Mutant G-CSF Cysteine Insertion Site-Specific PEGylation
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Solution Overview
Problem
Current G-CSF therapies face challenges with short in vivo retention time and stability due to non-specific chemical conjugation with biocompatible polymers, which reduces biological activity and increases immunogenicity.
Innovation Solution
A mutant G-CSF with a cysteine insertion at a specific site allows for specific conjugation with polyethylene glycol (PEG), maintaining biological activity and extending in vivo retention time through precise PEGylation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If G-CSF is chemically conjugated with biocompatible polymers to extend in vivo retention time, then the in vivo half-life is extended, but the biological activity is reduced and immunogenicity is increased
Solution Approach 1:
The patent applies local quality by introducing a specific cysteine residue at position 133 of the G-CSF molecule, creating a localized conjugation site. This specific site-directed conjugation with PEG maintains the overall protein structure and biological activity while extending in vivo half-life, resolving the contradiction between extended duration and maintained reliability.
2Duration of action of stationary object
If non-specific chemical conjugation is used to modify G-CSF, then the in vivo retention time is extended, but the manufacturing precision and biological activity are reduced
Solution Approach 1:
The invention introduces a specific cysteine residue at position 133 to create a localized conjugation site, enabling site-specific PEGylation. This approach provides manufacturing precision by ensuring consistent conjugation at a defined location while maintaining biological activity, overcoming the limitations of non-specific conjugation methods.
3Ease of manufacture
If multiple cysteine residues are present in G-CSF, then chemical conjugation is facilitated, but the manufacturing precision and biological activity are reduced due to non-specific conjugation
Solution Approach 1:
The patent extracts or removes the problematic multiple cysteine residues (at positions 17, 56, and 70) by substituting them with serine or alanine, leaving only a single cysteine at position 133. This elimination of alternative conjugation sites ensures manufacturing precision by forcing conjugation to occur only at the desired location, while still maintaining ease of manufacture through the preserved cysteine conjugation capability.
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
The mutant G-CSF conjugates exhibit increased in vivo half-life and stability, with enhanced neutrophil activation effects, superior to existing Filgrastim formulations, by ensuring accurate PEG attachment without reducing biological activity.
Implementation Method 1
a mutant of human granulocyte-colony stimulating factor (G-CSF) modified by substitution or insertion of an amino acid at a specific site on the G-CSF and chemical conjugate thereof with a nonprotein polymer such as polyethylene glycol (PEG)
Data Source
AI summary
Provided are mutants of human granulocyte-colony stimulating factor (G-CSF) designed for specific chemical conjugation, and chemical conjugates thereof for use as an adjuvant in the treatment of cancer. The present invention provides a mutant of a G-CSF in which a threonine (Thr) residue at position 133 of G-CSF comprising the amino acid sequence identified in SEQ ID NO: 1 is substituted with a cysteine (Cys) residue. In addition, the invention provides a mutant of a G-CSF in which a cysteine (Cys) residue is inserted between a glycine (GIy) residue at position 135 and an alanine (Ala) residue at position 136 of G-CSF. Further, the invention provides a chemically conjugated mutant G-CSF to which biocompatible polymer such as polyethylene glycol (PEG) was attached at the cysteine residue, which was introduced by the substitution or insertion mutation, increasing its in vivo retention time without reducing in vivo biological activity due to the conjugation with the biocompatible polymer, thereby ultimately extending the in vivo biological activity.