N‐glycosylation is defined as a key quality attribute for the majority of complex biological therapeutics. Despite many N‐glycan engineering efforts, the demand to generate desired N‐glycan profiles that may vary for different proteins in a reproducible manner is still difficult to fulfill in many cases. Stable production of homogenous structures with a more demanding level of processing, for instance high degrees of branching and terminal sialylation, is particularly challenging. Among many other influential factors, the level of productivity can steer N‐glycosylation towards less mature N‐glycan structures. Recently, we introduced an mRNA transfection system capable of elucidating bottlenecks in the secretory pathway by stepwise increase of intracellular model protein mRNA load. Here, this system was applied to evaluate engineering strategies for enhanced N‐glycan processing. The tool proves to indeed be valuable for a quick assessment of engineering approaches on the cellular N‐glycosylation capacity at high productivity. The gene editing approaches tested include overexpression of key Golgi‐resident glycosyltransferases, partially coupled with multiple gene deletions. Changes in galactosylation, sialylation, and branching potential as well as N‐acetyllactosamine formation were evaluated.

中文翻译：

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转染糖蛋白编码 mRNA 以快速评估 N-聚糖工程策略。

N-糖基化被定义为大多数复杂生物疗法的关键质量属性。尽管进行了许多 N-聚糖工程设计，但在许多情况下仍难以满足产生所需 N-聚糖谱的需求，这些谱可能以可重复的方式因不同蛋白质而异。具有更高加工水平的同质结构的稳定生产，例如高度支化和末端唾液酸化，尤其具有挑战性。在许多其他影响因素中，生产力水平可以将 N-糖基化导向不太成熟的 N-聚糖结构。最近，我们引入了一种 mRNA 转染系统，能够通过逐步增加细胞内模型蛋白 mRNA 负载来阐明分泌途径中的瓶颈。这里，该系统用于评估增强 N-聚糖加工的工程策略。事实证明，该工具对于快速评估高生产率下细胞 N-糖基化能力的工程方法确实很有价值。测试的基因编辑方法包括关键高尔基体糖基转移酶的过度表达，部分与多个基因缺失相结合。评估了半乳糖基化、唾液酸化和分支电位以及 N-乙酰乳糖胺形成的变化。