Chromatographic process for producing high purity fibrinogen and thrombin
Inventors
NUVULA, ASHOK KUMAR • Samaddar, Mitali • VADDE, NEELIMA • CHAKRABORTY, ZINIA • DUGGINENI, SWAPNA SAGAR • Komath, Uma Devi
Assignees
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Publication Number
US-9932388-B2
Publication Date
2018-04-03
Expiration Date
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.
Core Innovation
The invention relates to a chromatographic process for the isolation and purification of fibrinogen and thrombin obtained from human plasma. The process uses gel filtration chromatography to obtain three fractions (Fraction I, Fraction II and Fraction III), and does not use ethanol precipitation. The process provides fibrinogen and thrombin that contain plasminogen in amounts less than 1 ug/mL.
After gel filtration, Fraction II and Fraction III are subjected to additional chromatography steps for purification. Viral inactivation is performed, and isolated purified fibrinogen is recovered from Fraction II while isolated purified thrombin is recovered from Fraction III. The structure is directed to maintaining low plasminogen levels while separating and purifying the two components from human plasma.
Claims Coverage
The independent claim covers a chromatographic process that isolates and purifies fibrinogen and thrombin from human plasma without ethanol precipitation, while maintaining plasminogen below 1 ug/mL and incorporating viral inactivation and fraction-based recovery. Dependent claims further define purification steps for fibrinogen and thrombin, and they add fibrin sealant kit constraints such as omission of aprotinin and formulation concentration ranges.
Chromatographic isolation of fibrinogen and thrombin from human plasma without ethanol precipitation
A chromatographic process for the isolation and purification of fibrinogen and thrombin, obtained from human plasma, without the use of ethanol precipitation, containing plasminogen in amounts less than 1 ug/mL.
Gel filtration fractionation into three fractions from human plasma
Subjecting the human plasma to gel filtration chromatography to obtain three fractions (Fraction I, Fraction II and Fraction III).
Purification of Fraction II and Fraction III with viral inactivation and fraction recovery
Subjecting Fraction II and Fraction III to additional chromatography steps for purification, and subjecting to viral inactivation and recovering isolated purified fibrinogen from Fraction II and recovering isolated purified thrombin from Fraction III.
Fibrin sealant kit omission of aprotinin based on low plasminogen products
The purified fibrinogen and thrombin having plasminogen in amounts less than 1 ug/mL eliminate use of aprotinin in a fibrin sealant kit.
Fibrin sealant kit fibrinogen concentration range
The fibrin sealant kit includes fibrinogen at a concentration of 25–150 mg/ml.
Fibrin sealant kit calcium chloride amount range
The fibrin sealant kit further specifies that the amount of calcium chloride is within 20 to 60 µM.
Overall claim coverage centers on a chromatography-based isolation and purification workflow that separates fibrinogen and thrombin from human plasma via gel filtration into Fraction I–III, applies further purification to Fraction II and Fraction III, and performs viral inactivation while maintaining plasminogen below 1 ug/mL. Dependent claims link the low-plasminogen products to a fibrin sealant kit that omits aprotinin and specifies fibrinogen and calcium chloride concentration ranges.
Stated Advantages
Provides fibrinogen and thrombin containing plasminogen in amounts less than 1 ug/mL.
Eliminates use of aprotinin in a fibrin sealant kit.
Documented Applications
A fibrin sealant kit including purified fibrinogen and thrombin obtained by the chromatographic process, configured to omit aprotinin.
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