A multi‐pronged investigation into the effect of glucose starvation and culture duration on fed‐batch CHO cell culture

Fan, Yuzhou; Val, Ioscani Jiménez del; Müller, Christian; Lund, Anne Mathilde; Sen, Jette W.; Rasmussen, Steen; Kontoravdi, Cleo; Baycin-Hizal, Deniz; Betenbaugh, Michael J.; Weilguny, Dietmar; Andersen, Mikael Rørdam

doi:10.1002/bit.25620

Cited by 57 publications

(66 citation statements)

References 43 publications

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“…This is supported by the current literature, in which galactosylation levels of IgG are typically reported to be lower than 60%. This suggests that the speed of the glycoproteins through the Golgi, and therefore, the time the enzyme can interact with the glycan, might be critical (Fan et al, 2015). Gramer et al (2011) galactosylated species and, therefore, confirmed the possibility of obtaining high galactosylation levels in vitro.…”

Section: Galactosylation and Sialylationmentioning

confidence: 54%

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Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

Ehret

Zimmermann

Eichhorn

et al. 2019

Biotech & Bioengineering

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Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half‐life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed‐batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high‐mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2‐F‐peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.

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Section: Galactosylation and Sialylationmentioning

confidence: 54%

“…This was calculated for both the exponential and the stationary phase following the equation published by Fan et al (2015): This was calculated for both the exponential and the stationary phase following the equation published by Fan et al (2015):…”

Section: Purification and Analysis Of The Glycosylation Profilementioning

confidence: 99%

Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

Ehret

Zimmermann

Eichhorn

et al. 2019

Biotech & Bioengineering

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“…This bottleneck is due to a slowdown in the transfer of galactose and sialic acid residues to the glycoproteins and is unlikely to be caused by loss of productivity (which was nearly identical on day 10 and 14). Fan et al () demonstrated that a slowdown in glycan processing occurred with increased culture duration despite an abundance of nucleotide sugar donors, however this may not necessarily be true in our case due to differences in cell line and cell culture conditions used. It is also plausible that the bottleneck is caused either by an imbalance in the level of expression of glycosyltransferases or breakdown of relevant complexes (Kellokumpu et al, ) due to changes in cell culture conditions.…”

Section: Discussionmentioning

confidence: 67%

Site‐specific monitoring of N‐Glycosylation profiles of a CTLA4‐Fc‐fusion protein from the secretory pathway to the extracellular environment

Oliveira¹,

Spencer²,

Barton³

et al. 2017

Biotech & Bioengineering

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Glycosylation often plays a key role in the safety and efficacy of therapeutic proteins to patients, thus underlying the need for consistent control of this important post-translational modification during biologics production. In this study, we profiled the site-specific evolution of N-glycans on a CTLA4-Fc-fusion protein, from the intracellular secretory pathway to the conditioned medium (CM) in fed-batch cell culture. For this, we developed an approach that combined sub-cellular fractionation with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The study revealed that there was a significant amount of heterogeneity in the glycans displayed amongst the three distinct N-glycosylation sites. Furthermore, 54-60% of the intracellular protein was characterized by Man8 and Man9 glycans on day 10, when the cell density peaks, indicative of a significant bottleneck between the endoplasmic reticulum (ER) and the cis-Golgi. At longer culture duration, the accumulation of intracellular protein with bi-antennary-fucosylated GlcNAc-terminated residues identified the formation of another bottleneck in the medial and trans-Golgi compartments, which subsequently led to a decrease in sialylated species in the secreted protein. Glucose deprivation caused a reduction in the Man8 and Man9 glycans in favor of Man5 glycans and bi-antennary-fucosylated GlcNAc-terminated residues in the organellar pool of the Fc-fusion protein. However, transient deprivation of glucose did not lead to major differences in the glycan profile of proteins secreted into the CM. The approach developed here allows us to probe the secretory pathway and sheds light on the site-specific intracellular processing of glycans during fed-batch cell culture, thus serving as an initial step towards their rational control. Biotechnol. Bioeng. 2017;114: 1550-1560. © 2017 Wiley Periodicals, Inc.

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“…process information (such as viable cell density, productivity) to increase the mechanistic knowledge by integrating further important characteristics besides the media composition in the framework of glycosylation modulation 122,[280][281][282] . At later stages of process development, such additional (dynamic) characteristics are likely to play a key role in building predictive process models.…”

Section: Discussionmentioning

confidence: 99%

Tailoring recombinant protein quality by rational media design

Brühlmann

Jordan

Hemberger³

et al. 2015

Biotechnology Progress

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Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C‐ & N‐terminal modifications), aggregates, low‐molecular‐weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high‐throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015

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A multi‐pronged investigation into the effect of glucose starvation and culture duration on fed‐batch CHO cell culture

Cited by 57 publications

References 43 publications

Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

Site‐specific monitoring of N‐Glycosylation profiles of a CTLA4‐Fc‐fusion protein from the secretory pathway to the extracellular environment

Tailoring recombinant protein quality by rational media design

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