Claims for Patent: 9,932,388

Summary for Patent: 9,932,388

Title:	Chromatographic process for producing high purity fibrinogen and thrombin
Abstract:	The present invention relates to a chromatographic process for obtaining purified fibrinogen and thrombin from human plasma. The purified fibrinogen and thrombin preparations contain plasminogen in amounts less than 1 ug/mL. The low levels of plasminogen eliminates use of a proteolytic inhibitor, such as aprotinin in fibrin sealant kits which are used for human therapeutic applications.
Inventor(s):	Nuvula; Ashok Kumar (Hyderabad, IN), Samaddar; Mitali (Hyderabad, IN), Vadde; Neelima (Hyderabad, IN), Chakraborty; Zinia (Hyderabad, IN), Duggineni; Swapna Sagar (Hyderabad, IN), Komath; Uma Devi (Hyderabad, IN)
Assignee:	Hemarus Therapeutics Limited (Hyderabad, IN)
Application Number:	14/883,574
Patent Claims:	1. A chromatographic process for the isolation and purification of fibrinogen and thrombin, obtained from human plasma, without the use of ethanol precipitation, that contains plasminogen in amounts less than 1 ug/mL, comprising: i. subjecting said human plasma to gel filtration chromatography to obtain three fractions (Fraction I, Fraction II and Fraction III); ii. subjecting Fraction II and Fraction III to additional chromatography steps for purification; and iii. subjecting to viral inactivation and recovering isolated purified fibrinogen from Fraction II and recovering isolated purified thrombin from Fraction III. 2. The chromatographic process of claim 1, wherein purification of the fibrinogen from said Fraction II, further comprises: a) adding ammonium sulphate to Fraction II to a concentration of 0.05M to 0.5M; b) loading onto a hydrophobic interaction chromatography (HIC) column to obtain eluate with plasminogen in amounts of less than 1 ug/mL; c) conducting solvent/detergent (S/D) virus inactivation for the eluate obtained from step b; and d) subjecting the treated eluate from step c to anion exchange chromatography for recovering purified fibrinogen. 3. The chromatographic process of claim 1, wherein purification of thrombin from said Fraction III, further comprises: a) loading the Fraction III onto an anion exchange column equilibrated with a buffer comprising an acetate or citrate, with a molarity of 0.01M to 0.1M in the pH range of 6.5 to 8.5, eluting a partially purified prothrombin with buffer containing sodium chloride, to obtain prothrombin with plasminogen in amounts of less than 1 ug/mL; b) conducting solvent-detergent (S/D) virus inactivation and loading onto an affinity column for the capture of prothrombin and removal of S/D; c) collecting purified prothrombin eluate by elution with a buffer containing increasing amount of sodium chloride ranging from 0 to 0.5M; d) subjecting the eluate from step c to an anion exchange in the pH range of 6 to 9 in a buffer containing 1 to 50 mM concentration of calcium chloride, at 4.degree. C. to 28.degree. C. temperature for 50 to 70 hours for the conversion of prothrombin to thrombin; and e) subjecting the treated eluate obtained from step d to nanofiltration for virus removal and recovering purified thrombin. 4. The chromatographic process of claims 1-3, wherein the purified fibrinogen and thrombin having plasminogen in amounts of less than 1 ug/mL, eliminates use of aprotinin, a proteolytic inhibitor, in a fibrin sealant kit. 5. The fibrin sealant kit of claim 4, wherein the concentration of fibrinogen is 25-150 mg/ml. 6. The fibrin sealant kit of claim 4, wherein the concentration of thrombin is 400 to 800 IU/ml. 7. The fibrin sealant kit of claim 6, wherein the fibrin sealant kit optionally comprises an additional vial of thrombin at a lower strength in the range of 2 to 10 IU/ml. 8. The fibrin sealant kit of claim 5, further comprising a source of calcium, which is calcium chloride. 9. The fibrin sealant kit of claim 8, wherein the amount of calcium chloride is in the range of 20 to 60 uM.

Details for Patent 9,932,388

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Bayer Healthcare Pharmaceuticals Inc.	TRASYLOL	aprotinin	Injection	020304	12/29/1993		2034-11-13