PH20 Hyaluronidase Variants for Thermal Stability and Expression
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Solution Overview
Problem
Conventional recombinant PH20 hyaluronidase exhibits insufficient thermal stability and expression levels, leading to issues with protein aggregation and reduced efficacy in high-concentration pharmaceutical applications.
Innovation Solution
Development of PH20 variants with specific amino acid substitutions, deletions, and insertions in the alpha-helix and linker regions, along with selective N- and C-terminal deletions, to enhance thermal stability and enzymatic activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If recombinant PH20 hyaluronidase is produced using conventional methods, then enzymatic activity is achieved, but thermal stability is low and expression levels are reduced
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (e.g., L342W, L345W, L348W substitutions) to alter the protein's thermal stability and expression characteristics. These point mutations change the physical-chemical parameters of the enzyme, improving its stability without sacrificing enzymatic activity.
Solution Approach 2:
The patent creates composite structures by fusing the PH20 hyaluronidase coding sequence with affinity tag sequences (such as hexa-histidine tag). This composite design allows the enzyme to maintain its functional properties while enabling improved purification and stability characteristics.
2Reliability
If recombinant PH20 hyaluronidase is produced using conventional methods, then enzymatic activity is achieved, but immunogenicity is increased
Solution Approach 1:
The patent extracts and removes the immunogenic signal peptide sequence from the native PH20 hyaluronidase. By deleting this N-terminal region that is responsible for immunogenicity while retaining the catalytic domain, the enzyme maintains its enzymatic activity but exhibits reduced immunogenicity, allowing repeated administrations.
Solution Approach 2:
The patent replaces the native immunogenic protein structure with a modified version that has reduced immunogenicity. The truncated PH20 variant acts as a functional substitute that achieves the same therapeutic effect (hyaluronic acid degradation) without triggering strong immune responses.
3Reliability
If amino acid substitutions are made to improve thermal stability, then thermal stability increases, but enzymatic activity may be reduced
Solution Approach 1:
The patent applies local quality changes by making specific amino acid substitutions at particular positions (e.g., positions 342, 345, 348 in the alpha-helix region) that locally enhance thermal stability without disrupting the overall catalytic function. These localized modifications preserve enzymatic activity while improving global stability.
Solution Approach 2:
The patent carefully selects amino acid substitutions that change local structural parameters (hydrophobicity, steric bulk, hydrogen bonding capacity) to enhance thermal stability. For example, introducing tryptophan residues at specific positions strengthens hydrophobic interactions and stabilizes the protein structure without blocking the active site.
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 PH20 variants demonstrate improved protein expression levels, increased thermal stability, and maintained enzymatic activity, with enhanced protein refolding and reduced immunogenicity, suitable for repeated administration.
Implementation Method 1
Hyaluronidases are enzymes that degrade hyaluronic acid present in the extracellular matrix
Implementation Method 2
The catalytic amino acids of PH20 are D111 and E113
Implementation Method 3
the PH20 variants or fragments thereof according to the present invention have improved protein refolding so that they are re-natured faster than the mature wild-type PH20
Data Source
AI summary
Disclosed are novel PH20 variants or fragments thereof with improved thermal stability and enzymatic activity of human hyaluronidase, which is an enzyme that hydrolyzes hyaluronic acid, and more particularly novel PH20 variants or fragments thereof including one or more amino acid residue substitutions in the variant having the amino acid sequence of SEQ ID NO: 3, wherein one or more amino acid residues at the N-terminus and/or the C-terminus are optionally further deleted.