Recombinant NGF and Mutein Production with Mixed-Mode Purification
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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 impurities, making commercially viable production challenging.
Innovation Solution
A process involving recombinant expression in E. coli, followed by protease digestion without prior chromatographic purification, and subsequent use of mixed mode chromatography for purification, including Capto® MMC and SP sepharose, to obtain high-purity NGF or muteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant expression in bacteria is used to produce NGF, then production cost is reduced and scalability is improved, but the protein forms biologically inactive aggregates with inefficient folding
Solution Approach 1:
The patent applies preliminary action by performing protease digestion immediately after cell lysis and before any chromatographic purification steps. This preliminary proteolytic treatment cleaves the precursor peptide from the mature NGF protein while the protein is still in the crude extract, preventing aggregate formation and enabling proper folding before purification begins. The digestion step is performed in situ without isolating the protein first, which resolves the contradiction between scalable bacterial production and proper protein folding.
2Manufacturing precision
If traditional purification methods are used after bacterial expression, then protein purity is improved, but yield is reduced due to losses during multiple purification steps
Solution Approach 1:
The patent merges the protease digestion step with the crude extract preparation step, performing both operations in the same solution without intermediate isolation. The protease is added directly to the crude bacterial extract, and digestion proceeds during the purification process itself. This merging eliminates separate processing steps that would otherwise cause yield losses, while still achieving high purity through subsequent mixed-mode chromatography.
Solution Approach 2:
The patent maintains continuity of useful action by performing protease digestion continuously during the purification process rather than as a separate batch step. The digestion occurs while the protein flows through the purification system, ensuring that the mature NGF is generated and purified in a continuous manner, minimizing losses and maximizing yield while maintaining high purity.
3Manufacturing precision
If protease digestion is performed before purification, then mature NGF is generated for purification, but impurities from crude extract may interfere with the process
Solution Approach 1:
The patent uses the mixed-mode chromatography resin as an intermediary that selectively binds the mature NGF protein while allowing crude extract impurities to pass through or be washed away. The resin's dual-mode binding mechanism (ion-exchange and hydrophobic interaction) provides selective recognition of the mature NGF structure, enabling it to act as a mediator that separates the desired product from impurities generated during protease digestion in the crude extract.
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 high-purity and high-yield production of NGF or muteins, overcoming the limitations of existing technologies by ensuring efficient protease digestion and effective purification, suitable for therapeutic applications.
Implementation Method 1
purification on a mixed mode stationary phase
Implementation Method 2
mixed mode chromatography comprises the use of a stationary phase having a charged group, preferably negatively charged group
Implementation Method 3
exposure to a protease
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.