim 5, wherein the prescreening includes prescreening the hydrophobic resin based on solubilized phospholipid recovery in the range of 75% to 95%.

8. The method of claim 1 further comprising the steps of:

measuring the total phospholipid concentration of the essentially detergent-free TF/phospholipid vesicle mixture; and

determining acceptability of the essentially detergent-free TF/phospholipid vesicle mixture for use as a tissue factor-based PT reagent based on the measured total phospholipid concentration.

9. The method of claim 8, wherein the measuring step measures the phospholipid concentration in terms of inorganic phosphate concentration and the determining step includes accepting for use tissue factor-based PT reagent with a measured total phospholipid concentration in the range of 2.5 to 5.0 mM in terms of inorganic phosphate concentration.

10. The method of claim 1, wherein the TF activity is measured by a fluorogenic TF activity assay.

11. The method of claim 10, wherein the fluorogenic TF activity assay utilizes a 6-peptidylamino-1-naphthalenesulfonamide fluorogenic substrate.

12. The method of claim 11, wherein the combining step combines a predetermined quantity of TF such that a TF activity of the TF/phospholipid mixture is in the range of 9,000 units to 18,000 units per liter.

13. A method for manufacturing a recombinant tissue factor-based prothrombin time (PT) reagent, the method comprising:

combining predetermined quantities of recombinant tissue factor (rTF), phospholipid and detergent-containing buffer to create a rTF/phospholipid mixture, by adding the predetermined quantity of rTF to a mixture of phospholipid and detergent-containing buffer, wherein;

the predetermined quantity of rTF is based on a measured rTF activity thereof, the rTF activity having been measured using a fluorogenic-based rTF assay employing a 6-peptidylamino-1-naphthalenesulfonamide fluorogenic substrate;

adding a prescreened hydrophobic resin to the rTF/phospholipid mixture;

incubating the rTF/phospholipid mixture and the added prescreened resin until the rTF/phospholipid mixture is essentially free of detergent; and

removing said hydrophobic resin from the rTF/phospholipid mixture, thereby providing an essentially detergent-free rTF/phospholipid vesicle mixture;

measuring the total phospholipid concentration of the essentially detergent-free rTF/phospholipid vesicle mixture; and

determining acceptability of the essentially detergent-free rTF/phospholipid mixture for use as a recombinant tissue factor-based PT reagent based on the measured total phospholipid concentration.

14. The method of claim 13, wherein the combining step combines a predetermined quantity of rTF such that the rTF activity of the rTF/phospholipid mixture is in the range of 9,000 units to 18,000 units per liter.

15. The method of claim 13, wherein the prescreening includes prescreening the hydrophobic resin based on solubilized phospholipid recovery.

16. The method of claim 14, wherein the prescreening includes prescreening the hydrophobic resin based on solubilized phospholipid recovery in the range of 75% to 95%.