Relaxin-2 Synthesis via Segmented Chain Assembly
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Solution Overview
Problem
Current methods for synthesizing relaxin-2 are inefficient, resulting in low yields and high impurity levels, making it difficult to produce a highly pure form suitable for medicinal applications, particularly for cardiovascular and renal system treatments.
Innovation Solution
A process involving the synthesis of A and B chains with protective groups, followed by chromatographic purification and simultaneous folding in ammonium hydrogencarbonate buffer, using specific amino acid derivatives and resin supports, to produce highly pure relaxin-2, which can be formulated into galenic preparations such as lyophilized forms or liposomal encapsulations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If recombinant synthesis is used to prepare relaxin-2, then purity can be maintained, but the process is tedious and yields are low
Solution Approach 1:
The relaxin-2 molecule is divided into two separate chains (A chain and B chain) that are synthesized independently using solid-phase peptide synthesis. This segmentation allows for more efficient production of each chain separately, which are then combined to form the complete relaxin-2 molecule, thereby improving overall yield while maintaining purity through controlled assembly.
2Manufacturing precision
If conventional chemical synthesis with selective introduction of disulfide bridges is used, then structural accuracy can be achieved, but enormous chemical expenditure and multiple chromatographic purification steps are required
Solution Approach 1:
Cysteine residues are pre-protected with trityl groups during the solid-phase peptide synthesis of chains A and B. This preliminary protection prevents premature disulfide bond formation and allows for controlled introduction of disulfide bridges only during the final assembly step, thereby simplifying the overall process while ensuring structural accuracy.
Solution Approach 2:
The synthesis of chains A and B is combined with the formation of disulfide bridges in a single simultaneous folding step. This merging of operations eliminates the need for separate purification steps after each disulfide bridge formation, reducing process complexity while maintaining structural integrity.
3Manufacturing precision
If multiple chromatographic purification steps are performed after each disulfide bridge formation, then purity can be maintained, but considerable loss of substance occurs
Solution Approach 1:
Multiple purification operations are merged into a single chromatographic purification step performed after the complete assembly of relaxin-2. This consolidation maintains purity while minimizing material loss by reducing the number of times the product must be processed and handled.
Solution Approach 2:
The synthesis process is designed to maintain continuous production flow without interrupting for multiple purification cycles. The single final purification step allows the synthesis to proceed continuously from raw materials to final product, minimizing material loss while ensuring purity.
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 process achieves a yield of 20-30% with >98% purity, enabling effective treatment of various cardiovascular, renal, and pulmonary disorders, including heart failure and diabetes, with stable and side-effect-free formulations.
Implementation Method 1
effecting a chromatographic purification of the individual chains A and B after the solid state synthesis
Implementation Method 2
the simultaneous folding and combination of the individual chains A and B in ammonium hydrogencarbonate buffer at pH 7.9 to 8.4
Data Source
AI summary
A process for preparing human relaxin-2 having the following amino acid sequence:A chain:(SEQ ID NO: 1)pGlu-Leu-Tyr-Ser-Ala-Leu-Ala-Asn-Lys-Cys-Cys-His-Val-Gly-Cys-Thr-Lys-Arg-Ser-Leu-Ala-Arg-Phe-CysB chain:(SEQ ID NO: 2)Asp-Ser-Trp-Met-Glu-Glu-Val-Ile-Lys-Leu-Cys-Gly-Arg-Glu-Leu-Val-Arg-Ala-Gln-Ile-Ala-Ile-Cys-Gly-Met-Ser-Thr-Trp-Ser;comprising the following steps:providing the amino acids necessary for the synthesis of the A and B chains with usual protective groups, wherein the cysteines are employed as trityl-protected amino acids (L-Cys(Trt)-OH);effecting a chromatographic purification of the individual chains A and B after the solid state synthesis;followed by the simultaneous folding and combination of the individual chains A and B in ammonium hydrogencarbonate buffer at pH 7.9 to 8.4; andsubsequent purification of the relaxin-2 formed.