High pH Protein Refolding Without Denaturing Agents
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Solution Overview
Problem
Current methods for refolding denatured proteins, particularly those in inclusion bodies, are inefficient and require large volumes of denaturing agents and specific optimization for each protein, making them challenging and costly for manufacturing.
Innovation Solution
A method involving suspending denatured proteins in a suspension solution, followed by solubilization with a strongly alkaline buffer and subsequent refolding with a buffer of specific pH ranges, without the use of significant denaturing or reducing agents, to achieve efficient protein refolding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current refolding methods are used for denatured proteins in inclusion bodies, then refolding can be achieved, but large volumes of denaturing agents are required and the process requires extensive optimization for each protein
Solution Approach 1:
The invention changes the pH parameter of the buffer solution to strongly alkaline conditions (pH 10-13), which fundamentally alters the refolding mechanism. This parameter change enables refolding without requiring large volumes of traditional denaturing agents, directly resolving the contradiction between refolding efficiency and reagent volume requirements
Solution Approach 2:
The invention replaces expensive, complex denaturing agent systems with simple, inexpensive alkaline buffers that can be readily prepared and discarded. This substitution reduces both the volume and cost of reagents while maintaining effective refolding, addressing the contradiction between productivity and quantity of substance
2Manufacturing precision
If traditional refolding processes are used, then protein refolding can occur, but the processes require very low protein concentrations and consequently large volumes
Solution Approach 1:
By changing to strongly alkaline pH conditions, the invention enables refolding at much higher protein concentrations than traditional methods. This parameter change allows the same refolding accuracy to be achieved in smaller volumes, directly resolving the contradiction between manufacturing precision and refolding volume
3Reliability
If refolding is performed to restore protein from inclusion bodies, then biologically active conformation can be achieved, but aggregation and precipitation losses occur
Solution Approach 1:
The strongly alkaline pH conditions create a unique chemical environment that prevents aggregation and precipitation during refolding. This parameter change maintains high protein activity recovery while minimizing substance loss, directly resolving the contradiction between reliability and loss of substance
4Manufacturing precision
If Fc fusion proteins are refolded using conventional methods, then some disulfide bonds may form, but the six disulfide bonds required for native Fc structure are difficult to correctly form
Solution Approach 1:
The strongly alkaline pH conditions fundamentally change the chemistry of disulfide bond formation, enabling all six disulfide bonds of Fc fusion proteins to form correctly. This single parameter change simplifies the refolding process while achieving high manufacturing precision for disulfide bond formation, resolving the contradiction between manufacturing precision and device complexity
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 allows for high-yield refolding of proteins with reduced aggregation and precipitation, minimizing the need for large volumes and expensive reagents, and effectively forming disulfide bonds in Fc fusion proteins.
Implementation Method 1
combining the composition comprising suspended denatured proteins with a solubilization buffer having a pH in the range of 10.5 to 13 to thereby obtain a composition comprising solubilized denatured proteins
Implementation Method 2
combining the composition comprising solubilized denatured proteins with a refold buffer having a pH in the range of 9 to 11 to thereby obtain a composition comprising refolded proteins
Implementation Method 3
The Fc region of human IgG1 antibodies contains six disulfide bonds... the refolding process must correctly form the six disulfide bonds that exist in the native form of the protein
Data Source
AI summary
Provided herein are methods for refolding denatured protein (e.g., from inclusion bodies) that do not require the use of a denaturing agent. Exemplary methods use a high pH for solubilizing denatured protein, followed by a decrease in pH for refolding the proteins.