HV2-Lys47 Hirudin Purification With Membrane and Single-Column Separation
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Solution Overview
Problem
Existing methods for purifying hirudin require multiple chromatographic separations, leading to environmental pollution and high costs, while natural hirudin productivity is limited, and traditional anticoagulants affect biochemical tests.
Innovation Solution
A method involving membrane technologies and a single molecular sieve column chromatography with water elution is used to purify hirudin mutant HV2-Lys47, reducing waste and suitable for industrial scale-up, with high anticoagulant activity and minimal interference in blood tests.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple chromatographic separations are used to purify hirudin, then purity is improved, but environmental pollution and production costs increase
Solution Approach 1:
The patent extracts and utilizes specific properties of hirudin (anticoagulant activity, molecular size, charge characteristics) to selectively separate it from fermentation broth components through a single chromatographic step, eliminating the need for multiple purification steps and reducing waste generation
Solution Approach 2:
The patent optimizes chromatographic parameters (mobile phase composition, flow rate, temperature, pH) to achieve high purity separation in a single step, transforming the purification process from multiple stages to one efficient stage that reduces environmental impact
2Manufacturing precision
If multiple chromatographic separations are used to purify hirudin, then purity is improved, but production time and costs increase
Solution Approach 1:
The patent combines multiple purification functions (desalting, concentration, and purity separation) into a single chromatographic step, merging what were previously separate sequential operations into one integrated process that reduces both time and resource consumption
Solution Approach 2:
The patent performs preliminary concentration and pretreatment of the fermentation broth before chromatography, preparing the sample in advance to enable efficient single-step purification and eliminating the need for subsequent refinement steps
3Reliability
If natural hirudin is used, then anticoagulant activity is achieved, but productivity is limited
Solution Approach 1:
The patent replaces natural extraction methods with recombinant DNA technology, substituting biological extraction from leeches with bacterial expression systems that can be scaled up indefinitely, transforming a resource-limited process into an industrially scalable one
Solution Approach 2:
The patent modifies the hirudin gene sequence to create mutants with improved expression characteristics in E. coli, changing biological parameters to enhance productivity while maintaining or improving anticoagulant activity
4Reliability
If traditional anticoagulants are used in blood collection tubes, then anticoagulant effect is achieved, but interference in biochemical tests occurs
Solution Approach 1:
The patent produces highly pure hirudin mutant through optimized chromatography, ensuring that only the active anticoagulant component is present in the final product, eliminating impurities that would otherwise interfere with biochemical measurements while maintaining the desired anticoagulant effect
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 method achieves high purity and anticoagulant activity, suitable for industrial scale-up, with minimal environmental impact and no interference in blood testing, reducing labor and waste in medical settings.
Implementation Method 1
subjecting the filtrate to molecular sieve column chromatography only once, eluting the same with water
Implementation Method 2
treating the resultant fermentation broth from the ceramic membrane or the centrifuge by an ultrafiltration membrane to remove impurity proteins; treating the resultant fermentation broth from the ceramic membrane or the centrifuge, or the resultant fermentation broth from the ultrafiltration membrane by a nanofiltration membrane to obtain a concentrated solution
Implementation Method 3
treating the sterilized fermentation broth by a ceramic membrane or by a centrifuge to remove the bacteria
Implementation Method 4
treating the sterilized fermentation broth by a ceramic membrane or by a centrifuge to remove the bacteria
Data Source
AI summary
Provided are a method for extracting a hirudin mutant, HV2-Lys47, from a fermentation broth of the hirudin mutant HV2-Lys47 produced by the fermentation of Escherichia coli, and purifying same, the use of the hirudin mutant HV2-Lys47 in an anticoagulated blood collection tube, and an anticoagulated blood collection tube containing the hirudin mutant HV2-Lys47. In the purification method, a membrane technology and a primary column chromatography technology are used to obtain the hirudin mutant HV2-Lys47.