Subcellular Fractionation Enables Assessment of Nucleotide Sugar Donors Inside the Golgi Apparatus as a Prerequisite for Unraveling Culture Impacts on Glycoforms of Antibodies

IF 3.2 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Niklas Regett, Marcel Dieterle, Fleur Peters, Max Deuring, Kaja Stegmaier, Attila Teleki, Ralf Takors
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引用次数: 0

Abstract

Glycosylation is a critical quality attribute in biopharmaceuticals that influences crucial properties, such as biological activity and blood clearance. Current methods for modeling glycosylation typically rely on imprecise or limited data on nucleotide sugar donor (NSD) dynamics. These methods use in vitro transporter kinetics or flux balance analysis, which overlook the key aspects of metabolic regulation. We devised an integrative workflow for absolute subcellular NSD quantification in both cytoplasm and secretory organelles. Using subcellular fractionation, exhaustive sample extraction, and liquid chromatography triple-quadrupole tandem mass spectrometry, we accurately measured NSD concentrations ranging from 1.6 amol/cell to 3 fmol/cell.

As expected, NSD concentration profiles aligned closely with the glycan distributions on antibodies, particularly after nutrient pulsing to stimulate NSD production, showcasing method validity. This method enables empirical observation of compartment-specific NSD dynamics. Thus, this study provides novel insights indicating that N-glycosylation, which governs NSD supply, is primarily regulated within the Golgi apparatus (GA). This method offers a novel tool to obtain sophisticated data for a more efficient optimization of glycosylation processes in production cell lines.

Abstract Image

糖基化是生物制药的关键质量属性,会影响生物活性和血液清除率等重要特性。目前的糖基化建模方法通常依赖于不精确或有限的核苷酸糖供体(NSD)动态数据。这些方法使用体外转运体动力学或通量平衡分析,忽略了代谢调控的关键方面。我们设计了一种综合工作流程,用于对细胞质和分泌细胞器中的亚细胞核苷酸供体进行绝对定量。正如预期的那样,NSD浓度曲线与抗体上的聚糖分布密切吻合,特别是在营养脉冲刺激NSD产生之后,显示了方法的有效性。这种方法能对特定区室的 NSD 动态进行经验观察。因此,这项研究提供了新的见解,表明支配 NSD 供应的 N-糖基化主要在高尔基体(GA)内调节。这种方法提供了一种新工具,可用于获取复杂的数据,从而更有效地优化生产细胞系中的糖基化过程。
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来源期刊
Biotechnology Journal
Biotechnology Journal Biochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
8.90
自引率
2.10%
发文量
123
审稿时长
1.5 months
期刊介绍: Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances. In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office. BTJ promotes a special emphasis on: Systems Biotechnology Synthetic Biology and Metabolic Engineering Nanobiotechnology and Biomaterials Tissue engineering, Regenerative Medicine and Stem cells Gene Editing, Gene therapy and Immunotherapy Omics technologies Industrial Biotechnology, Biopharmaceuticals and Biocatalysis Bioprocess engineering and Downstream processing Plant Biotechnology Biosafety, Biotech Ethics, Science Communication Methods and Advances.
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