Low Glycosylated KLK1 Mutants with PEG Modification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current KLK1 products face challenges with glycosylation control, stability, short half-life, and immunogenicity, which affect their efficacy and safety as pharmaceutical agents.
Innovation Solution
Development of low glycosylated KLK1 mutants with reduced N-glycosylation at specific sites, combined with pegylation to enhance stability and reduce immunogenicity, using polyethylene glycol modification to improve pharmacokinetic properties and homogeneity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If high glycosylated KLK1 is produced through conventional expression systems, then the product shows moderate enzyme activity, but the glycosylation level is difficult to control and product homogeneity is poor
Solution Approach 1:
The patent removes the problematic NFS glycosylation motif from the KLK1 sequence, extracting the source of uncontrolled glycosylation. This allows production of homogeneous low-glycosylated KLK1 without needing to optimize complex expression conditions to control glycosylation levels.
Solution Approach 2:
The patent changes the amino acid sequence parameter at the NFS motif to prevent N-glycosylation. By mutating the asparagine residue in the NFS motif to a non-glycosylatable amino acid, the product consistently shows low glycosylation levels with high homogeneity, eliminating the need for complex expression system optimization.
2Ease of manufacture
If low glycosylated KLK1 is produced without motif mutation, then purification is simpler, but enzyme activity and stability are insufficient
Solution Approach 1:
The patent removes the NFS glycosylation motif to simplify purification, but additionally introduces point mutations in the catalytic domain to enhance enzyme activity and stability. This combination maintains manufacturing simplicity while improving product reliability.
Solution Approach 2:
The patent changes amino acid residues in the catalytic domain through point mutations to optimize enzyme activity and stability. These parameter changes in the active site compensate for the reduced glycosylation, maintaining or enhancing therapeutic efficacy while keeping purification simple.
3Duration of action of moving object
If conventional KLK1 products are used, then they show immunogenicity and short half-life, but pegylation increases molecular weight and may affect enzyme activity
Solution Approach 1:
The patent uses polyethylene glycol (PEG) as an intermediary molecule attached to KLK1. This PEGylation modifies the molecular structure to reduce immunogenicity and extend half-life, while the low-glycosylated variant maintains better enzyme activity compared to highly glycosylated forms.
Solution Approach 2:
The patent creates a composite structure by combining KLK1 with PEG chains. This composite material approach produces a conjugate that combines the therapeutic benefits of KLK1 with the pharmacokinetic advantages of PEG, including reduced immunogenicity and extended circulation half-life.
4Productivity
If animal-sourced KLK1 is extracted, then production is straightforward, but raw material availability is limited and safety concerns exist
Solution Approach 1:
The patent creates a recombinant human KLK1 copy expressed in CHO cells, replacing animal-sourced extraction. This recombinant approach ensures unlimited production capacity while guaranteeing human sequence identity and reduced immunogenicity, addressing both productivity and safety concerns.
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 low glycosylated KLK1 mutants demonstrate higher activity and stability, with pegylation leading to longer-acting, safer, and more controllable pharmaceutical formulations, reducing administration frequency and improving patient compliance for conditions like acute ischemic stroke and diabetic nephropathy.
Implementation Method 1
polyethylene glycol modification to improve pharmacokinetic properties
Implementation Method 2
KLK1 is a serine protease with two major classes... plays a series of biological roles by converting kininogens into kinins
Data Source
AI summary
Provided are low glycosylated kallikrein I with no or a small amount of glycosylation at the NFS sequence and polyethylene glycol modified product and pharmaceutical applications. KLK1 with lower glycosylation at NFS is more active than KLK1 with higher glycosylation at NFS. A recombinant KLK1 mutant without N-glycosylation at the NFS sequence is also provided, containing only two N-glycosylation sites at NMS and NHT. Glycosylation of the recombinant KLK1 mutant is relatively more consistent, the molecular weight of the product is relatively more homogeneous, the yield is higher, the purification process is simpler, and the biological activity is higher, and the quality is more controllable.