Hyal1 Variant Engineering for Neutral pH Hyaluronic Acid Hydrolysis
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Solution Overview
Problem
Human hyaluronidase Hyal1 is limited to functioning effectively only in acidic conditions, making it difficult to decompose hyaluronic acid in neutral pH environments, which hampers its utility in drug delivery applications.
Innovation Solution
A variant of Hyal1 is engineered by substituting specific amino acids adjacent to its catalytic amino acids with acidic or polar amino acids, forming ionic bonds to enhance activity at neutral pH, including substitutions like Asp129 to Asp or Glu, and basic amino acids like Arg or His to form ionic bonds with adjacent amino acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Hyal1 is used in acidic conditions (pH 3-4), then it exhibits catalytic activity to hydrolyze hyaluronic acid, but it cannot function effectively in neutral pH environments (pH 7.0-7.5) such as subcutaneous tissue
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of Hyal1 to alter its pH activity profile. Specifically, substitutions are made in the region surrounding the catalytic domain (residues 120-140) to shift the enzyme's optimal pH from acidic (3-4) to neutral (7.0-7.5), enabling it to function in subcutaneous tissue environments while maintaining catalytic activity.
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions in specific regions of the Hyal1 protein, particularly around the catalytic domain (residues 120-140), while leaving the rest of the protein structure intact. This localized modification approach changes the pH sensitivity of the catalytic region without affecting other functional properties of the enzyme.
2Adaptability or versatility
If PH20 is used for subcutaneous injection, then it can hydrolyze hyaluronic acid in neutral pH, but it limits the utility of other hyaluronidases like Hyal1
Solution Approach 1:
The patent applies universality by creating a modified Hyal1 variant that can perform the same function as PH20 in neutral pH environments. This engineered Hyal1 maintains its identity as a distinct enzyme while acquiring the ability to hydrolyze hyaluronic acid in subcutaneous tissue, reducing dependency on PH20 and expanding the utility of the Hyal1 enzyme family.
3Adaptability or versatility
If amino acid substitutions are made in the catalytic region of Hyal1, then neutral pH activity is achieved, but the enzyme structure and stability may be affected
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions in specific regions of the Hyal1 protein, particularly around the catalytic domain (residues 120-140), while leaving the rest of the protein structure intact. This localized modification approach changes the pH sensitivity of the catalytic region without affecting other functional properties of the enzyme.
Solution Approach 2:
The patent applies parameter changes by modifying the amino acid sequence of Hyal1 to alter its pH activity profile. Specifically, substitutions are made in the region surrounding the catalytic domain (residues 120-140) to shift the enzyme's optimal pH from acidic (3-4) to neutral (7.0-7.5), enabling it to function in subcutaneous tissue environments while maintaining catalytic activity.
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 Hyal1 variant can efficiently hydrolyze hyaluronic acid across a broad pH range from acidic to neutral, enabling effective drug delivery and use in various preparations such as subcutaneous administration, intravenous injection, and ophthalmological applications.
Implementation Method 1
the substituted basic amino acid can form an ionic bond with an acidic or polar amino acid adjacent to the catalytic amino acids
Implementation Method 2
Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid
Implementation Method 3
Hyal1 mainly functions as a catalyst in acidic conditions
Data Source
AI summary
Provided is a Hyal1 variant in which at least one amino acid adjacent to the catalytic amino acids in wild-type human hyaluronidase Hyal1, comprising aspartate and glutamate as catalytic amino acids, is substituted with an acidic or polar amino acid; a method for preparing the Hyal1 variant; a nucleic acid, an expression vector, and a host cell which can be applied to the preparation of the Hyal1 variant; and a formulation or use of the Hyal1 variant. In addition, provided is a Hyal1 variant in which at least one amino acid adjacent to the catalytic amino acids in the tertiary structure is substituted with a basic amino acid; a method for preparing the Hyal1 variant; a nucleic acid, an expression vector, and a host cell which can be applied to the preparation of the Hyal1 variant; and a formulation or use of the Hyal1 variant.