Fmoc Solid-Phase Synthesis for High-Purity ACTH Peptide
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Solution Overview
Problem
Current methods for synthesizing adrenocorticotropic hormone (ACTH) suffer from low yield, low purity, and difficulties in purification due to incomplete reactions and the presence of impurities, particularly in the Boc solid-phase synthesis and liquid-phase fragment synthesis techniques.
Innovation Solution
The use of Fmoc solid-phase synthesis technology with optimized synthesis and post-treatment conditions to achieve high-purity ACTH (human sequence) or its analogue, with a purity of ≥99% and ≤1% total impurities, by coupling amino acids from the C-terminal to the N-terminal and employing a cleavage cocktail and precipitation reagent for purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Boc solid-phase synthesis or liquid-phase fragment synthesis is used to manufacture ACTH, then the product can be obtained, but the yield is low and purity is low due to incomplete reactions and presence of impurities
Solution Approach 1:
The patent changes the synthesis methodology from Boc solid-phase synthesis or liquid-phase fragment synthesis to Fmoc solid-phase synthesis. This parameter change in the synthesis approach enables complete reactions and effective removal of impurities, achieving both high purity (≥99%) and high yield (≥63%) simultaneously, thereby resolving the contradiction between manufacturing precision and productivity.
2Manufacturing precision
If Boc solid-phase synthesis or liquid-phase fragment synthesis is used to manufacture ACTH, then the product can be obtained, but purification is difficult due to incomplete reactions and impurities
Solution Approach 1:
The patent employs Fmoc solid-phase synthesis which enables complete reactions and generates minimal impurities, making purification straightforward. The complete reaction ensures that the crude peptide obtained after cleavage has high purity, and subsequent purification by reversed-phase high-pressure liquid chromatography efficiently removes any remaining impurities to achieve ≥99% purity, greatly simplifying the purification process compared to previous methods.
3Manufacturing precision
If Fmoc solid-phase synthesis with optimized conditions is used, then purity is high (≥99%) and yield is high (≥63%), but the synthesis process requires optimization of multiple parameters
Solution Approach 1:
The patent performs preliminary optimization of synthesis conditions including coupling reagents, solvents, and reaction parameters before large-scale production. By establishing optimized protocols in advance for Fmoc solid-phase synthesis, the process achieves consistent high purity and high yield results without requiring complex adjustments during production, thereby reducing operational complexity while maintaining excellent product quality.
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 significantly improves the chromatographic purity and yield of ACTH, simplifies the reaction and purification processes, and is suitable for both small-scale laboratory and large-scale industrial production, reducing the complexity and cost of production while maintaining high product stability.
Implementation Method 1
Treatment of the peptidyl-resin of ACTH (human sequence) or analogue thereof with protective groups by cleavage cocktail, a solution containing ACTH (human sequence) or analogue thereof is obtained after cleaving ACTH (human sequence) or analogue thereof from the resin and removing all the protective groups from the peptide chain
Implementation Method 2
The solution containing ACTH (human sequence) or analogue thereof is treated with precipitation reagent to obtain crude product of ACTH (human sequence) or analogue thereof
Implementation Method 3
The crude product of the ACTH (human sequence) or analogue thereof is purified by liquid chromatography to obtain a composition containing the ACTH (human sequence) or analogue thereof
Data Source
AI summary
The invention belongs to the technical field of polypeptide preparation methods, and in particular relates to a high-purity ACTH (human sequence) or analogue and large-scale preparation method thereof. The main steps include: amino acids are coupled from the C-terminal to the N-terminal by Fmoc solid-phase synthesis method to obtain the crude ACTH (human sequence) or analogue peptidyl-resin with protective groups, wherein the reaction temperature of C-15 peptide synthesis is 40-60° C. After cleavge and precipitation, the crude product of ACTH (human sequence) or analogue is obtained, and then the high-purity product is obtained by liquid chromatography. The chromatographic purity of ACTH (human sequence) or analogue prepared by the invention is more than 99%, the stability is good, and the yield of the target peptide is ≥63%.