An efficient method to control high mannose and core fucose levels in glycosylated antibody production using deoxymannojirimycin

Levanon, Sagit Shalel; Aharonovitz, Orit; Maor-Shoshani, Ayelet; Abraham, Gita; Kenett, Dan; Aloni, Yehoshua

doi:10.1016/j.jbiotec.2018.04.006

Cited by 8 publications

(13 citation statements)

References 14 publications

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“…The dose‐dependent decrease in complex glycans is likely due to the inhibitor preventing subsequent glycosylation steps. The level of afucosylated glycans was equivalent to high‐mannose glycans as seen in other mannosidase I inhibitors, indicating that tris most likely did not inhibit the fucosylation glycoenzyme but only reduced the G0 and G0‐GlcNAc substrate availability 20,45 …”

Section: Resultsmentioning

confidence: 78%

“…The level of afucosylated glycans was equivalent to high-mannose glycans as seen in other mannosidase I inhibitors, indicating that tris most likely did not inhibit the fucosylation glycoenzyme but only reduced the G0 and G0-GlcNAc substrate availability. 20,45 Tris concentrations exceeding 20 mM resulted in reduced VCD, viability, and titer likely due to tris toxicity ( Figure 6). Toxicity of tris has been demonstrated for bacteria and various mammalian tissue cultures in the range of 10-50 mM likely due to disruption of cell membranes.…”

Section: Dose-dependent Response Of Novel Inhibitormentioning

confidence: 99%

“…36 GDP-Fuc was not quantified due to the lack of reference standard. [19][20][21][22]37,38 Cultures exposed to 10 mM of novel inhibitor AMPD or serinol resulted in increased peak VCD of 5 million/ml compared to control.…”

Section: Monoclonal Antibody Measurementmentioning

confidence: 99%

“…[46][47][48][49] The results are comparable to titration studies of other mannosidase I inhibitors (kifunensine and DMJ) in CHO cells, which resulted in decreased harvest VCD, viability, and titer with an increase in high-mannose glycans. 20,50 The fed-batch shake flasks studies have shown tris as a lever for modulating high mannose glycans similarly to kifunensine and DMJ.…”

Section: Dose-dependent Response Of Novel Inhibitormentioning

confidence: 99%

“…Kifunensine, a potent inhibitor of alpha‐mannosidase I, has been used extensively at ranges of 0.1–2.5 μg/ml in various mammalian (CHO and HEK), plant (rice cell culture), and bacteria (agrobacterium) cells and results in up to 95% high‐mannose glycans 5,16‐18 . Deoxymannojirimycin (DMJ) has also been shown to inhibit mannosidase I in CHO, yeast, and MDCK cells at 0.8–80 μg/ml resulting in 94% high‐mannose glycans 15,19,20 . However, kifunensine and DMJ readily degrade at room temperature, and stock solutions require long‐term storage at −20°C and short‐term at 4°C to prevent decomposition.…”

Section: Introductionmentioning

confidence: 99%

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A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

Brantley

Mitchelson

Khattak

2020

Biotechnology Progress

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N-linked glycosylation of therapeutic monoclonal antibodies is an important product quality attribute for drug safety and efficacy. An increase in the percent of high mannose N-linked glycosylation may be required for drug efficacy or to match the glycosylation profile of the innovator drug during the development of a biosimilar. In this study, the addition of several chemical additives to a cell culture process resulted in high mannose N-glycans on monoclonal antibodies produced by Chinese hamster ovary (CHO) cells without impacting cell culture performance. The additives, which include known mannosidase inhibitors (kifunensine and deoxymannojirimycin) as well as novel inhibitors (tris, bis-tris, and 1-amino-1-methyl-1,3-propanediol), contain one similar molecular structure: 2-amino-1,3-propanediol, commonly referred to as serinol. The shared chemical structure provides insight into the binding and inhibition of mannosidase in CHO cells. One of the novel inhibitors, tris, is safer compared to kifunensine, 35x as cost-effective, and stable at room temperature. In addition, tris and bis-tris provide multiple low-cost alternatives to kifunensine for manipulating glycosylation in monoclonal antibody production in a cell culture process with minimal impact to productivity or cell health.

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Section: Resultsmentioning

confidence: 78%

Section: Dose-dependent Response Of Novel Inhibitormentioning

confidence: 99%

Section: Monoclonal Antibody Measurementmentioning

confidence: 99%

Section: Dose-dependent Response Of Novel Inhibitormentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

Brantley

Mitchelson

Khattak

2020

Biotechnology Progress

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Strategies for controlling afucosylation in monoclonal antibodies during upstream manufacturing

Rish

Siddiquee

Huang

et al. 2023

Biotechnology Journal

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Core fucosylation is a highly prevalent and significant feature of N-glycosylation in therapeutic monoclonal antibodies produced by mammalian cells where its absence (afucosylation) plays a key role in treatment safety and efficacy. Notably, even slight changes in the level of afucosylation can have a considerable impact on the antibody-dependent cell-mediated cytotoxicity. Therefore, implementing control over afucosylation levels is important in upstream manufacturing to maintain consistent quality across batches of product, since standard downstream processing does not change afucosylation. In this review, the influences and strategies to control afucosylation are presented. In particular, there is emphasis on upstream manufacturing culture parameters and media supplementation, as these offer particular advantages as control strategies over alternative approaches such as cell line engineering and chemical inhibitors. The review discusses the relationship between the afucosylation influences and the underlying cellular metabolism to promote increased process understanding. Also, briefly highlighted is the value of empirical and mechanistic models in evaluating and designing control methods for core fucosylation.

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Modulation of high mannose levels in N‐linked glycosylation through cell culture process conditions to increase antibody‐dependent cell‐mediated cytotoxicity activity for an antibody biosimilar

Rameez

Gowtham

Nayar

et al. 2021

Biotechnology Progress

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The regulatory approval of a biosimilar product is contingent on the favorable comparability of its safety and efficacy to that of the innovator product. As such, it is important to match the critical quality attributes of the biosimilar product to that of the innovator product. The N‐glycosylation profile of a monoclonal antibody (mAb) can influence effector function activities such as antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity. In this study, we describe efforts to modulate the high‐mannose (HM) levels of a biosimilar mAb produced in a Chinese hamster ovary cell fed‐batch process. Because the HM level of the mAb was observed to impact ADCC activity, it was desirable to match it to the innovator mAb's levels. Several cell culture process related factors known to modulate the HM content of N‐glycosylation were investigated, including osmolality, ammonium chloride (NH4Cl) addition, glutamine concentration, monensin addition, and the addition of alternate sugars and amino sugars to the feed medium. The process conditions evaluated varied in impact on HM levels, process performance and product quality. One condition, the addition of alternate sugars and amino sugars to feed medium, was identified as the preferred method for increasing HM levels with minimal disruptions to process performance or other product quality attributes. Interestingly, a secondary interaction between sugar and amino sugar supplemented feeds and osmolality was observed during process scale‐up. These studies demonstrate sugar and amino sugar concentrations and osmolality are critical variables to evaluate to match HM content in biosimilar and their innovator mAbs.

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An efficient method to control high mannose and core fucose levels in glycosylated antibody production using deoxymannojirimycin

Cited by 8 publications

References 14 publications

A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

Strategies for controlling afucosylation in monoclonal antibodies during upstream manufacturing

Modulation of high mannose levels in N‐linked glycosylation through cell culture process conditions to increase antibody‐dependent cell‐mediated cytotoxicity activity for an antibody biosimilar

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