G-CSF Refolding via Stepwise Dilution and Mild Denaturants

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Solution Overview

Problem

Current methods for isolating and purifying recombinant granulocyte colony-stimulating factor (G-CSF) from inclusion bodies in prokaryotic cells are complex, costly, and inefficient, often requiring strong denaturing agents and heavy metals, which are challenging to scale up and can result in low recovery of biologically active protein.

Innovation Solution

A method involving solubilization of G-CSF with a denaturing agent followed by a sequential stepwise dilution process using a folding buffer with a reduced thiol redox pair to achieve correctly folded and purified G-CSF, avoiding strong denaturants and heavy metals, and utilizing techniques like affinity chromatography for recovery.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If existing purification processes are used to isolate G-CSF from inclusion bodies, then G-CSF can be obtained, but the process becomes complex, lengthy and costly with low recovery rates

Engineering Contradiction:

ImproveG-CSF recovery rateVSAvoidpurification process complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent changes the chemical parameters of the solubilization process by using mild denaturing agents (urea or guanidine hydrochloride) instead of strong denaturants, combined with specific reducing agents (beta-mercaptoethanol or DTT) at controlled concentrations and pH levels (pH 7.5-8.5), to achieve efficient solubilization and folding while simplifying the overall process and improving recovery rates

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent introduces intermediary substances including mild denaturing agents (urea or guanidine hydrochloride), reducing agents (beta-mercaptoethanol or DTT), and folding promoters (zymolyase or lysozyme) that mediate the solubilization and refolding process, enabling efficient G-CSF recovery without complex procedures

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If strong denaturing agents and heavy metals are used in the purification process, then G-CSF can be solubilized from inclusion bodies, but the process becomes caustic and costly at large production scale

Engineering Contradiction:

Improvescalability of production processVSAvoidcaustic effect of denaturing agents

Core Design Contradiction:

Ease of manufactureVSObject-affected harmful factors

Solution Approach 1:

The patent replaces expensive and caustic heavy metal-based denaturing agents with cheaper, biodegradable, and safer alternatives such as urea or guanidine hydrochloride combined with reducing agents, making the process more economical and environmentally friendly for large-scale production

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Solution Approach 2:

The patent converts the potentially harmful effect of strong denaturants into a beneficial mild denaturation process using urea or guanidine hydrochloride, which effectively solubilize inclusion bodies without the caustic effects and environmental hazards of heavy metals, while still achieving high G-CSF recovery

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Ease of manufacture

If G-CSF is recombinantly produced in prokaryotic cells, then production cost is reduced, but the G-CSF is expressed in inactive form with limited solubility

Engineering Contradiction:

Improveproduction costVSAvoidbiological activity of G-CSF

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent applies preliminary solubilization treatment using mild denaturing agents and reducing agents on the inclusion bodies before refolding, ensuring that the G-CSF is properly unfolded and prepared for correct folding, thereby converting the inactive form into biologically active form while maintaining cost-effectiveness of prokaryotic production

Inventive Principle:

Principle #10Preliminary action

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 method achieves biologically active G-CSF with purity greater than 80% and improved functional activity, reducing production costs and simplifying the process while maintaining high recovery rates, making it suitable for commercial production.

Implementation Method 1

solubilizing the G-CSF contained in the IBs with a solubilization buffer including a denaturing agent

Methodology Applied

Scientific EffectDenaturation:

Implementation Method 2

initiating folding of the solubilized G-CSF by diluting, via a sequential stepwise dilution process, the solubilizate from (a) with a folding buffer including only the reduced form of a thiol redox pair

Methodology Applied

Scientific EffectRedox reaction: Redox Reactions

Data Source

PatentUS20220195002A1Process for preparing granulocyte-colony stimulating factor

Publication Date: 2022.06.23 TANVEX BIOPHARMA USA INC

AI summary

Provided herein, inter alia, are compositions and methods for the isolation and/or purification of granulocyte colony-stimulating factor (G-CSF) from inclusion bodies (IBs). Some embodiments of the disclosure relate to a method for preparing biologically active and correctly folded G-CSF with improved purity and/or functional activity by optimizing the folding of recombinant G-CSF contained in the IBs. Also provided are G-CSF obtained by such methods, pharmaceutical compositions containing the same, as well as methods for the treatment and/or prevention of a disease in a subject in need thereof.