HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

Floc'h, F. Le; Tessier, B.; Chenuet, Sébastien; Guillaume, Jean-Marc; Cans, Pierre; Marc, Annie; Goergen, Jean‐Louis

doi:10.1021/bp0343211

Cited by 21 publications

(15 citation statements)

References 48 publications

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“…4c). As already published (Le Floch et al 2004) the EPO glycosylation patterns determined all over the process were very similar to those of EPO collected at the beginning of the previous culture meaning that desialylation observed in the previous experiment did not occur for these serum-free conditions using adapted cells. In addition to the previously published results, we performed analysis of EPO glycans collected at the end of the cell growth phase and at the end of the culture (after 100 and 260 h) in presence of serum, but with cells previously adapted to serum-free conditions (culture #2).…”

Section: Kinetics Of Cells Adapted and Non-adapted To Serum-free Condsupporting

confidence: 81%

“…Figure 4a presents the HPCE patterns of complete and desialylated N-glycans of EPO produced at the beginning and at the end of the culture (after 40 and 300 h) performed in presence of serum, with the non-adapted cells (culture #1). As shown already (Le Floch et al 2004), the HPCE patterns of non-desialylated and chemically desialylated N-glycans from EPO revealed that, except for the sialylation degree, glycosylation of EPO was quite constant all over the culture.…”

Section: Kinetics Of Cells Adapted and Non-adapted To Serum-free Condsupporting

confidence: 63%

“…In a previous paper (Le Floch et al 2004) it was shown that glycosylation and especially desialylation of EPO produced by CHO cells was dependent on the presence of serum in the culture medium. As the culture of CHO cells in serum-free conditions required the adaptation of those cells, two different cell lines might emerge : (i) the original cell line growing in serumcontaining medium and (ii) the adapted CHO cells capable to grow without serum in the culture medium.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, to discriminate between the potential effect of serum starvation and cell adaptation procedure to serum-free medium, we have used the previously adapted HPCE-LIF technique (Le Floch et al 2004) to monitor EPO glycosylation during the course of the different batch cultures. A dramatical EPO desialylation was registered when produced by non-adapted cells cultivated with serum (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A previous paper regarding recombinant EPO produced by CHO cells in serum-free conditions deals with differences in productivity and glycosylation structure obtained in presence or in absence of serum (Le Floch et al 2004). In fact, modifications of cell performances could result either from the serum removal or from the modification of the glycosylation mechanism during the adaptation procedure.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Kinetics Of Cells Adapted and Non-adapted To Serum-free Condsupporting

confidence: 81%

Section: Kinetics Of Cells Adapted and Non-adapted To Serum-free Condsupporting

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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Protein Glycosylation: Analysis, Characterization, and Engineering

Andersen

Nam

Sharfstein

2011

Encyclopedia of Industrial Biotechnology

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Protein glycosylation is critically important in vivo; current estimates are that more than half of the proteins in the SWISS‐PROT database are glycoproteins. Glycosylation plays a substantial role wide a range of physiological and pathological processes including development, immunology, cancer, and infectious disease. Protein glycosylation is also vitally important in the development of therapeutic bioproducts. Currently, more than 165 recombinant protein pharmaceuticals are approved for human use, with another 500 in preclinical and clinical trials. Of these, approximately 70% are glycosylated proteins. Glycosylation affects the structure, activity, immunogenicity, protease sensitivity, stability, and biological clearance of glycoproteins. Hence, an understanding of the mechanisms by which proteins are glycosylated, and strategies for analyzing and controlling glycoforms has become increasingly important in the development of biopharmaceuticals. Advances in chromatography and mass spectrometry have permitted more detailed identification of glycans, while cellular and protein engineering strategies have allowed manipulation of the glycoforms. In this chapter, we review the biology of protein glycosylation, methods for identifying and characterizing glycans and glycoproteins, and the effects of host cell line, culture conditions, and cellular engineering on the glycoforms of recombinant glycoproteins, providing a comprehensive overview of glycosylation of recombinant protein therapeutics.

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Single‐step affinity purification of enzyme biotherapeutics: A platform methodology for accelerated process development

Brower¹,

Ryakala²,

Bird³

et al. 2014

Biotechnology Progress

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Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).

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HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

Cited by 21 publications

References 48 publications

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

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

Protein Glycosylation: Analysis, Characterization, and Engineering

Single‐step affinity purification of enzyme biotherapeutics: A platform methodology for accelerated process development

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