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Published on: December 28, 2015
Efficient Expression of Functionally Active Aflibercept with Designed N-glycans
Tahereh Keshvari1,2, Stanislav Melnik1, Lin Sun1
1Institute of Plant Biotechnology and Cell Biology, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences BOKU Vienna, 1190 Vienna, Austria.
Researchers engineered plants to produce aflibercept, a vascular endothelial growth factor receptor (VEGFR) fusion protein, with controlled glycosylation. This plant-based production yielded high amounts of functional aflibercept variants, paving the way for optimized biopharmaceutical development.
Area of Science:
- Biotechnology
- Molecular Biology
- Glycobiology
Background:
- Aflibercept is a crucial therapeutic fusion protein targeting VEGFR.
- Its production in mammalian cells leads to complex glycosylation heterogeneity, complicating analysis and optimization.
- N-glycosylation sites on aflibercept can influence its structure and function.
Purpose of the Study:
- To develop a plant-based system for producing aflibercept with controlled N-glycosylation profiles.
- To express and characterize aflibercept variants with specific glycan structures (AFLI GnGn and AFLI Sia).
- To assess the functional activity and binding potency of the glycoengineered aflibercept variants.
Main Methods:
- Utilized *Nicotiana benthamiana* for transient expression of aflibercept.
- Employed glycoengineering strategies to introduce terminal GlcNAc and sialic acid residues.
- Performed mass spectrometry for site-specific glycosylation analysis.
- Assessed VEGF165 binding potency of the produced variants.
Main Results:
- Achieved high-level expression of aflibercept variants (2 g/kg fresh leaf material) in plant leaves.
- Demonstrated proper dimer assembly and consistent site-specific glycosylation patterns.
- Identified incomplete occupancy at some glycosylation sites.
- Observed similar binding potency to VEGF165 for both AFLI GnGn and AFLI Sia, with a slight decrease in binding for more sialylated variants.
Conclusions:
- Successfully produced functionally active aflibercept in significant quantities using a plant-based system.
- Demonstrated the ability to control and design N-glycosylation profiles in plant-expressed aflibercept.
- The findings support further research towards optimized biopharmaceutical production with tailored glycosylation.
