Fusion Protein Refolding for Myasthenia Gravis Treatment
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Solution Overview
Problem
Current treatments for myasthenia gravis are limited in effectiveness and often cause serious side effects, as they primarily focus on symptom relief rather than addressing the root cause of the autoimmune attack on the acetylcholine receptor (AChR), leading to a significant unmet need for more effective treatment options.
Innovation Solution
A method for producing a fusion protein between the extracellular domain of the nicotine acetylcholine receptor subunit alpha 1 (nAChRα1) and a solubility-enhancing peptide, which is refolded into a monomeric form to restore tolerance to the immune system, specifically targeting the AChR proteins incorrectly attacked in myasthenia gravis, using a solubilization and refolding process involving specific pH adjustments and the use of ion-exchange resin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current symptom-relieving therapies (acetylcholinesterase inhibitors or immunosuppressive therapy) are used, then muscle weakness and fatigue are temporarily relieved, but the root cause of the autoimmune attack on AChR is not addressed and serious side effects occur
Solution Approach 1:
The patent extracts the extracellular domain of the nAChRα1 subunit (amino acids 1-58, 84-235) as a fusion protein to specifically target and neutralize the autoimmune attack on AChR. This extracted domain serves as a tolerogen that induces immune tolerance without causing the side effects of conventional therapies.
Solution Approach 2:
The fusion protein acts as an intermediary substance that mediates between the immune system and the AChR. By introducing the extracellular domain as a tolerogen, it creates immune tolerance to the native AChR, thereby stopping the autoimmune attack without requiring immunosuppressive drugs that cause serious side effects.
2Productivity
If the fusion protein is produced in inclusion bodies, then high expression levels are achieved, but the protein requires complex solubilization and refolding processes to achieve monomeric form
Solution Approach 1:
The patent employs systematic parameter changes throughout the purification process: pH adjustment from basic (pH 11-12) during solubilization to neutral/acidic conditions during refolding, temperature control at 4°C, and controlled dilution ratios. These parameter changes enable the transformation from inclusion bodies to monomeric fusion protein while maintaining high expression levels.
Solution Approach 2:
The fusion protein is first expressed in inclusion bodies as a concentrated intermediate form, then systematically solubilized and refolded through controlled pH adjustment and dilution. This preliminary action of expressing in inclusion bodies allows high productivity while the subsequent controlled refolding process achieves the desired monomeric form.
3Ease of manufacture
If the fusion protein is solubilized at high pH (pH 11-12), then inclusion bodies are effectively dissolved, but the protein may aggregate or misfold during refolding
Solution Approach 1:
The patent employs periodic action through stepwise pH adjustment and controlled dilution. The protein is first solubilized at high pH, then systematically refolded through controlled pH reduction and dilution steps. This periodic action allows complete solubilization while preventing aggregation and misfold4ing during the refolding process.
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
The fusion protein effectively reduces muscle weakness and fatigue in myasthenia gravis patients by creating or restoring tolerance to the body's own AChR proteins, offering a potentially more effective treatment with fewer side effects compared to existing therapies.
Implementation Method 1
solubilizing inclusion bodies comprising the fusion protein in a solubilization solution having a pH less than 11
Implementation Method 2
loading the solubilized fusion proteins onto an ion-exchange resin
Implementation Method 3
diluting the pH-adjusted fusion protein eluate in a refolding solution having a pH of no more than 9 to form a refolded monomeric form of the fusion protein
Data Source
AI summary
The invention relates to a method of producing a fusion protein between an extracellular domain of neuronal acetylcholine receptor subunit alpha 1 (nAChR&1) and a solubility enhancing peptide. The method comprises solubilizing inclusion bodies comprising the fusion protein in a solubilization solution having a pH of at least 11 to form solubilized fusion proteins. The method also comprises diluting the solubilized fusion protein in a refolding solution having a pH of no more than 9 to form a refolded monomeric form of the fusion protein.