Insulin Refolding Process for Correct Disulfide Bonding
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Solution Overview
Problem
Current processes for producing insulin from genetically engineered E. coli cells result in precursor molecules with incorrectly bonded cysteine bridges, requiring complex and inefficient refolding steps with high purification losses.
Innovation Solution
A process involving solubilization with urea, dithiothreitol, and ethanolamine, followed by dilution with a refold solution containing cystamine, to achieve correct cysteine bridge bonding in insulin precursor molecules, including enzymatic processing and purification steps to obtain correctly bonded insulin or insulin analogs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional refolding processes are used for proinsulin from E. coli, then cysteine bridges can be formed, but the process requires many steps and results in high purification losses
Solution Approach 1:
The patent combines multiple refolding steps into a single optimized process by using a specific solvent system (urea, DTT, ethanolamine) that simultaneously achieves denaturation, disulfide bond reduction, and refolding in one continuous operation, eliminating the need for separate sequential steps required by conventional methods
Solution Approach 2:
The invention employs specific concentration parameters (3.8-4.3 M urea, 2.2-2.8 mM DTT, 265-365 mM ethanolamine) and pH control (pH 10.5) to optimize the refolding process, where precise parameter control enables correct disulfide bond formation while minimizing purification losses and reducing overall process complexity
2Manufacturing precision
If conventional refolding processes are used for proinsulin from E. coli, then cysteine bridges can be formed, but purification losses are high
Solution Approach 1:
The patent uses optimized concentration parameters (3.8-4.3 M urea, 2.2-2.8 mM DTT, 265-365 mM ethanolamine) and pH control (pH 10.5) to maximize the efficiency of correct disulfide bond formation during refolding, thereby reducing purification losses by minimizing incorrect folding that would require extensive purification
Solution Approach 2:
The invention converts the potentially harmful effect of high urea concentration (which can cause aggregation) into a benefit by combining it with specific ratios of DTT and ethanolamine that protect against aggregation while promoting correct refolding, thus reducing substance loss
3Quantity of substance
If inclusion bodies are solubilized with conventional methods, then precursor molecules can be obtained, but incorrect cysteine bridge bonding occurs
Solution Approach 1:
The patent employs a specific solvent system with precisely controlled parameters (3.8-4.3 M urea, 2.2-2.8 mM DTT, 265-365 mM ethanolamine at pH 10.5) that simultaneously achieves complete solubilization of inclusion bodies and correct refolding with accurate disulfide bond formation, eliminating the need for separate refolding steps
Solution Approach 2:
The invention uses ethanolamine as an intermediary substance that mediates between the denaturing urea environment and the refolding process, protecting cysteine residues from incorrect bonding while maintaining solubilization conditions, thereby ensuring correct disulfide bridge formation
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 method reduces the number of process steps, minimizes purification losses, and achieves high yields of correctly bonded insulin or insulin analogs with improved efficiency and concentration handling.
Implementation Method 1
solubilizing the inclusion bodies by adding urea, dithiothreitol (DTT), and ethanolamine to the suspension
Implementation Method 2
solubilizing the inclusion bodies by adding urea, dithiothreitol (DTT), and ethanolamine to the suspension
Implementation Method 3
incubating the refold solution with stirring to provide aeration for 6 to 12 hours to provide the precursor of insulin or insulin analog having correctly bonded cysteine bridges
Data Source
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AI summary
A process for solubilization and refolding of precursor insulin or insulin analogs from inclusion body isolates for use in the production of insulin or insulin analog is described.