Engineered Yeast Humanized Glycoprotein Production
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Solution Overview
Problem
Current methods for producing therapeutic proteins in yeast result in non-human glycosylation phenotypes, leading to unwanted immune responses due to high mannose residues, which reduces the therapeutic value of glycoproteins intended for applications beyond immune response enhancement.
Innovation Solution
Genetically engineered yeast cells are developed to produce humanized glycoproteins with defined O-linked glycans by expressing exogenous enzymes that mimic human glycosylation pathways, reducing high mannose structures and enabling the production of glycoproteins with human-like O-glycans, and fusion proteins containing mannose residues for targeted immune responses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If yeast cells are used to produce glycoproteins, then productivity and ease of manufacture are improved, but the glycosylation phenotype becomes non-human with high mannose residues, leading to unwanted immune responses
Solution Approach 1:
The patent changes the glycosylation parameters of yeast cells by introducing exogenous glycosyltransferase enzymes and modifying the glycosylation pathway. This transforms the default high-mannose glycosylation phenotype into a human-like glycosylation pattern with reduced mannose residues and addition of human-specific sugar structures, thereby eliminating the unwanted immune response while maintaining yeast productivity
Solution Approach 2:
The patent uses exogenous glycosyltransferase enzymes as intermediaries to bridge the gap between yeast's native glycosylation system and human-like glycosylation requirements. These enzymes act as catalysts that redirect the glycosylation pathway to produce human-compatible glycans on therapeutic proteins, resolving the conflict between using yeast for production and avoiding immune responses
2Reliability
If exogenous glycosyltransferase enzymes are introduced to humanize glycans, then therapeutic value is improved, but device complexity and manufacturing complexity increase
Solution Approach 1:
The patent applies preliminary action by pre-modifying the yeast host cells with the necessary exogenous glycosyltransferase genes before protein production. This creates a stable, genetically engineered yeast strain that automatically performs human-like glycosylation during routine fermentation processes, thereby eliminating the need for complex post-production glycosylation modifications and simplifying overall manufacturing
Solution Approach 2:
The patent merges the glycosylation modification step with the protein production process itself. By integrating the humanizing glycosyltransferase enzymes into the yeast's native glycosylation pathway, the system combines protein synthesis and glycan modification into a single unified process, reducing manufacturing complexity compared to separate production and modification steps
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 approach allows for the production of glycoproteins with human-like glycosylation patterns, enhancing therapeutic value by avoiding immune responses and improving targeting capabilities through mannose-binding receptors, thereby increasing the efficacy of glycoproteins for various therapeutic uses.
Implementation Method 1
The cell is engineered to express one or more exogenous N-acetylgalactosaminyltransferase
Implementation Method 2
The mannose-binding receptors include the macrophage mannose receptor (MMR; CD206)... Both the MMR and DC-SIGN have the capacity to direct internalized antigens into endocytic pathways
Data Source
AI summary
Cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the processing of glycoproteins in humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their human counterparts. The lower eukaryotes, which ordinarily produce high-mannose containing N-glycans, including unicellular and multicellular fungi are modified to produce O-glycans or other structures along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains which: do not express certain enzymes which create the undesirable complex structures characteristic of the fungal glycoproteins, which express exogenous enzymes selected either to have optimal activity under the conditions present in the fungi where activity is desired, or which are targeted to an organelle where optimal activity is achieved, and combinations thereof wherein the genetically engineered eukaryote expresses multiple exogenous enzymes required to produce "human-like" glycoproteins.