Protein Refolding via Rapid Mixing and Dilution
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Solution Overview
Problem
Current methods for refolding recombinant proteins from denatured inclusion bodies are inefficient, leading to high aggregation and low yields due to non-ideal mixing conditions and concentration-dependent misfolding, especially in large-scale industrial processes.
Innovation Solution
A method involving rapid mixing of a denatured protein feed solution with a refolding buffer under ideal mixing conditions, achieving mixing times of 1 msec to 10 sec and dilution rates of 1:1 to 1:100000, which minimizes aggregation by maintaining low protein concentrations and ensuring homogeneity, followed by a plug flow reactor for further stabilization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional batch or fed-batch refolding methods are used, then the process is simple to operate, but the yield of biologically active protein is low due to high aggregation
Solution Approach 1:
The patent applies parameter changes by controlling mixing time (1 msec to 10 sec) and dilution rate (1:1 to 1:100000) to optimize the refolding process. By adjusting these parameters, the method achieves ideal mixing conditions that minimize aggregation while maximizing the yield of biologically active protein, directly resolving the contradiction between productivity and harmful aggregation effects.
Solution Approach 2:
The patent employs preliminary action by ensuring rapid mixing and immediate dilution of the denatured protein feed solution before aggregation can occur. The mixing step precedes the refolding process, creating homogeneous conditions that prevent intermolecular misfolding from the outset, thereby preventing aggregation before it can significantly reduce yield.
2Productivity
If rapid mixing with high dilution rate is applied, then aggregation is minimized and yield increases, but the device complexity increases due to specialized mixing equipment
Solution Approach 1:
The patent utilizes hydraulic principles through continuous flow mixing systems where feed solution and refolding buffer are pumped through mixing devices at controlled flow rates. This hydraulic approach enables precise control of mixing time and dilution rate without requiring complex mechanical mixing equipment, thus achieving high yield while limiting device complexity.
Solution Approach 2:
The patent replaces complex mechanical mixing systems with flow-based mixing where the movement and mixing of solutions are achieved through fluid dynamics rather than mechanical agitators. This substitution simplifies the device complexity while maintaining the rapid mixing conditions necessary to minimize aggregation and maximize yield.
3Productivity
If protein concentration is kept high to improve productivity, then the refolding process is more efficient, but intermolecular misfolding and aggregation increase
Solution Approach 1:
The patent applies parameter changes by optimizing the dilution rate (1:1 to 1:100000) to achieve the appropriate protein concentration during refolding. This parameter control ensures that the protein concentration remains low enough to prevent intermolecular misfolding and aggregation, while the overall process efficiency is maintained through continuous operation and optimized flow rates.
Solution Approach 2:
The patent implements local quality by creating different concentration zones: the initial mixing zone maintains low protein concentration to prevent aggregation, while subsequent zones allow for progressive refolding. This spatial variation in concentration ensures high folding accuracy in the critical early stages while maintaining overall productivity through continuous processing.
4Object-generated harmful factors
If mixing time is extended to ensure homogeneity, then aggregation is reduced, but the productivity decreases due to longer processing time
Solution Approach 1:
The patent applies parameter changes by optimizing mixing time to the specific range of 1 msec to 10 sec, which is sufficient to achieve the homogeneity needed to minimize aggregation. This optimized parameter prevents excessive processing time while still achieving the necessary mixing quality to prevent aggregation, thus balancing productivity and aggregation reduction.
Solution Approach 2:
The patent employs continuous flow mixing and refolding processes where the useful action of mixing and refolding continues without interruption. This continuity eliminates idle time between mixing and refolding steps, ensuring that the minimal necessary mixing time (1-10 sec) is充分利用, thereby preventing aggregation while maximizing productivity through continuous operation.
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 significantly increases the yield of biologically active recombinant proteins by reducing aggregation and ensuring complete refolding, with improved homogeneity and efficiency compared to conventional batch or fed-batch methods.
Implementation Method 1
mixing a feed solution containing the protein in its denatured form and/or its biologically inactive intermediate forms with a refolding buffer under conditions that approximate ideal mixing
Implementation Method 2
followed by a plug flow reactor for further stabilization
Data Source
AI summary
A method for refolding a protein by mixing a protein solution with a refolding buffer at mixing conditions that approximate ideal mixing. The method can be carried out batch wise, in a fed-batch mode or continuously with on-line solubilization of inclusion bodies.