Endo-Si Enzyme Mutations for Antibody Sugar Chain Remodeling
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Solution Overview
Problem
Current methods for producing antibodies with homogeneous sugar chains are inefficient and costly, as they require multiple steps and specific enzymes with balanced hydrolysis and transglycosylation activities, which are not adequately provided by existing endo-β-N-acetylglucosaminidases like EndoS and Endo-F3.
Innovation Solution
A novel endo-β-N-acetylglucosaminidase, Endo-Si, cloned from Streptococcus iniae, with specific mutations at positions 241, 190, 311, and 360, exhibits enhanced hydrolysis and transglycosylation activities, allowing for efficient sugar chain remodeling of antibodies, reducing production costs and increasing purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional endo-β-N-acetylglucosaminidases (EndoS, Endo-F3) are used for sugar chain remodeling, then the process can be performed, but the production cost increases and purity decreases due to inefficient hydrolysis and transglycosylation activities
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at positions 241, 190, 311, and 360 in the endo-β-N-acetylglucosaminidase enzyme sequence. These mutations alter the enzyme's catalytic properties to optimize both hydrolysis and transglycosylation activities, achieving efficient sugar chain remodeling with homogeneous results. The mutations modify the enzyme's active site or structural parameters to enhance its functional performance.
2Manufacturing precision
If multiple enzyme steps are used to achieve homogeneous sugar chains, then sugar chain homogeneity improves, but the process complexity and production cost increase
Solution Approach 1:
The patent applies universality by creating a single endo-β-N-acetylglucosaminidase enzyme that performs multiple functions: it efficiently catalyzes both hydrolysis of complex-type sugar chains and transglycosylation reactions. This multi-functional enzyme replaces the need for multiple separate enzymatic steps, simplifying the overall process while maintaining sugar chain homogeneity. The enzyme can work with various substrate types including N297-linked sugar chains in antibodies.
3Ease of manufacture
If wild type endo-β-N-acetylglucosaminidase is used, then the enzyme is readily available, but it lacks optimized hydrolysis and transglycosylation activities required for efficient production
Solution Approach 1:
The patent applies this principle by using recombinant DNA technology to express the mutated endo-β-N-acetylglucosaminidase in host cells such as E. coli or CHO cells. This allows production of the optimized enzyme at scale through standard biotechnology processes, making the enhanced enzyme readily available and cost-effective. The enzyme can be produced as a purified protein product for industrial applications.
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
Endo-Si efficiently hydrolyzes and remodels N297-linked sugar chains in antibodies, enabling the production of antibodies with homogeneous sugar chains, reducing production costs and improving the quality of the final product by enhancing transglycosylation activity and reducing hydrolysis activity.
Implementation Method 1
The transglycosylation reaction is a multiple step process carried out in vitro and consisting of cleaving a sugar chain (hydrolysis reaction) and adding of another sugar chain by condensation
Implementation Method 2
a one-pot method of directly transferring a sugar chain to a GlcNAc acceptor by use of two types of ENGases
Data Source
AI summary
The present invention provides endo-β-N-acetylglucosaminidase (Endo-Si) cloning from a strain belonging to Streptococcus iniae and a mutant enzyme thereof, a gene encoding the enzyme, a recombinant plasmid, a transformant obtained by transformation of a cell by the plasmid and use thereof, and a method for producing e.g., a sugar chain remodeled antibody using the enzyme. A polypeptide having an amino acid sequence at amino acid positions 34 to 928 in SEQ ID NO: 2 or an amino acid sequence having the same amino acid sequence except containing one or mutations at more amino acid positions selected from the group consisting of amino acids at position 241 (D241), 190 (T190), 311 (Q311) and 360 (E360), said polypeptide exhibiting a sugar chain hydrolysis activity and/or transglycosylation activity.