Dual-Function Fc-FGF21 Protein Production Without Protease Clipping
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Solution Overview
Problem
Existing methods for producing recombinant proteins face challenges such as protease-mediated clipping, leading to heterogeneity and instability, which complicates production and purification, and conventional protease inhibitors are not effective due to cytotoxicity and removal difficulties.
Innovation Solution
A culture method using dextran sulfate in the medium for producing a recombinant dual function protein, comprising a fibroblast growth factor 21 (FGF21) mutant protein and an Fc region of an immunoglobulin, with specific mutations to enhance stability and reduce aggregation, is employed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If protease inhibitors are added to the culture medium to prevent protease-mediated clipping, then protein stability is improved, but cytotoxicity increases and product purification becomes more difficult
Solution Approach 1:
The patent removes harmful protease inhibitors from the culture medium and instead extracts/eliminates the source of proteases by selecting host cells that do not secrete proteases during culture, thereby preventing protein clipping without introducing cytotoxic substances
Solution Approach 2:
The patent introduces an intermediary solution by using specific host cell lines (CHO-DG44, CHO-K1, NSO, HEK293) that serve as a biological mediator to prevent protease-mediated clipping naturally, replacing the need for chemical protease inhibitors
2Stability of the object's composition
If protease inhibitors are used during production, then protein homogeneity is improved, but the complexity of the production process increases due to removal requirements
Solution Approach 1:
The patent eliminates the need for protease inhibitor removal steps from the production process by selecting host cells that do not secrete proteases, thereby maintaining protein homogeneity while simplifying the overall production workflow
Solution Approach 2:
The host cell system is selected to naturally prevent protease-mediated clipping through its own biological properties (lack of protease secretion), making the system self-regulating and eliminating the need for external inhibitor management
3Adaptability or versatility
If a universal method is applied to all target proteins, then adaptability is improved, but manufacturing precision may be compromised due to lack of optimization
Solution Approach 1:
The patent establishes a universal production method using mammalian host cells (CHO-DG44, CHO-K1, NSO, HEK293) that can produce various recombinant proteins with Fc regions or other heterologous regions without protease-mediated clipping, applicable across different target proteins while maintaining high production precision
Data Source
AI summary
A method for producing a dual function protein includes a biologically active protein and an FGF21 mutant protein. The method allows stable production of a target protein by effectively preventing decomposition of the target protein, and thus has a high potential for commercial usage.