Production of nerve growth factor (NGF) and of muteins thereof
Inventors
Assignees
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Publication Number
US-12378293-B2
Publication Date
2025-08-05
Expiration Date
Abstract
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.
Core Innovation
The invention relates to production of a mutein of nerve growth factor (NGF), wherein the mutein is characterized by more than 90% sequence identity with SEQ ID NO: 2 and comprises at least one mutation at any of positions 95-101 of SEQ ID NO: 2. The process obtains a precursor of the mutein in a non-properly folded form, re-folds the non-properly folded form to produce a re-folded precursor, and proteolytically cleaves the re-folded precursor prior to purification to produce the mutein of NGF.
Purification comprises, in order, capturing using mixed mode chromatography and polishing using cation exchange chromatography. Mixed mode chromatography uses a mixed mode chromatography stationary phase, with stationary-phase options characterized by aromatic interactions and electrostatic interactions, including Capto Blue, Capto MMC, and MEP HyperCel Mixed-Mode Chromatography Sorbent.
The mixed mode stationary phase is associated with charged ligand behavior such that the ligand exhibits a negative net charge at pH 7. The document contrasts mixed-mode capture with conventional ion exchange in the strict sense and notes that ion exchange directly after protease digestion is inefficient, while mixed-mode capture with low-conductivity eluate enables direct polishing without buffer exchange.
Claims Coverage
The independent claim coverage includes three main inventive areas: the NGF mutein definition, the production sequence with re-folding and proteolytic cleavage prior to purification, and the ordered purification scheme using mixed mode chromatography for capturing followed by cation exchange chromatography for polishing. Dependent claims further specify particular mutein substitutions, precursor form, protease options, and mixed-mode stationary phase behavior.
NGF mutein definition with 95-101 mutations
The mutein of nerve growth factor (NGF) is characterized by more than 90% sequence identity with SEQ ID NO: 2 and comprises at least one mutation at any of positions 95-101 of SEQ ID NO: 2.
Non-properly folded precursor re-folding followed by proteolytic cleavage prior to purification
The process obtains a precursor of the mutein in a non-properly folded form, re-folds the non-properly folded form to produce a re-folded precursor, and proteolytically cleaves the re-folded precursor prior to purification to produce the mutein of NGF.
Purification workflow: mixed mode capturing then cation exchange polishing
Purifying the mutein of NGF comprises, in order, capturing using mixed mode chromatography and polishing using cation exchange chromatography.
Mixed mode chromatography stationary phase
Capturing using mixed mode chromatography comprises using a mixed mode chromatography stationary phase.
Precursors obtained in inclusion bodies
The precursor of the mutein is obtained in inclusion bodies.
Proteolytic cleavage using trypsin or furin
The proteolytic cleavage is performed using a protease selected from trypsin or furin.
Substitution at position 100
The mutein comprises an arginine substitution at position 100, such as R100E.
Substitution replacing Pro61
The mutein includes a substitution replacing the proline at position 61 of SEQ ID NO: 2 with another amino acid, such as P61S.
The claims cover an NGF mutein defined by more than 90% identity to SEQ ID NO: 2 and mutations in positions 95-101, together with a production route that includes re-folding a non-properly folded precursor, proteolytic cleavage prior to purification, and mixed mode capture followed by cation exchange polishing. Dependent claims further narrow specific substitutions and process features.
Stated Advantages
Improved purity and yield, with reduction of impurities including charge variants such as des-nona degradation product.
Mixed-mode capture is contrasted with ion exchange in the strict sense, and ion exchange directly after protease digestion is described as experimentally inefficient.
Mixed-mode capture with low-conductivity eluate enables direct polishing without buffer exchange.
Documented Applications
Production of a mutein of nerve growth factor (NGF), including purification using a capture step based on mixed mode chromatography followed by polishing using cation exchange chromatography.
Biological and assay context is described, including TrkA and p75/LNGFR/p75NTR recognition and use in a TFI proliferation assay, alongside nociceptive activity, detectability, and selective recognition by immunoreagents as characterized in the document summary.
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