Generation of site‐distinct N‐glycan variants for in vitro bioactivity testing
| Autor: | David Brühlmann, Jürgen Hemberger, Zeynep Manco, Alessandra D'Angelo, Eugenio Galano, Jonathan Souquet, Thomas Vuillemin, Abhijeet Satwekar, Markus Sauer, Angelo Palmese, Martin Jordan, Hervé Broly |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Glycan Glycosylation medicine.drug_class Population Bioengineering CHO Cells Monoclonal antibody 01 natural sciences Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound Cricetulus Polysaccharides 010608 biotechnology medicine Animals education Fucosylation education.field_of_study biology Chinese hamster ovary cell Receptors IgG Antibodies Monoclonal Biological activity Immunoglobulin Fc Fragments Sialic acid carbohydrates (lipids) 030104 developmental biology chemistry Biochemistry biology.protein Mannose Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 116:1017-1028 |
| ISSN: | 1097-0290 0006-3592 |
| Popis: | Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity. |
| Databáze: | OpenAIRE |
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