Surface-Displayed Endoglycosidase for Recombinant Protein Deglycosylation
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Solution Overview
Problem
Recombinant proteins produced in Pichia pastoris are highly glycosylated, leading to immunogenicity concerns, and existing methods for deglycosylation are inefficient and costly, requiring additional purification steps to remove endoglycosidase and ensuring sufficient contact time between the protein and enzyme.
Innovation Solution
Engineered eukaryotic cells with a surface-displayed catalytic domain of endoglycosidase, anchored via a cell surface protein, effectively deglycosylate secreted glycoproteins without releasing the enzyme into the culture medium, eliminating the need for additional purification and reducing manufacturing time and expense.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If endoglycosidase is added to culture medium for deglycosylation, then glycosylation is reduced, but enzyme contamination and purification complexity increase
Solution Approach 1:
The catalytic domain of endoglycosidase is extracted and fused to a cell surface anchor protein, displaying the enzymatic function on the cell surface while leaving the cell itself. This allows the enzyme to be spatially separated from the culture medium, providing deglycosylation activity without enzyme contamination in the final product
Solution Approach 2:
A cell surface anchor protein serves as an intermediary, tethering the catalytic domain to the cell surface. This mediator enables the enzyme to remain stationary on cells while processing secreted glycoproteins in the culture medium, eliminating the need for enzyme removal from the final product
2Manufacturing precision
If endoglycosidase is used for deglycosylation, then glycosylation is reduced, but manufacturing time increases due to required contact time
Solution Approach 1:
The surface-displayed endoglycosidase continuously processes secreted glycoproteins as they are released into the culture medium. The enzyme remains actively displayed on cell surfaces throughout the culture period, providing ongoing deglycosylation without requiring separate incubation steps or extended contact times
Solution Approach 2:
The endoglycosidase is pre-displayed on cell surfaces before protein secretion occurs. When glycoproteins are secreted, they immediately encounter the active enzyme on the cell surface, eliminating the need for subsequent deglycosylation steps or extended contact times
3Manufacturing precision
If endoglycosidase is added to culture medium, then deglycosylation occurs, but cost increases due to enzyme removal requirements
Solution Approach 1:
The catalytic function is extracted and displayed on cell surfaces, separating the enzymatic activity from the culture medium. This eliminates the need for enzyme removal steps and associated costs, as the enzyme remains bound to cells and does not contaminate the final product
Solution Approach 2:
The engineered cells with surface-displayed endoglycosidase autonomously perform deglycosylation of secreted glycoproteins in the culture medium. The system is self-sufficient, requiring no additional enzyme additions or removal steps, thereby reducing manufacturing complexity and cost
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 surface-displayed endoglycosidase efficiently deglycosylates secreted proteins, reducing glycosylation levels and avoiding contamination with the enzyme, thus enhancing the efficiency and cost-effectiveness of protein production for human or animal use.
Implementation Method 1
surface displayed catalytic domain of endoglycosidase... effectively deglycosylate secreted glycoproteins
Data Source
AI summary
The present disclosure provides engineered eukaryotic cells comprising a surface displayed catalytic domain of an endoglycosidase and methods of use.