Progress toward rapid, at-line N-glycosylation detection and control for recombinant protein expression

Mao, Leran; Schneider, James W.; Robinson, Anne S.

doi:10.1016/j.copbio.2022.102788

Cited by 7 publications

(6 citation statements)

References 57 publications

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“…Harvested IgG was purified with a 96‐well protein A plate (Thermofisher) as described previously. [ 100 ] The plate was pre‐equilibrated by washing three times with PBS followed by centrifuging at 300 x g for 2 min. Then, clarified cell culture media was added to each well and incubated for 5–10 min.…”

Section: Methodsmentioning

confidence: 99%

“…Quantification was described as previously reported by gating cells with positive staining as compared to the control (unsupplemented) condition. [ 100 ]…”

Section: Methodsmentioning

confidence: 99%

“…[39] Protein glycosylation is particularly important for protein therapies because it contributes to their stability, half-life, as well as potency. [41] Previously, RA-supplemented cultures resulted in a deviation of glycosylation patterns from a reference standard, [20] although the authors did not report a change in monoclonal antibody (mAb) potency. In that case, the target mAb (an IgG4) possesses a single glycosylation site and is much simpler in structure compared to other mAbs; thus, changes to its glycosylation pattern have minimal effect on the protein's function.…”

Section: Ra Prolongs the Viable Period Of Cell Culture Enhances Antib...mentioning

confidence: 99%

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Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway

Mao,

Schneider,

Robinson

2023

Biotechnology Journal

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Rosmarinic acid (RA) has gained attraction in bioprocessing as a media supplement to improve cellular proliferation and protein production. Here, we observe up to a two‐fold increase in antibody production with RA‐supplementation, and a concentration‐dependent effect of RA on cell proliferation for fed‐batch CHO cell cultures. Contrary to previously reported antioxidant activity, RA increased the reactive oxygen species (ROS) levels, stimulated endoplasmic reticulum (ER) stress, activated the unfolded protein response (UPR), and elicited DNA damage. Despite such stressful events, RA appeared to maintained cell health via mTOR pathway activation; both mTORC1 and mTORC2 were stimulated in RA‐supplemented cultures. By reversing such mTOR pathway activity through either chemical inhibitor addition or siRNA knockdown of genes regulating the mTORC1 and mTORC2 complexes, antibody production, UPR signaling, and stress‐induced DNA damage were reduced. Further, the proliferative effect of RA appeared to be regulated selectively by mTORC2 activation and have reproduced this observation by using the mTORC2 stimulator SC‐79. Analogously, knockdown of mTORC2 strongly reduced XBP1 splicing, which would be expected to reduce antibody folding and secretion, sugging that reduced mTORC2 would correlate with reduced antibody levels. The crosstalk between mTOR activation and UPR upregulation may thus be related directly to the enhanced productivity. Our results show the importance of the mTOR and UPR pathways in increasing antibody productivity, and suggest that RA supplementation may obviate the need for labor‐intensive genetic engineering by directly activating pathways favorable to cell culture performance.This article is protected by copyright. All rights reserved

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Section: Methodsmentioning

confidence: 99%

“…Quantification was described as previously reported by gating cells with positive staining as compared to the control (unsupplemented) condition. [ 100 ]…”

Section: Methodsmentioning

confidence: 99%

Section: Ra Prolongs the Viable Period Of Cell Culture Enhances Antib...mentioning

confidence: 99%

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Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway

Mao,

Schneider,

Robinson

2023

Biotechnology Journal

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“…Glycoproteins produced in CHO cells resemble those produced in the native human body due to a lack of α‐galactose residues, making clinical transferability considerably more efficient. Despite their widespread use, the ability to measure and control post‐translational modifications during a bioprocess remains limited 4,5 …”

Section: Introductionmentioning

confidence: 99%

“…Although many tools are available for analyzing glycan microheterogeneity, 4 glycan macroheterogeneity is frequently characterized towards the end of the downstream process by mass spectrometry (MS), which requires expert knowledge and is time intensive. Thus, a simpler and faster screening platform that allows characterization of both glycan micro‐ and macroheterogeneity is needed; such a rapid analysis platform will be essential for screening process conditions that affect antibody quality.…”

Section: Introductionmentioning

confidence: 99%

Use of single analytic tool to quantify both absolute N‐glycosylation and glycan distribution in monoclonal antibodies

Mao

Schneider

Robinson

2023

Biotechnology Progress

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Recombinant proteins represent almost half of the top selling therapeutics—with over a hundred billion dollars in global sales—and their efficacy and safety strongly depend on glycosylation. In this study, we showcase a simple method to simultaneously analyze N‐glycan micro‐ and macroheterogeneity of an immunoglobulin G (IgG) by quantifying glycan occupancy and distribution. Our approach is linear over a wide range of glycan and glycoprotein concentrations down to 25 ng/mL. Additionally, we present a case study demonstrating the effect of small molecule metabolic regulators on glycan heterogeneity using this approach. In particular, sodium oxamate (SOD) decreased Chinese hamster ovary (CHO) glucose metabolism and reduced IgG glycosylation by 40% through upregulating reactive oxygen species (ROS) and reducing the UDP‐GlcNAc pool, while maintaining a similar glycan profile to control cultures. Here, we suggest glycan macroheterogeneity as an attribute should be included in bioprocess screening to identify process parameters that optimize culture performance without compromising antibody quality.

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Artificial Intelligence Applications for Producing Glycosylated Biopharmaceutical Drug Modalities

von Horsten

2024

Management for Professionals

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Progress toward rapid, at-line N-glycosylation detection and control for recombinant protein expression

Cited by 7 publications

References 57 publications

Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway

Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway

Use of single analytic tool to quantify both absolute N‐glycosylation and glycan distribution in monoclonal antibodies

Artificial Intelligence Applications for Producing Glycosylated Biopharmaceutical Drug Modalities

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