Engineered A1M Protein Variants for Enhanced Stability

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Solution Overview

Problem

Recombinant human alpha-1-microglobulin (A1M) expressed in E.coli lacks glycosylation and has poor stability and solubility, limiting its use as a therapeutic agent due to difficulties in obtaining highly concentrated solutions and long-term storage, especially at physiological pH and salt conditions.

Innovation Solution

Site-directed mutagenesis and N-terminal extensions were used to engineer A1M variants with improved stability and solubility, including specific amino acid substitutions and charged hydrophilic extensions, to enhance thermostability and solubility without compromising physiological functions.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If recombinant human A1M is expressed in E.coli, then production is achieved, but stability and solubility are poor

Engineering Contradiction:

Improveproduction capabilityVSAvoidstability

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent applies parameter changes by modifying amino acid residues at specific positions (N17, N96, R66) in the A1M protein sequence. These parameter changes at the molecular level alter the protein's physical-chemical properties, resulting in improved stability and solubility while maintaining production capability in E.coli expression systems.

Inventive Principle:

Principle #35Parameter changes

2Ease of manufacture

If recombinant human A1M is expressed in E.coli, then production is achieved, but solubility is poor

Engineering Contradiction:

Improveproduction capabilityVSAvoidsolubility

Core Design Contradiction:

Ease of manufactureVSStability of the object's composition

Solution Approach 1:

The patent modifies specific amino acid parameters (N17, N96, R66 positions) to change the protein's solubility characteristics. These parameter changes reduce aggregation and improve solubility of the recombinant A1M produced in E.coli, enabling better formulation and storage.

Inventive Principle:

Principle #35Parameter changes

3Quantity of substance

If high concentration solutions are prepared, then therapeutic efficacy is improved, but storage becomes difficult

Engineering Contradiction:

ImproveconcentrationVSAvoidstorage stability

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent applies parameter changes to the A1M protein structure (amino acid substitutions at N17, N96, R66) that enable the protein to maintain stability even at high concentrations. This resolves the contradiction by allowing high concentration formulations to be stored long-term without precipitation or aggregation.

Inventive Principle:

Principle #35Parameter changes

4Adaptability or versatility

If physiological pH and salt conditions are used, then therapeutic applicability is improved, but protein stability deteriorates

Engineering Contradiction:

Improvephysiological compatibilityVSAvoidprotein stability

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent modifies the protein's intrinsic parameters (amino acid sequence at N17, N96, R66) to enhance its stability under physiological conditions. This allows the protein to maintain both physiological compatibility and stability, resolving the contradiction between therapeutic applicability and protein stability.

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentEP3430032B1Novel alpha-1-microglobulin derived proteins and their use

Publication Date: 2021.09.01 GUARD THERAPEUTICS INT AB

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AI summary

This invention relates to an alpha-1-microglobulin derived protein for medical use.