Multi-Step Chromatography for Fibrinogen and Thrombin Purification
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Solution Overview
Problem
Current methods for purifying fibrinogen and thrombin for fibrin sealant kits often result in high levels of plasminogen contamination, leading to the need for aprotinin, which can cause hypersensitivity reactions and hinder wound healing, and lack detailed purity levels and viral safety assurance.
Innovation Solution
A multi-step chromatography process is employed to purify fibrinogen and thrombin from human plasma, eliminating plasminogen to undetectable levels, thereby eliminating the need for aprotinin and ensuring high purity and viral safety, using a combination of gel filtration, hydrophobic interaction, anion exchange, and solvent/detergent treatment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional purification methods (precipitation by organic solvents or salts) are used to prepare fibrinogen and thrombin, then the process is simple and cost-effective, but plasminogen contamination occurs at high levels
Solution Approach 1:
The purification process is divided into multiple sequential chromatography steps, each targeting specific contaminants. Fibrinogen undergoes hydrophobic interaction chromatography followed by anion exchange chromatography, while thrombin undergoes cation exchange chromatography followed by heparin affinity chromatography. This segmentation allows systematic removal of plasminogen and other contaminants at different stages.
Solution Approach 2:
Chromatography media serve as intermediary substances that selectively bind to and remove contaminants. The hydrophobic interaction column, anion exchange column, cation exchange column, and heparin affinity column each act as intermediaries with specific binding characteristics, enabling selective purification of fibrinogen and thrombin while removing plasminogen.
2Reliability
If aprotinin is added to prevent fibrin clot degradation, then clot stability is improved, but hypersensitivity reactions and interference with wound healing occur
Solution Approach 1:
The invention extracts and removes plasminogen, the source of fibrinolytic activity, through multiple chromatography steps. By achieving undetectable levels of plasminogen in the purified fibrinogen and thrombin preparations, the need for aprotinin as a protective agent is eliminated, thereby removing the source of hypersensitivity reactions and wound healing interference.
Solution Approach 2:
The invention converts the harmful presence of plasminogen into a benefit by using it as a target for selective removal. The chromatography process is designed to specifically capture and remove plasminogen, transforming what was previously a harmful contaminant requiring chemical inhibition into an eliminated impurity, resulting in a safer product.
3Manufacturing precision
If multiple chromatography steps are employed to achieve high purity, then plasminogen is reduced to undetectable levels, but the process complexity and manufacturing time increase
Solution Approach 1:
The purification processes for fibrinogen and thrombin are merged into a coordinated multi-step chromatography scheme. Both proteins undergo similar sequences of chromatography steps using different media, allowing parallel optimization and reducing overall processing time compared to separate purification approaches.
Solution Approach 2:
Hydrophobic interaction chromatography is performed as a preliminary step before anion exchange chromatography for fibrinogen purification. This preliminary step removes bulk contaminants and concentrates the fibrinogen, making subsequent anion exchange chromatography more efficient and reducing the time required for the final purification step.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The process achieves fibrinogen and thrombin preparations with over 95% clottable protein and undetectable plasminogen levels, enhancing purity and safety, reducing the risk of hypersensitivity reactions, and providing a fibrinolysis inhibitor-free fibrin sealant kit.
Implementation Method 1
The thawed and pooled plasma after filtration is loaded onto a gel filtration column, packed with any of the commonly used resins
Implementation Method 2
The eluate containing fibrinogen from the HIC column of Step 1(c), is subjected to a Solvent/Detergent (S/D) treatment with 1% TRITON™ X 100 and 0.3% TNBP
Implementation Method 3
The eluate containing fibrinogen from the HIC column of Step 1(c), is subjected to a Solvent/Detergent (S/D) treatment with 1% TRITON™ X 100 and 0.3% TNBP [tri (n-butyl) phosphate] for 4 to 6 hrs for viral inactivation
Data Source
AI summary
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.