BOKU - Universität für Bodenkultur Wien - Forschungsinformationssystem
Gewählte Publikation:
Schriebl, K; Trummer, E; Lattenmayer, C; Weik, R; Kunert, R; Müller, D; Katinger, H; Vorauer-Uhl, K.
(2006):
Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells.
Protein Expr Purif. 2006; 49(2):265-275
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- Abstract:
- One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fe part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG(1) molecule. In the present study, culture supernatant of a stable clone was collected and purified by affinity chromatography prior characterization. We emphasized product quality aspects regarding the fusion protein itself and in addition, post-translational characterization of the subunits in comparison to human antibodies and rhEpo. However, overproduction of recombinant proteins in mammalian cells is well established, analysis of product quality of complex products for different purposes, such as product specification, purification issues, batch to batch consistency and therapeutical consequences, is required. Besides product quantification by ELISA, N-acetylneuraminic acid quantification in microtiterplates, quantitative isoform pattern and entire glycan profiling was performed. By using these techniques for the characterization of the recombinant human Epo-Fc (rhEpo-Fc) molecule itself and furthermore, for the separate characterization of both subunits, we could clearly show that no significant differences in the core glycan structures compared to rhEpo and human antibody N-glycans were found. The direct comparison with other rhEpo-Fc fusion proteins failed, because no appropriate data were found in the literature. (c) 2006 Elsevier Inc. All rights reserved.
- Autor*innen der BOKU Wien:
- Katinger Hermann
- Kunert Renate
- Trummer-Gödl Evelyn
- Vorauer-Uhl Karola
- Find related publications in this database (using NML MeSH Indexing)
- Animals -
- CHO Cells -
- Carbohydrate Conformation -
- Cricetinae -
- Enzyme-Linked Immunosorbent Assay -
- Erythropoietin, Recombinant - chemistry
- Gene Expression - chemistry
- Glycosylation - chemistry
- Humans - chemistry
- Immunoglobulin Fc Fragments - biosynthesis
- Protein Modification, Translational - biosynthesis
- Recombinant Fusion Proteins - biosynthesis
- Find related publications in this database (Keywords)
- CHO cells
- rhEpo-Fc fusion protein
- N-glycan
- N-acetylneuraminic acid
- quantitative isoform pattern
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