Characterization of Erythropoietin Dimerization

DePaolis, Anneli M.; Advani, Javher V.; Sharma, Basant

doi:10.1002/jps.2600841105

Cited by 28 publications

(9 citation statements)

References 19 publications

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“…In contrast the intramolecular disulfide bond between Cys 34 -Cys 188 seems to be crucial for the preservation of the molecular structure 51 . Nevertheless studies with an initial reduction of Cys 34 -Cys 188 disulfide bond showed a partial dimerization of EPO monomers exhibiting biological activity 52 , 53 . It might be possible that the cell-free produced termination product with an unoccupied cysteine residue, showed a similar behavior in our experimental setup.…”

Section: Discussionmentioning

confidence: 99%

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

Zemella

Thoring

Hoffmeister

et al. 2018

Sci Rep

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As one of the most complex post-translational modification, glycosylation is widely involved in cell adhesion, cell proliferation and immune response. Nevertheless glycoproteins with an identical polypeptide backbone mostly differ in their glycosylation patterns. Due to this heterogeneity, the mapping of different glycosylation patterns to their associated function is nearly impossible. In the last years, glycoengineering tools including cell line engineering, chemoenzymatic remodeling and site-specific glycosylation have attracted increasing interest. The therapeutic hormone erythropoietin (EPO) has been investigated in particular by various groups to establish a production process resulting in a defined glycosylation pattern. However commercially available recombinant human EPO shows batch-to-batch variations in its glycoforms. Therefore we present an alternative method for the synthesis of active glycosylated EPO with an engineered O-glycosylation site by combining eukaryotic cell-free protein synthesis and site-directed incorporation of non-canonical amino acids with subsequent chemoselective modifications.

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Section: Discussionmentioning

confidence: 99%

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

Zemella

Thoring

Hoffmeister

et al. 2018

Sci Rep

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“…The EPO molecule has been well-characterized [5][6][7][8][9] and is a stable molecule that remains predominantly in monomeric form when stored at 2-8 • C. However, when the product is exposed to higher temperatures or to certain stress conditions, dimer and higher order aggregates of r-HuEPO can be formed [10,11]. As with many other marketed biopharmaceuticals, to protect the active protein against denaturation or aggregate formation, nonionic surfactants such as polysorbate 80 are included as stabilizers [12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates

Gunturi¹,

Ghobrial²,

Sharma³

2007

Journal of Pharmaceutical and Biomedical Analysis

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“…monograph Erythropoietin concentrated solution (1316) prescribes the determination of dimers and higher order oligomers in EPO products and stipulates that dimer content should not exceed 2% by area [ 2 ]. Studies have suggested that EPO dimers may form as a result of an initial reduction of internal disulfide bond with subsequent disulfide bond re-oxidation between EPO molecules [ 3 ]. Most reference preparations are free from dimers and as such, the analyst performing the test is required to generate their own system suitability sample to ensure that the performance of their method is able to discriminate the dimers and oligomers from monomeric EPO.…”

Section: Introductionmentioning

confidence: 99%

Development of a stable chemically cross-linked erythropoietin dimer for use in the quality control of erythropoietin therapeutic products

et al. 2019

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Erythropoietin (EPO) is a glycoprotein hormone which promotes red cell replenishment and is also a global biotherapeutic medicine widely used to treat anaemia resulting, for example, from chemotherapy. Requirements of the European Pharmacopoeia stipulate that the level of dimer must be quantified in clinical EPO products (with a limit of 2%). Quantification is hampered by the lack of reference preparations containing stable measurable levels of EPO dimer, but the reproducible generation of a stable dimerised EPO preparation is challenging. We describe here the development of a lyophilised, chemically cross-linked EPO preparation, which has good stability and may be used for calibration and system suitability assurance for the size exclusion chromatographic separation of EPO preparations. Graphical abstract

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Characterization of Erythropoietin Dimerization

Cited by 28 publications

References 19 publications

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin

Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates

Development of a stable chemically cross-linked erythropoietin dimer for use in the quality control of erythropoietin therapeutic products

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