Single-Chain Insulin Analog Thermal Stability via Amino Acid Substitution
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Solution Overview
Problem
Conventional insulin preparations are not stable enough for use in developing countries where refrigeration is limited, and they tend to fibrillate, leading to reduced potency and clogging issues in infusion devices.
Innovation Solution
A single-chain insulin (SCI) analog with enhanced thermal stability and resistance to fibrillation, deamidation, and chemical degradation, produced using recombinant methods and a recombinant vector construct, maintains insulin receptor selectivity and potency similar to native insulin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional insulin preparations are used, then they provide standard insulin therapy, but they exhibit poor thermal stability and fibrillation tendency at temperatures above 25°C
Solution Approach 1:
The patent modifies the amino acid sequence of insulin by substituting specific residues (e.g., B3Asn→Gln, A21Asn→Gln, B26Phe→Tyr) to change the chemical and physical parameters of the protein structure. These parameter changes enhance thermal stability and reduce fibrillation tendency without compromising insulin receptor binding activity, allowing the insulin analogue to remain stable at temperatures up to 25°C for extended periods
Solution Approach 2:
The patent creates a composite insulin analogue structure by combining modified A-chain and B-chain segments with specific amino acid substitutions. This composite structure integrates multiple stabilizing features including altered disulfide bonding patterns and modified surface properties that collectively enhance thermal stability and reduce aggregation while maintaining biological activity
2Ease of operation
If conventional insulin is stored without refrigeration, then accessibility is improved for developing countries, but stability and potency are reduced
Solution Approach 1:
The patent applies parameter changes by modifying the insulin molecule's amino acid composition to increase its thermal stability threshold. The specific substitutions (B3Asn→Gln, A21Asn→Gln, B26Phe→Tyr) alter the protein's resistance to thermal denaturation and chemical degradation, enabling the insulin analogue to maintain potency at ambient temperatures (up to 25°C) for periods exceeding 28 days without refrigeration
Solution Approach 2:
The patent implements beforehand cushioning by pre-modifying the insulin structure with stabilizing amino acid substitutions before storage. This structural cushioning protects the insulin molecule against thermal stress and chemical degradation that would otherwise occur during storage at elevated temperatures, ensuring potency is maintained throughout the extended storage period without refrigeration
3Stability of the object's composition
If insulin formulations are made more stable through modification, then thermal stability improves, but complexity of the molecular structure increases
Solution Approach 1:
The patent applies local quality by making targeted, localized amino acid substitutions at specific positions (B3, A21, B26) rather than globally modifying the entire insulin molecule. Each substitution is strategically placed to address specific stability issues (deamidation at B3 and A21, aggregation at B26) while leaving the rest of the molecular structure largely unchanged, thus minimizing overall structural complexity
Solution Approach 2:
The patent uses parameter changes by substituting specific amino acid residues with chemically similar alternatives that have improved stability properties. The substitutions (Asn→Gln at B3 and A21 to prevent deamidation, Phe→Tyr at B26 to reduce aggregation) modify local chemical parameters without fundamentally altering the overall molecular architecture, maintaining relatively simple structural changes
Data Source
AI summary
The subject matter of this invention is directed towards chemically and thermodynamically stable single-chain insulin (SCI) analogues that are resistant to deamidation and fibrillation. The invention further discloses improved methods for the recombinant expression, purification and refolding of SCI.