Redox Buffer Refolding Proteins at High Concentrations
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Solution Overview
Problem
Current methods for refolding complex proteins, such as antibodies and Fc fusion proteins, are limited by the need for low concentrations (typically below 2.0 g/L) due to issues with incorrectly paired disulfide bonds and protein aggregation, which complicates industrial-scale production.
Innovation Solution
A method involving a refold buffer with a redox component having a thiol-pair ratio between 0.001 and 100 and a redox buffer strength of 2 mM or greater, along with denaturants, aggregation suppressors, and protein stabilizers, is used to refold proteins at concentrations of 2.0 g/L or higher.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If proteins are refolded at typical low concentrations (0.01-0.5 g/L), then correct disulfide bond formation is achieved, but large volumes are required making industrial-scale production inefficient
Solution Approach 1:
The patent changes the redox parameters of the refolding buffer by incorporating a redox-active polymer with specific molecular weight and functional group density. This modifies the redox potential and disulfide exchange kinetics, enabling correct disulfide bond formation at high protein concentrations (2.0 g/L or greater) without requiring large volumes, thus resolving the contradiction between reliability of disulfide bonding and productivity
2Productivity
If refolding is performed at high concentrations (2.0 g/L or greater), then production efficiency improves, but incorrectly paired disulfide bonds and protein aggregation increase
Solution Approach 1:
The patent introduces a redox-active polymer as an intermediary substance that mediates disulfide bond formation. The polymer acts as a redox buffer and disulfide exchange catalyst, facilitating correct pairing of disulfide bonds even at high protein concentrations where direct protein-protein interactions would otherwise lead to mispairing and aggregation. This intermediary enables high productivity while maintaining reliability
3Quantity of substance
If large volumes are used for refolding industrial-scale protein production, then sufficient protein can be processed, but time and resources are significantly consumed
Solution Approach 1:
By changing the redox parameters through the use of redox-active polymers, the patent enables refolding at high protein concentrations (2.0 g/L or greater). This concentration increase directly reduces the volume required for refolding while maintaining correct disulfide bond formation, thereby reducing both the time and resources needed for industrial-scale protein production without sacrificing quantity or quality
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 enables efficient refolding of complex proteins at high concentrations, reducing the need for large volumes and minimizing protein aggregation, thereby enhancing production efficiency and cost-effectiveness on both small and industrial scales.
Implementation Method 1
refold buffer with a redox component having a thiol-pair ratio between 0.001 and 100 and a redox buffer strength of 2 mM or greater
Data Source
AI summary
A method of refolding proteins expressed in non-mammalian cells present in concentrations of 2.0 g/L or higher is disclosed. The method comprises identifying the thiol pair ratio and the redox buffer strength to achieve conditions under which efficient folding at concentrations of 2.0 g/L or higher is achieved and can be employed over a range of volumes, including commercial scale.