Clostripain Cleavage of Lysine-Substituted Insulin B-Chain
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Solution Overview
Problem
Conventional methods for producing active insulin analogues face challenges in efficiently converting insulin to an active form without cleaving the albumin binding domain, especially when using trypsin, which limits the production of long-acting insulin derivatives with extended in vivo half-life.
Innovation Solution
A method involving the substitution of arginine at position 22 of the insulin B-chain with lysine in an insulin analogue derivative, fused with an albumin binding domain, is reacted with clostripain to produce an active form without cleavage, utilizing specific enzymatic conditions and a linker for optimal conversion and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If trypsin is used to convert proinsulin to active insulin form, then the conversion efficiency is improved, but the albumin binding domain is cleaved resulting in loss of long-acting properties
Solution Approach 1:
The patent changes the amino acid parameter at position 22 of the B-chain from arginine to lysine. This parameter change prevents cleavage by clostripain while maintaining the ability to be converted to active form, thereby preserving the albumin binding domain integrity while achieving conversion efficiency.
Solution Approach 2:
The patent introduces clostripain as an intermediary enzyme to replace trypsin. Clostripain specifically cleaves at arginine residues but not at the modified position 22, allowing selective conversion without damaging the albumin binding domain. The intermediary enzyme mediates the conversion process with higher specificity.
2Device complexity
If arginine at position 22 of B-chain is maintained in insulin analogue, then the structure is simpler, but cleavage occurs when reacted with clostripain preventing active form production
Solution Approach 1:
The patent modifies the amino acid parameter at position 22 from arginine to lysine. This single parameter change prevents unwanted cleavage by clostripain while maintaining the overall simplicity of the insulin analogue structure and enabling efficient production of the active form.
3Ease of manufacture
If conventional insulin production methods are used, then the process is well-established, but the in vivo half-life is limited preventing long-acting formulation
Solution Approach 1:
The patent performs preliminary modification by fusing the albumin binding domain to the insulin analogue before the conversion step. This preliminary action ensures that when the protein is converted to active form, the albumin binding capability is already in place, enabling long-acting properties while using well-established production methods.
Solution Approach 2:
The patent creates a composite structure by fusing insulin analogue with albumin binding domain. This composite material combines the glucose-lowering activity of insulin with the extended circulation half-life provided by albumin binding, achieving long-acting formulation while maintaining ease of manufacture through established recombinant expression systems.
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 approach effectively produces a long-acting insulin analogue with increased in vivo half-life and stability, maintaining albumin binding properties and reducing impurities, thereby enhancing the production of a therapeutic agent for diabetes treatment.
Implementation Method 1
reacting an insulin analogue derivative which comprises an insulin analogue comprising an insulin-B chain variant represented by the amino acid sequence of SEQ ID NO: 2, arginine (Arg) at amino acid position 22 of an insulin-B chain is substituted with lysine (Lys) in native insulin, with clostripain
Data Source
AI summary
The present invention relates to a method for producing an active form of a long-acting insulin analogue derivative, in which the amino acid at position 22 of the insulin B-chain is substituted from arginine (Arg) to lysine (Lys), so that the insulin analogue can be converted to an active form without cleavage of the B-chain even when it is reacted with clostripain. In a conventional method of converting pro-insulin to an active form by use of trypsin, an albumin binding domain is cleaved, making it difficult to convert the long-acting insulin analogue derivative to an active form. The production method according to the present invention overcomes this difficulty, and thus it can be effectively used for the production of a long-acting therapeutic agent for treatment of diabetes.