Recombinant Plasminogen Purification via Ion Exchange Chromatography
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Solution Overview
Problem
The production of high-quality plasmin is hindered by contamination from plasminogen activators and the protease's susceptibility to autodegradation, making it challenging to achieve pharmaceutical purity and stability for clinical use.
Innovation Solution
A method involving cation-exchange and anion-exchange chromatography, along with refolding and diafiltration, is employed to purify recombinant plasminogen and convert it to plasmin, selectively binding and separating the activators while maintaining the protease's activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If plasmin is prepared by activating plasminogen with plasminogen activators, then plasmin can be produced, but the preparation is extensively contaminated by plasminogen activators which trigger systemic bleeding
Solution Approach 1:
The patent extracts and removes plasminogen activator contamination from the plasmin preparation through multiple purification steps including cation-exchange chromatography, affinity chromatography, and anion-exchange chromatography. The method systematically separates and eliminates the contaminating activators while retaining plasmin, achieving pharmaceutical purity.
Solution Approach 2:
The patent applies preliminary purification actions during the activation process itself, incorporating cation-exchange chromatography and affinity chromatography steps before final formulation. This preliminary removal of activators prevents contamination from the outset, ensuring plasma stability and safety.
2Productivity
If plasmin is prepared in large quantities, then sufficient yield is achieved, but the protease is highly prone to autodegradation and loss of activity
Solution Approach 1:
The patent converts the harmful effect of plasmin's broad proteolytic specificity into a benefit by using controlled activation conditions and purification steps that selectively stabilize the enzyme. The method transforms the potential for autodegradation into a controlled process, achieving stable plasmin with high yield through optimized formulation and purification.
Solution Approach 2:
The patent applies parameter changes in pH, temperature, and ionic strength during the activation and purification processes to control plasmin stability. By optimizing these parameters, the method prevents autodegradation while maintaining high yield and enzymatic activity.
3Device complexity
If conventional purification methods are used, then the process is simple, but the plasmin preparation is contaminated and lacks pharmaceutical purity
Solution Approach 1:
The patent segments the purification process into distinct stages: cation-exchange chromatography, affinity chromatography, and anion-exchange chromatography. Each stage targets specific contaminants, systematically achieving pharmaceutical purity through divided, manageable steps rather than a single complex process.
Solution Approach 2:
The patent uses intermediary purification media and reagents that facilitate selective binding and separation. These intermediaries enable precise control over the purification process, achieving high pharmaceutical purity through carefully selected chromatography media and buffer conditions.
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
This approach results in a highly purified and stable plasmin with enhanced stability and reduced contamination, facilitating safe and effective thrombolytic activity.
Implementation Method 1
contacting a composition comprising a plasminogen with a cation-exchange medium under a cation-exchange condition that is sufficient for the cation-exchange medium to bind the plasminogen
Implementation Method 2
contacting the plasmin with an anion-exchange medium under an anion-exchange condition such that the anion-exchange medium preferentially binds the plasminogen activator relative to the plasmin
Implementation Method 3
contacting the solubilized recombinant plasminogen inclusion body with a refolding solution under a refolding condition to obtain a composition comprising the recombinant plasminogen, wherein a PEG or ammonium sulfate is added to the refolding solution under a precipitation condition
Implementation Method 4
a PEG or ammonium sulfate is added to the refolding solution under a precipitation condition, wherein the refolding solution comprises the plasminogen and aggregated polypeptides, wherein the precipitation condition is sufficient to precipitate all or a substantial portion of the aggregated proteins
Data Source
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AI summary
Compositions and methods for preparing plasminogen, in particular recombinant plasminogen, and compositions and methods of utilizing same for preparing plasmin are provided.