Relationships Between the N-Glycan Structures and Biological Activities Of Recombinant Human Erythropoietins Produced Using Different Culture Conditions and Purification Procedures

Ct, Yuen; Pl, Storring; Rj, Tiplady; Izquierdo, Mabel; Wait, Robin; Ck, Gee; Gerson, Peter; Lloyd, Pauline; Ja, Cremata

doi:10.1007/0-387-25515-x_25

Cited by 12 publications

(9 citation statements)

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“…They are immature forms of N-linked glycans, and their proportion can vary with culture conditions (Yuen et al, 2003). In this study, the proportion of Man6P1 and Man5P1 in total was fairly constant in the range of 7.0-8.6% (Fig.…”

Section: Discussionmentioning

confidence: 96%

“…Phosphorylated oligomannosidic N-linked glycans, mannose-6-phosphate (Man6P1) and mannose-5-phosphate (Man5P1), have been described to occur on a recombinant EPO expressed in baby hamster kidney cells (Nimtz et al, 1995) and in CHO cells (Yuen et al, 2003). They are immature forms of N-linked glycans, and their proportion can vary with culture conditions (Yuen et al, 2003).…”

Section: Discussionmentioning

confidence: 98%

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Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

Ahn

Jeon

Jeong

et al. 2008

Biotech & Bioengineering

112

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To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 98%

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

Ahn

Jeon

Jeong

et al. 2008

Biotech & Bioengineering

112

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“…N-glycosylation has widely been reported to exert significant effects on several properties of glycoproteins, including antigenicity, solubility, protein folding, protease resistance, pharmacokinetics or biological activity (Schauer 1988;Kobata 1992;Yusa et al 2005). Moreover, sialylation is known to be able to influence the in vivo circulatory lifetime of these proteins, which is crucial to their therapeutic value (Dordal et al 1985;Takeuchi et al 1989;Imai et al 1990;Misaizu et al 1995;Koury 2003;Yuen et al 2003;Elliott et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

et al. 2006

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The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product, particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and nonadapted CHO cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when cells were adapted to SFX-CHO TM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in the enriched SFM TM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells.

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“…It is of utmost importance to assure that minimal degradation occurs during sample processing as the degree or extent of glycosylation, and sialylation in particular, are associated with several glycan processing related pathologies such as CDG [37], rheumatoid arthritis, fetal alcohol syndrome, or carbohydrate-mediated biomolecular interactions (viral infections, cancer metastasis, etc.). With respect to carbohydrate-mediated interactions it is known that the recombinant pharmaceuticals employed here are engineered and purified for a high sialic acid content to prevent interactions with the hepatic galactosyl-receptor and thus clearance from circulation [38]. Another structural element that has been suggested to confer a prolonged half-life to glycoprotein drugs refers to the O-acetylation of the sialic acid residues [39].…”

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confidence: 99%

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

Llop¹,

Gallego²,

Belalcazar³

et al. 2007

Proteomics

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The structure, function, and physico-chemical properties of many proteins are determined by PTM, being glycosylation the most complex. This study describes how a combination of typical proteomics methods (2-DE) combines with glycomics strategies (HPLC, MALDI-TOF-MS, exoglycosidases sequencing) to yield comprehensive data about single spot-microheterogeneity, providing meaningful information for the detection of disease markers, pharmaceutical industry, antidoping control, etc. Recombinant erythropoietin and its hyperglycosylated analogue darbepoetin-a were chosen as showcases because of their relevance in these fields and the analytical challenge they represent. The combined approach yielded good results in terms of sample complexity (mixture glycoforms), reproducibility, sensitivity (,25 pmoles of glycoprotein/spot), and identification of the underlying protein. Heterogeneity was present in all spots but with a clear tendency; spots proximal to the anode contained the highest amount of tetra-antennary tetrasialylated glycans, whereas the opposite occurred for spots proximal to the cathode with the majority of the structures being undersialylated. Spot microheterogeneity proved a consequence of the multiple glycosylation sites as they contributed directly to the number of possibilities to account for a discrete charge in a single spot. The interest of this combined glycoproteomics method resides in the efficiency for detecting and quantifying subtle dissimilarities originated from altered ratios of identical glycans including N-acetyl-lactosamine repeats, acetylation, or antigenic epitopes, that do not significantly contribute to the electrophoretic mobility, but affect the glycan microheterogeneity and the potential underlying related functionality.

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Relationships Between the N-Glycan Structures and Biological Activities Of Recombinant Human Erythropoietins Produced Using Different Culture Conditions and Purification Procedures

Cited by 12 publications

References 1 publication

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

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