NGF Mutein Purification After Protease Cleavage in E. Coli
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Solution Overview
Problem
Current methods for producing nerve growth factor (NGF) and its muteins in bacteria result in biologically inactive aggregates, with inefficient folding, purification, and high costs due to low yields and purity issues, particularly when using trypsin for cleavage.
Innovation Solution
A method involving recombinant expression of NGF or muteins in E. coli, followed by protease treatment without prior chromatographic purification, and subsequent purification using mixed mode chromatography with Capto MMC and SP sepharose to achieve high purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If trypsin is used for cleavage of pro-NGF, then cleavage efficiency is improved, but manufacturing precision deteriorates due to generation of degradation products
Solution Approach 1:
The patent modifies the cleavage conditions by controlling pH, temperature, and trypsin-to-substrate ratio to optimize the balance between cleavage efficiency and specificity. By carefully adjusting these parameters, the process achieves high cleavage efficiency while minimizing non-specific degradation of the NGF product.
2Manufacturing precision
If multiple chromatographic purification steps are performed, then purity of NGF is improved, but productivity deteriorates due to time-consuming sequential operations
Solution Approach 1:
The patent combines multiple chromatographic purification steps into a single integrated workflow where cleavage and purification occur in sequence without intermediate processing. The crude cleavage mixture is directly applied to the chromatography column, merging the cleavage and purification operations into an efficient continuous process that maintains high purity while reducing overall production time.
3Productivity
If NGF is produced in bacteria, then productivity is improved, but manufacturing precision deteriorates due to formation of inclusion bodies and misfolding
Solution Approach 1:
The patent performs proteolytic cleavage of the pro-NGF precursor while it is still in the inclusion body form or during early solubilization stages, before complete misfolding occurs. This preliminary cleavage action releases the mature NGF from the pro-sequence, allowing proper folding to occur subsequently during the chromatography process, thereby achieving both high yield and correct folding.
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 yields NGF or muteins with high purity and efficiency, overcoming the limitations of existing methods by ensuring effective cleavage and purification, thereby making commercially viable quantities available.
Implementation Method 1
wherein the precursor of NGF or of a mutein thereof is subjected to a step (c) exposure to a protease
Implementation Method 2
the purification comprises purification on a mixed mode stationary phase
Implementation Method 3
purification on a mixed mode stationary phase
Data Source
AI summary
The present invention relates to a process for production of nerve growth factor (NGF) and muteins thereof, in particular muteins of human NGF. The process of the present invention yields nerve growth factor (NGF) and muteins thereof, e.g. from recombinant sources, at high purity. Aspects related to the process of the present invention, such as muteins obtainable thereby, are also described. The respective muteins may be characterized e.g. by improved detectability and/or reduced nociceptive activity, compared to wildtype human NGF.