PH20 Polypeptide Position 324 Variants for Stable Hyaluronidase Activity
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Solution Overview
Problem
Existing hyaluronidases, such as those derived from bovine or ovine sources, are immunogenic for human treatment and lack sufficient stability under denaturing conditions, limiting their therapeutic efficacy.
Innovation Solution
Modified PH20 polypeptides with specific amino acid replacements or modifications that enhance stability and activity, including resistance to protein denaturation conditions like elevated temperature and preservatives, while maintaining at least 40% of the unmodified hyaluronidase activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing hyaluronidases from bovine or ovine sources are used for human treatment, then they can provide hyaluronidase activity, but they are immunogenic and lack sufficient stability under denaturing conditions
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the PH20 polypeptide through site-directed mutagenesis. Specific amino acid substitutions (e.g., P324L, P324A, P324D, P324E, P324Q, P324R, P324S, P324T, P324V, P324K, P324M, P324N, P324F, P324H, P324C, P324W, P324Y, P324G, P324P) are introduced to alter the protein's physical and chemical properties, thereby improving stability under denaturing conditions while eliminating immunogenicity through humanization of the sequence
Solution Approach 2:
The patent creates a humanized version of the hyaluronidase enzyme by copying the functional structure of bovine/ovine hyaluronidase but replacing it with a human PH20 polypeptide sequence. This copying approach maintains the essential catalytic function while changing the immunogenic identity of the protein, allowing it to be used safely in human treatments
2Stability of the object's composition
If amino acid modifications are introduced to enhance stability, then resistance to denaturation improves, but there is a risk of losing enzymatic activity
Solution Approach 1:
The patent applies local quality by introducing amino acid modifications at specific, strategically chosen positions in the PH20 polypeptide sequence. Rather than random modifications, the focus is on position 324 and other specific residues that are likely to influence stability without disrupting the catalytic core. This localized approach allows stability enhancement while preserving essential enzymatic function
Solution Approach 2:
The patent employs feedback through systematic screening and characterization of multiple PH20 variants. Each amino acid modification is evaluated for its effect on both stability and activity, allowing iterative optimization. The feedback loop involves measuring hyaluronidase activity, assessing stability under denaturing conditions, and selecting variants that meet both criteria, thereby resolving the trade-off between stability and functionality
3Reliability
If the PH20 polypeptide is modified to increase stability under denaturing conditions, then therapeutic efficacy is improved, but the complexity of production and characterization increases
Solution Approach 1:
The patent applies segmentation by focusing modifications on specific regions or positions of the PH20 polypeptide rather than attempting comprehensive optimization of the entire sequence. By targeting position 324 and other key residues individually, the patent simplifies the production process compared to generating and characterizing all possible variants, while still achieving the desired stability improvement
Data Source
AI summary
Modified PH20 hyaluronidase polypeptides, including modified polypeptides that exhibit increased stability and/or increased activity, are provided. Also provided are compositions and formulations and uses thereof.