Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation

Brühlmann, David; Muhr, Anais; Parker, Rebecca E.; Vuillemin, T.; Bucsella, Blanka; Kálmán, Franka; Torre, Serena; Neve, Fabio La; Lembo, A.; Haas, Tobias; Sauer, Markus; Souquet, Jonathan; Broly, Hervé; Hemberger, Jürgen; Jordan, Martin

doi:10.1016/j.jbiotec.2017.04.026

Cited by 27 publications

(14 citation statements)

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“…The literature also reported significantly lower concentrations with CHO cells expressing a human anti‐TEM b mAb: 100 ng/ml (corresponds to 0.4 µM; Zhou et al, ). Surprisingly, the effect of raffinose on the glycosylation metabolism of this cell line was significantly weaker than we found earlier with a different cell line (Brühlmann, Muhr, et al, ). Further investigations have to be conducted to understand whether this discrepancy was due to a raffinose lot‐to‐lot variability, as small trace element differences might have had an impact—as summarized in a review article (Arigony et al, )—or whether it is the result of substantially different behaviors of the two cell lines.…”

Section: Discussioncontrasting

confidence: 93%

“…We used well‐known additives to produce a diverse N‐glycan variant population including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Careful optimization of both the concentration of supplements (Brühlmann, Sokolov, et al, ) and the medium osmolality (Brühlmann, Muhr, et al, ) made it possible to extend substantially the glycosylation variation ranges. Kifunensine, a strong mannosidase I inhibitor, and raffinose, a trisaccharide, were selected to increase the abundance of high mannose glycans as previously reported (Brühlmann, Muhr, et al, ; Zhou et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…Careful optimization of both the concentration of supplements (Brühlmann, Sokolov, et al, ) and the medium osmolality (Brühlmann, Muhr, et al, ) made it possible to extend substantially the glycosylation variation ranges. Kifunensine, a strong mannosidase I inhibitor, and raffinose, a trisaccharide, were selected to increase the abundance of high mannose glycans as previously reported (Brühlmann, Muhr, et al, ; Zhou et al, ). 2F‐peracetyl fucose, a fucose analog specifically inhibiting fucosyltransferase (Rillahan et al, ) was selected to generate afucosylated glycans.…”

Section: Introductionmentioning

confidence: 99%

“…We used well-known additives to produce a diverse N-glycan variant population including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Careful optimization of both the concentration of supplements (Brühlmann, Sokolov, et al, 2017) and the medium osmolality (Brühlmann, Muhr, et al, 2017) made it possible to extend substantially the glycosylation variation ranges.…”

mentioning

confidence: 99%

“…Kifunensine, a strong mannosidase I inhibitor, and raffinose, a trisaccharide, were selected to increase the abundance of high mannose glycans as previously reported (Brühlmann, Muhr, et al, 2017;Zhou et al, 2008). 2F-peracetyl fucose, a fucose analog specifically inhibiting fucosyltransferase (Rillahan et al, 2012) was selected to generate afucosylated glycans.…”

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confidence: 99%

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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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Section: Discussioncontrasting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Brühlmann

Vuillemin²,

Satwekar

et al. 2019

Biotech & Bioengineering

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Mammalian Fed‐batch Cell Culture for Biopharmaceuticals

Yang

2019

Cell Culture Engineering

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Advancing Raman model calibration for perfusion bioprocesses using spiked harvest libraries

Romann

Kolar

Tobler

et al. 2022

Biotechnology Journal

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Background Raman spectroscopy has gained popularity to monitor multiple process indicators simultaneously in biopharmaceutical processes. However, robust and specific model calibration remains a challenge due to insufficient analyte variability to train the models and high cross‐correlation of various media components and artifacts throughout the process. Main Methods A systematic Raman calibration workflow for perfusion processes enabling highly specific and fast model calibration was developed. Harvest libraries consisting of frozen harvest samples from multiple CHO cell culture bioreactors collected at different process times were established. Model calibration was subsequently performed in an offline setup using a flow cell by spiking process harvest with glucose, raffinose, galactose, mannose, and fructose. Major Results In a screening phase, Raman spectroscopy was proven capable not only to distinguish sugars with similar chemical structures in perfusion harvest but also to quantify them independently in process‐relevant concentrations. In a second phase, a robust and highly specific calibration model for simultaneous glucose (root mean square error prediction [RMSEP] = 0.32 g L−1) and raffinose (RMSEP = 0.17 g L−1) real‐time monitoring was generated and verified in a third phase during a perfusion process. Implication The proposed novel offline calibration workflow allowed proper Raman peak decoupling, reduced calibration time from months down to days, and can be applied to other analytes of interest including lactate, ammonia, amino acids, or product titer.

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Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation

Cited by 27 publications

References 53 publications

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

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

Mammalian Fed‐batch Cell Culture for Biopharmaceuticals

Advancing Raman model calibration for perfusion bioprocesses using spiked harvest libraries

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