Improving the recombinant human erythropoietin glycosylationusing microsome supplementation in CHO cell-free system

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dc.contributor.authorGurramkonda, Chandrasekhar
dc.contributor.authorRao, Aniruddha
dc.contributor.authorBorhani, Shayan
dc.contributor.authorPilli, Manohar
dc.contributor.authorDeldari, Sevda
dc.contributor.authorGe, Xudong
dc.contributor.authorPezeshk, Niloufar
dc.contributor.authorTolosi, Michael
dc.contributor.authorKostov, Yordon
dc.contributor.authorTolosa, Leah
dc.contributor.authorFrey, Douglas D.
dc.contributor.authorRao, Govind
dc.date.accessioned2018-03-15T19:33:00Z
dc.date.available2018-03-15T19:33:00Z
dc.date.issued2018
dc.descriptionThis is the pre-peer reviewed version of the following article: Gurramkonda C, Rao A, Borhani S, et al. Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell-free system. Biotechnology and Bioengineering. 2018;1–12. https://doi.org/10.1002/bit.26554, which has been published in final form at https://doi.org/10.1002/bit.26554. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."
dc.description.abstractCell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the HO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25–28 kDa, and unglycosylated protein runs at 20 kDa on an SDS–PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have ∼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.en_US
dc.description.urihttp://onlinelibrary.wiley.com/doi/10.1002/bit.26554/full?wol1URL=/doi/10.1002/bit.26554/full&regionCode=US-MD&identityKey=cdcb031c-9694-4db7-9d62-8b1333523f4cen_US
dc.genrejournal articlesen_US
dc.identifierdoi:10.13016/M2J09W61Z
dc.identifier.citationGurramkonda C, Rao A, Borhani S, et al. Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell-free system. Biotechnology and Bioengineering. 2018;1–12. https://doi.org/10.1002/bit.26554en_US
dc.identifier.uri10.1002/bit.26554
dc.identifier.urihttp://hdl.handle.net/11603/7876
dc.language.isoen_USen_US
dc.publisherWileyen_US
dc.relation.ispartofUMBC Center for Advanced Sensor Technology (CAST)
dc.relation.ispartofUMBC Chemical, Biochemical & Environmental Engineering Department
dc.relation.ispartofUMBC Faculty Collection
dc.relation.ispartofUMBC Student Collection
dc.subjectcell-free protein synthesisen_US
dc.subjecterythropoietinen_US
dc.subjectglycosylationen_US
dc.subjectlectin-based assayen_US
dc.subjectmicrosomesen_US
dc.subjectPNGaseen_US
dc.titleImproving the recombinant human erythropoietin glycosylationusing microsome supplementation in CHO cell-free systemen_US
dc.typeTexten_US

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