Tuning a MAb glycan profile in cell culture: Supplementing N-acetylglucosamine to favour G0 glycans without compromising productivity and cell growth

Blondeel, Eric J. M.; Braasch, Katrin; McGill, Thomas E.; Chang, David D.; Engel, Christina; Spearman, Maureen; Butler, Michael; Aucoin, Marc G.

doi:10.1016/j.jbiotec.2015.09.014

Cited by 25 publications

(23 citation statements)

References 46 publications

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“…Various other groups have also observed similar adverse GlcN-induced effects with other CHO cell lines (Baker et al, 2001;Wong et al, 2010b;Yang and Butler, 2002). Blondeel et al (2015) recently attributed this GlcN-induced reduction in cell growth to the depletion of intracellular acetyl-CoA pools by the production of GlcNAc via GlcN acetylation. Consistent with the hypothesis that GlcN reduces cell growth by depleting intracellular acetyl-CoA, we observed a 5.4-fold increase in UDP-GlcNAc after adding GlcN to the hyperosmolar cultures ( Supplementary Fig.…”

Section: Relative Area (%)mentioning

confidence: 64%

“…S4). Blondeel et al (2015) recently attributed this GlcN-induced reduction in cell growth to the depletion of intracellular acetyl-CoA pools by the production of GlcNAc via GlcN acetylation. Blondeel et al (2015) recently attributed this GlcN-induced reduction in cell growth to the depletion of intracellular acetyl-CoA pools by the production of GlcNAc via GlcN acetylation.…”

Section: Relative Area (%)mentioning

confidence: 99%

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Understanding of decreased sialylation of Fc‐fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N‐glycosylation gene expression and N‐linked glycan antennary profile

Lee

Jeong

Kim

et al. 2017

Biotech & Bioengineering

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To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (q ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After 3 days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. Biotechnol. Bioeng. 2017;114: 1721-1732. © 2017 Wiley Periodicals, Inc.

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Section: Relative Area (%)mentioning

confidence: 64%

Section: Relative Area (%)mentioning

confidence: 99%

Understanding of decreased sialylation of Fc‐fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N‐glycosylation gene expression and N‐linked glycan antennary profile

Lee

Jeong

Kim

et al. 2017

Biotech & Bioengineering

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“…Our results highlight that cell culture medium supplementation is a powerful tool to induce meleolic changes affecting the glycosylation pathway. These small molecules are not only suitable for glycosylation profile tuning as many authors have reported (Blondeel et al, ; Gramer et al, ; Hossler et al, ), but also have the potential to create extreme glycan variants. Applying them at appropriate concentration ranges limited detrimental effects on the culture performance.…”

Section: Discussionmentioning

confidence: 99%

Generation of site‐distinct N‐glycan variants for in vitro bioactivity testing

Brühlmann

Vuillemin²,

Satwekar

et al. 2019

Biotech & Bioengineering

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Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed‐batch cell culture batches yielded consistent glycoprofiles of a Fc‐fusion antibody comprizing three different N‐glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N‐glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site‐specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell‐based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N‐glycan structures, a higher degree of antennarity, and a site‐specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc‐site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N‐glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

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“…Instead of inhibiting one of the glycosylation transformation enzymes in the endoplasmic reticulum (ER) or the Golgi apparatus, the substrate generation may be targeted. Cultures supplemented with N-acetylglucosamine (GlcNAc) at the millimolar level resulted in reduced complexity of glycan profiles, hence favoring the G0 glycoform 204 . Media supplementation with kifunensine, a potent α-mannosidase I inhibitor entailed an increase of oligomannose containing monoclonal antibodies (mAb) 91 .…”

Section: Introductionmentioning

confidence: 99%

“…Targeted metabolic engineering approaches have been successfully applied to tune mAb glycan profile in cell culture. N-acetylglucosamine supplementation in cell culture favored non-galactosylated glycans (FA2) 204 . Exact and specific control of antibody galactosylation was achieved by feeding of uridine, manganese chloride and galactose, primarily shifting FA2 to FA2G1 species 52 .…”

Section: Introductionmentioning

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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Tuning a MAb glycan profile in cell culture: Supplementing N-acetylglucosamine to favour G0 glycans without compromising productivity and cell growth

Cited by 25 publications

References 46 publications

Understanding of decreased sialylation of Fc‐fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N‐glycosylation gene expression and N‐linked glycan antennary profile

Understanding of decreased sialylation of Fc‐fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N‐glycosylation gene expression and N‐linked glycan antennary profile

Generation of site‐distinct N‐glycan variants for in vitro bioactivity testing

Tailoring recombinant protein quality by rational media design

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