FGF-2 Muteins Enhancing Thermostability and Activity

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Solution Overview

Problem

Fibroblast growth factor-2 (FGF-2) is highly labile at 37°C, leading to rapid loss of biological activity, which limits its usage in cell culture due to instability and aggregation, necessitating frequent additions to maintain effective concentrations.

Innovation Solution

Development of FGF-2 muteins with specific amino acid substitutions, such as Q56I, N102G, K119N, A75C, C69S, and C87S, which enhance thermostability, protease resistance, and biological activity, allowing for reduced frequency and concentration of FGF-2 additions in cell culture media.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If wild-type FGF-2 is used in cell culture at 37°C, then biological activity is initially present, but activity is rapidly lost due to lability and aggregation

Engineering Contradiction:

Improvebiological activity stabilityVSAvoidduration of biological activity

Core Design Contradiction:

ReliabilityVSDuration of action of stationary object

Solution Approach 1:

The patent applies amino acid substitutions (Q56I, N102G, K119N, A75C, C69S, C87S) to modify the molecular parameters of FGF-2, transforming it from a labile protein to a thermostable mutein that maintains biological activity at 37°C for extended periods without aggregation

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent replaces the unstable wild-type FGF-2 (short-lived at 37°C) with engineered muteins that have extended half-life, eliminating the need for frequent media changes and protein replenishment in cell culture applications

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

2Reliability

If FGF-2 is added frequently to maintain effective concentrations, then cell culture conditions are maintained, but experimental complexity and time increase

Engineering Contradiction:

Improvecell culture condition stabilityVSAvoidtime for frequent additions

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent incorporates stability-enhancing amino acid mutations into the FGF-2 sequence beforehand, creating muteins that are pre-adapted to withstand 37°C incubation without degradation, thereby eliminating the need for subsequent frequent additions during cell culture

Inventive Principle:

Principle #10Preliminary action

3Reliability

If FGF-2 is used at higher concentrations to compensate for rapid loss, then initial activity is sufficient, but total protein usage and cost increase

Engineering Contradiction:

Improvebiological activity effectivenessVSAvoidtotal FGF-2 consumption

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The patent modifies the thermal stability parameter of FGF-2 through site-directed mutagenesis, creating muteins that maintain potency at lower concentrations over extended periods, thereby reducing total protein consumption while maintaining cultural effectiveness

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS10336799B2Fibroblast growth factor muteins with increased activity

Publication Date: 2019.07.02 MILTENYI BIOTEC BV & CO KG

AI summary

The present invention provides FGF-2 polypeptides that differ from the wild-type FGF-2 (SEQ ID NO:1) at least at amino acid position 56, 102 and 119, wherein the differences are Q56I, N102G, K119N substitutions, leading to FGF-2 polypeptides with higher thermostability, higher biological activity and higher resistance to proteolytic degradation. These polypeptides can be used in cell culture media.