Mannose-Terminated Glycoprotein Production in CHO Cells
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Solution Overview
Problem
Current methods for producing recombinant glucocerebrosidase and other glycoproteins with mannose-terminated N-glycans are inefficient and costly due to the need for extensive post-expression processing, and alternative expression systems like Lec 1 cells have low productivity and are not suitable for large-scale production.
Innovation Solution
A method involving Chinese Hamster Ovary (CHO) cells with a mutation in the GnT 1 gene, selected using Ricinus communis agglutinin I (RCA-I), that express recombinant proteins with mannose-terminated glycan structures without the need for additional enzymatic processing, using the JW152 cell line for high productivity and scalability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If exoglucosidases are used to trim glycan structures to terminal mannose structures, then mannose-terminated N-glycans are achieved for macrophage targeting, but production cost increases and process complexity increases
Solution Approach 1:
The patent applies preliminary action by genetically modifying CHO cells to constitutively produce mannose-terminated N-glycans on recombinant proteins during expression. This eliminates the need for subsequent enzymatic trimming with exoglucosidases, as the desired glycan structure is already present before purification. The GnT1 knockout prevents complex glycan formation, while overexpression of GnTII and MANII enzymes ensures mannose termination is established during protein synthesis.
Solution Approach 2:
The patent extracts the problematic post-expression processing step (exoglucosidase treatment) by redesigning the expression system to produce the final desired product directly. The enzymatic trimming step is completely removed from the workflow, as the CHO cells are engineered to secrete recombinant proteins with pre-formed mannose-terminated N-glycans, requiring only standard purification.
2Reliability
If exoglucosidases are used to trim glycan structures to terminal mannose structures, then mannose-terminated N-glycans are achieved for macrophage targeting, but manufacturing complexity increases
Solution Approach 1:
The patent applies preliminary action by genetically modifying CHO cells to constitutively produce mannose-terminated N-glycans on recombinant proteins during expression. This eliminates the need for subsequent enzymatic trimming with exoglucosidases, as the desired glycan structure is already present before purification. The GnT1 knockout prevents complex glycan formation, while overexpression of GnTII and MANII enzymes ensures mannose termination is established during protein synthesis.
Solution Approach 2:
The patent extracts the problematic post-expression processing step (exoglucosidase treatment) by redesigning the expression system to produce the final desired product directly. The enzymatic trimming step is completely removed from the workflow, as the CHO cells are engineered to secrete recombinant proteins with pre-formed mannose-terminated N-glycans, requiring only standard purification.
3Ease of manufacture
If alternative expression systems like Lec 1 cells are used, then mannose-terminated N-glycans are produced without enzymatic processing, but productivity decreases and scalability is limited
Solution Approach 1:
The patent applies parameter changes by modifying the glycosylation pathway parameters within CHO cells through genetic engineering. Specifically, GnT1 is knocked out to prevent complex glycan formation, while GnTII and MANII are overexpressed to ensure mannose termination. This creates a balanced enzymatic environment that produces mannose-terminated N-glycans constitutively during protein expression, achieving both high productivity and desired glycan structure without enzymatic processing.
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
This approach allows for the efficient and cost-effective production of recombinant proteins with mannose-terminated N-glycans, such as glucocerebrosidase, that can be effectively targeted to macrophages, enabling effective treatment of Gaucher's disease and other conditions without the need for post-expression processing.
Implementation Method 1
selected using Ricinus communis agglutinin I (RCA-I)
Data Source
AI summary
We describe a method of expressing a recombinant protein comprising mannose-terminated N-glycans from a host cell, the method comprising: (a) introducing a nucleic acid encoding a recombinant protein into a Chinese Hamster Ovary (CHO) cell comprising a mutation in the GnT 1 gene (GenBank Accession Number AF343963) leading to loss of GnT 1 function; and (c) expressing the recombinant protein from the host cell, in which the expressed recombinant protein comprises a mannose-terminated glycan structure, and in which the method does not include a step of introducing functional GnT-I into the host cell. The method may be used for producing recombinant glucocerebrosidase with a mannose-terminated glycan structure, suitable for treatment or prevention of Gaucher's Disease.