Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells

Schriebl, Kornelia; Trummer, Evelyn; Lattenmayer, Christine; McCarley, Robert W.; Kunert, Renate; Müller, Dethardt; Katinger, Hermann; Vorauer-Uhl, Karola

doi:10.1016/j.pep.2006.05.018

Cited by 56 publications

(37 citation statements)

References 28 publications

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“…Furthermore, a similar dose (40 lg kg -1 ) of both rhEPO and cEPO was neuroprotective in a rat model of spinal cord hemisection [30]. The cEPO-FC molecule used is a fusion protein comprising two rhEPO molecules connected with the Fc domain of a human antibody IgG1 [14]. Thereafter, this whole complex is carbamylated until no erythropoietic potency remains.…”

Section: Experimental Protocolmentioning

confidence: 99%

“…All data on cEPOrelated organ protection against I/R injury, however, originate from nonresuscitated rodent models which did not integrate standard resuscitation measures such as fluid resuscitation and/or vasopressor support to guarantee wellmaintained systemic hemodynamics. Therefore, we tested the hypothesis that a newly developed carbamylated EPO-FC fusion protein, consisting of two EPO molecules fused to the FC part of IgG1 (cEPO-FC) [14], protects against ischemic spinal cord dysfunction and neuronal damage comparably to rhEPO in swine undergoing thoracic aortic balloon occlusion-induced I/R injury [4].…”

Section: Introductionmentioning

confidence: 99%

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Comparison of carbamylated erythropoietin-FC fusion protein and recombinant human erythropoietin during porcine aortic balloon occlusion-induced spinal cord ischemia/reperfusion injury

et al. 2011

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Section: Experimental Protocolmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparison of carbamylated erythropoietin-FC fusion protein and recombinant human erythropoietin during porcine aortic balloon occlusion-induced spinal cord ischemia/reperfusion injury

et al. 2011

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“…After labeling, the glycans were analyzed by normal-phase HPLC using a TSK-Gel Amide-80 column (4.6 9 250 mm, 5 lm; TOSOH Bioscience, Stuttgart, Germany). The retention times of the detected peaks were compared to the retention times of known commercial glycan standards from Dextra Laboratories (Reading, UK) and Glyko (Novato, CA, USA) [36].…”

Section: Carbohydrate Structure Analysismentioning

confidence: 99%

Influence of a Reduced CO2 Environment on the Secretion Yield, Potency and N-Glycan Structures of Recombinant Thyrotropin from CHO Cells

et al. 2008

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A consistent increase of approximately 60% in the secretion yield of CHO-derived hTSH was observed by changing cell culture CO2 conditions from 5% CO2 to an air environment. The overall quality of the products obtained under both conditions was evaluated in comparison with a well-known biopharmaceutical (Thyrogen). The N-glycans identified were of the complex type, presenting di-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing approximately 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more di-antennary structures obtained in the absence of CO2 and 7-9% more tri-antennary structures in its presence. No remarkable difference in charge isomers was observed between the three preparations, the isoelectric focusing profiles showing six distinct bands in the 5.39-7.35 pI range. A considerably different distribution, with more forms in the acidic region, was observed, however, for two native pituitary preparations. All recombinant preparations showed a higher in vivo bioactivity when compared to native hTSH. Different production processes apparently do not greatly affect N-glycan structures, charge isomer distribution or bioactivity of CHO-derived hTSH.

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“…With protein-free generated clones, the problem of clonal changes and instability is circumvented and relevant parameters allowing the prediction of clonal performance can be applied earlier in clonal development. In conclusion, we could successfully demonstrate that the basic requirements for transfection of protein-free adapted host cells, namely, comparable production rates as well as product qualities (Schriebl et al, 2006) …”

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confidence: 85%

Protein‐free transfection of CHO host cells with an IgG‐fusion protein: Selection and characterization of stable high producers and comparison to conventionally transfected clones

Lattenmayer¹,

Loeschel

Schriebl³

et al. 2006

Biotech & Bioengineering

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In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.

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Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells

Cited by 56 publications

References 28 publications

Comparison of carbamylated erythropoietin-FC fusion protein and recombinant human erythropoietin during porcine aortic balloon occlusion-induced spinal cord ischemia/reperfusion injury

Comparison of carbamylated erythropoietin-FC fusion protein and recombinant human erythropoietin during porcine aortic balloon occlusion-induced spinal cord ischemia/reperfusion injury

Influence of a Reduced CO2 Environment on the Secretion Yield, Potency and N-Glycan Structures of Recombinant Thyrotropin from CHO Cells

Protein‐free transfection of CHO host cells with an IgG‐fusion protein: Selection and characterization of stable high producers and comparison to conventionally transfected clones

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