Tangential Flow Filtration Protein Refolding
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
E. coli expression systems face challenges in refolding larger proteins like Fc fusions due to the lack of chaperone proteins and a reducing environment, leading to protein aggregation and low expression levels, especially when proteins are produced as inclusion bodies, which require extensive optimization for each case and large volumes of reagents.
Innovation Solution
A method involving the use of tangential flow filtration (TFF) to refold denatured proteins, where denatured proteins are solubilized with a denaturing agent, diafiltered with a refold buffer, and then incubated with an oxidizing buffer to achieve partial and complete refolding, reducing the need for large volumes and optimizing each process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If proteins are expressed in E. coli inclusion bodies, then expression levels are high, but refolding is difficult and requires extensive optimization
Solution Approach 1:
The patent applies preliminary action by performing diafiltration before final refolding to gradually remove denaturing agents and exchange buffers. This preparatory step creates optimal conditions for subsequent refolding, reducing aggregation and improving recovery. The TFF process exchanges buffers in a controlled sequence (first with refolding buffer, then with oxidizing buffer) to prepare the protein for each stage of refolding.
Solution Approach 2:
The patent employs parameter changes by systematically varying buffer composition, pH, and redox conditions during the refolding process. The use of different buffers (refolding buffer with reducing agents, oxidizing buffer with oxidizing agents) and controlling parameters like pH and agent concentrations enables effective refolding from inclusion bodies without requiring extensive case-by-case optimization.
2Reliability
If large volumes of buffer are used for refolding, then protein refolding can be achieved, but reagent costs increase and processing becomes less efficient
Solution Approach 1:
The patent applies the extraction principle by removing excess buffer and denaturing agents through diafiltration with TFF. The concentrated protein solution is processed to extract unwanted components while retaining the protein, thereby reducing the volume of expensive reagents needed for subsequent refolding steps and lowering overall reagent consumption.
Solution Approach 2:
The TFF diafiltration process discards large volumes of denaturing buffer and recovers the concentrated protein in a smaller volume suitable for refolding. This recovery step reduces reagent consumption by eliminating the need to use large volumes of expensive refolding buffers while maintaining refolding effectiveness.
3Stability of the object's composition
If refolding is performed with reducing agents, then disulfide bonds are prevented from forming, but protein aggregation increases
Solution Approach 1:
The patent applies periodic action by using reducing agents in the first buffer exchange step, then removing them in subsequent steps, and finally introducing oxidizing agents in a controlled sequence. This periodic switching between reducing and oxidizing conditions allows disulfide bonds to form at the appropriate stage while minimizing aggregation, achieving both disulfide bond control and reduced aggregation.
Solution Approach 2:
The TFF diafiltration process maintains continuous buffer exchange to gradually transition from reducing to oxidizing conditions. This continuous action ensures that reducing agents are completely removed before oxidizing agents are introduced, preventing premature disulfide bond formation and aggregation while maintaining protein stability throughout the process.
4Stability of the object's composition
If refolding is performed without oxidizing agents, then protein structure is maintained, but disulfide bonds do not form correctly
Solution Approach 1:
The patent applies preliminary action by first exchanging buffers to remove denaturing agents and introduce refolding conditions, then in a second stage introducing oxidizing agents to promote disulfide bond formation. This preliminary preparation ensures the protein is in the correct conformational state before oxidizing agents are introduced, enabling correct disulfide bond formation while maintaining structural stability.
Solution Approach 2:
The patent employs parameter changes by controlling the oxidation-reduction potential through sequential buffer exchange. The transition from reducing to oxidizing conditions is achieved by changing buffer composition and redox state, enabling correct disulfide bond formation while maintaining protein structural stability through controlled parameter variation.
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 efficient refolding of proteins with higher recovery rates and shorter processing times, effectively addressing the challenges of protein aggregation and low expression levels in E. coli systems.
Implementation Method 1
diafiltering the first protein composition comprising solubilized denatured protein with 2-4 diavolumes of a refold buffer
Implementation Method 2
Diafiltering the first protein composition may be conducted at least in part with a tangential flow filtration (TFF) device
Implementation Method 3
incubating the second protein composition comprising partially refolded protein with a refold/oxidizing buffer to obtain a third protein composition comprising the protein in a refolded state
Data Source
AI summary
Provided herein are methods for refolding proteins that are denatured. Exemplary methods comprise solubilizing the denatured protein with a denaturing agent, e.g., a chaotropic agent, and renaturing the protein using a buffer exchanging system, e.g., tangential flow filtration (TFF).