Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity

Konno, Yoshinobu; Kobayashi, Yuki; Takahashi, Ken; Takahashi, Eiji; Sakae, Shinji; Wakitani, Masako; Yamano, Kazuya; Suzawa, Toshiyuki; Yano, Keiichi; Ohta, Toshio; Koike, Masamichi; Wakamatsu, Kaori; Hosoi, Shinji

doi:10.1007/s10616-011-9377-2

Cited by 41 publications

(35 citation statements)

References 77 publications

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“…genetic engineering of recombinant cell lines (Louie et al, 2016;Yamane-Ohnuki et al, 2004); (b) addition of enzyme inhibitors (Allen et al, 2016;Okeley et al, 2015); (c) modification of the levels of cofactors and substrates involved in glycosylation, including supplementation with alternative sugars (Hossler et al, 2014;Hossler et al, 2017); and (d) fine-tuning of cell culture process parameters (Konno et al, 2012). Of the many cell culture process parameters known to impact glycosylation previously (Hossler, Khattak, & Li, 2009), osmolality was shown to affect afucosylation (Konno et al, 2012).…”

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confidence: 99%

Interaction of cell culture process parameters for modulating mAb afucosylation

Dang¹,

Mun²,

Rose³

et al. 2019

Biotech & Bioengineering

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The extent of afucosylation, which refers to the absence of core fucose on Fc glycans, can correlate positively with the antibody-dependent cellular cytotoxicity (ADCC) activity of a monoclonal antibody (mAb). Therefore, it is important to maintain consistent afucosylation during cell culture process scale-up in bioreactors for a mAb with ADCC activity. However, there is currently a lack of understanding about the impact of partial pressure of carbon dioxide (pCO 2 )-a parameter that can vary with bioreactor scale-on afucosylation. Using a small-scale (3 L) bioreactor model that can modulate pCO 2 levels through modified configurations and gassing strategies, we identified three cell culture process parameters that influence afucosylation of a mAb produced by a recombinant Chinese Hamster Ovary (CHO) cell line: pCO 2 , media hold duration (at 37°C), and manganese. These three-independent parameters demonstrated a synergistic effect on mAb afucosylation; increase in pCO 2 , media hold duration, and manganese consistently increased afucosylation. Our investigations into the underlying mechanisms through proteomic analysis indicated that the synergistic interactions downregulated pathways related to guanosine diphosphatefucose synthesis and fucosylation, and upregulated manganese transport into the CHO cells. These new findings highlight the importance of considering potential differences in culture environment and operations across bioreactor scales, and understanding the impact of their interactions on product quality.

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mentioning

confidence: 99%

Interaction of cell culture process parameters for modulating mAb afucosylation

Dang¹,

Mun²,

Rose³

et al. 2019

Biotech & Bioengineering

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“…In these biotherapeutic molecules, N-glycans are linked to the two conserved asparagine residues (Asn 297) in the CH2 domain of the Fc region 61 . A large number of reports describe various impacts of glycosylation on the quality attributes of biologics including in vivo efficacy [62][63][64] , pharmacokinetics (PK) 11 , antibody-dependent cellular cytotoxicity (ADCC) 63,65 and complementdependent cytotoxicity (CDC) activities 66 , stability and overall structure of the molecule 63 , clearance and half-life in vivo 10 as well as immunogenicity 10,67 . Non-fucosylated therapeutic antibodies exhibit 50 to 1,000-fold higher efficacy than their fucosylated counterparts 65 due, in most cases, to enhanced ADCC activity 68 .…”

Section: Glycosylationmentioning

confidence: 99%

“…A large number of reports describe various impacts of glycosylation on the quality attributes of biologics including in vivo efficacy [62][63][64] , pharmacokinetics (PK) 11 , antibody-dependent cellular cytotoxicity (ADCC) 63,65 and complementdependent cytotoxicity (CDC) activities 66 , stability and overall structure of the molecule 63 , clearance and half-life in vivo 10 as well as immunogenicity 10,67 . Non-fucosylated therapeutic antibodies exhibit 50 to 1,000-fold higher efficacy than their fucosylated counterparts 65 due, in most cases, to enhanced ADCC activity 68 . Given the abundance of research efforts in this area, it is no surprise that many have concluded that glycosylation is one of the main areas requiring development 69 to improve efficacy 70 and safety 10,70 of next generation therapeutics 61 .…”

Section: Glycosylationmentioning

confidence: 99%

“…It is not surprising that increasing effector functions by means of Fc-glycosylation engineering has gained considerable interest 196 . As a consequence, the pharmaceutical industry has allocated remarkable research efforts to glycan engineering, aiming to increase ADCC of mAbs, in particular by limiting core fucosylation 48,65,91,[308][309][310][311][312][313] . Many reports have described the significant increase of ADCC whenever the fucose attachment to the glycan backbone was precluded 42,196,308,312 .…”

Section: Introductionmentioning

confidence: 99%

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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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“…Glycosylation is one of very important post-translational modifications because it can have significant impact on antibody-dependent cell-mediated cytotoxicity (ADCC) activity [3,4], complement-dependent cytotoxicity (CDCC) activity [5], clearance [6] and immunogenicity [7,8]. Protein glycosylation can be significantly impacted by the host cell line, clone, media composition, feeding strategy and downstream processing conditions [9][10][11]. Therefore, N-glycosylation analysis of monoclonal antibody is widely performed in the biopharmaceutical development, and in some cases is used as release assay when glycosylation is established as a critical quality attribute [12].…”

Section: Introductionmentioning

confidence: 99%

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS

Wang¹,

Peng²,

Sosic³

et al. 2017

J Anal Bioanal Tech

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Rapid and sensitive product quality analysis is important for real-time monitoring during biopharmaceutical development and manufacturing. However, low level of protein concentration and complex cell culture matrix pose challenges for product quality characterization at early stages of cell line selection and process development. Here, we describe a fast and simple microfluidic ZipChip CE-MS method to measure quality attributes of monoclonal antibody protein directly from cell culture supernatant. Cell culture supernatant samples were characterized with charge-based separation using microfluidic capillary electrophoresis coupled to a high-resolution mass spectrometer. Under sample reducing conditions, multiple protein glycosylation attributes were determined on the heavy chain, whereas titer information was obtained from comparison of light chain signal intensity following sample spiking-in with heavy labeled mAb. Therefore, the protein expression and product quality can be monitored using the same method with a single microfluidic device. A total volume of ten to fifty microliter of cell culture supernatant is needed, whereas analysis time is within three minutes per sample. In addition, comparison of new method with traditional RP-LC-MS method using a set of time-course bioreactor cell culture samples has been performed. A good correlation of the levels of N-glycosylation attributes between ZipChip CE-MS of crude samples and RPLC-MS analysis following Protein A (ProA) purification step has been demonstrated.

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Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity

Cited by 41 publications

References 77 publications

Interaction of cell culture process parameters for modulating mAb afucosylation

Interaction of cell culture process parameters for modulating mAb afucosylation

Tailoring recombinant protein quality by rational media design

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS

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