Upstream R&D, Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany.
Cells. 2023 Jan 16;12(2):334. doi: 10.3390/cells12020334.
Thiamin is susceptible to heat and oxidation, which is a concern for the development of concentrated and room temperature stable feeds used to produce recombinant proteins. Hence, it is critical to understand the reactivity and necessity of the vitamin in liquid feeds to be able to either develop mitigation strategies to stabilize the vitamin or to remove thiamin from formulations if it is unnecessary. LC-MS/MS was used to investigate thiamin stability in different liquid feed formulations and to identify thiamin degradation products. Results indicate oxidation of thiamin and interaction with amino acids, keto acids, and sulfur containing components. Thiamin necessity in feed was assessed during a fed batch experiment, focusing on cell performance and critical quality attributes of the produced recombinant proteins. The impact of thiamin depletion in the feed on the intra- and extracellular metabolome was investigated using untargeted LC-MS/MS. Results indicate that thiamin can be removed from the feed without affecting the performance or the intra- and extracellular metabolome of the tested cell lines. Overall, profound insights on thiamin reactivity and necessity are presented in this study, suggesting the removal of the dispensable and instable vitamin as a simple means for the development of next generation feeds used to produce therapeutic biological entities.
硫胺素易受热和氧化影响，这是开发用于生产重组蛋白的浓缩和室温稳定饲料时需要关注的问题。因此，了解液体饲料中维生素的反应性和必要性至关重要，以便能够制定减轻维生素稳定性的策略，或者在不需要时从配方中去除硫胺素。LC-MS/MS 用于研究不同液体饲料配方中硫胺素的稳定性，并确定硫胺素降解产物。结果表明，硫胺素发生氧化反应，并与氨基酸、酮酸和含硫成分相互作用。在分批补料实验中评估了饲料中硫胺素的必要性，重点关注细胞性能和生产的重组蛋白的关键质量属性。使用非靶向 LC-MS/MS 研究了饲料中硫胺素耗尽对细胞内和细胞外代谢组的影响。结果表明，可以从饲料中去除硫胺素而不影响测试细胞系的性能或细胞内和细胞外代谢组。总的来说，本研究对硫胺素的反应性和必要性有了更深入的了解，表明去除可有可无且不稳定的维生素是开发用于生产治疗性生物实体的下一代饲料的一种简单方法。
