Hydrocortisone

蛋白质的糖基化是指寡糖链通过共价键与肽链结合，是蛋白质重要的翻译后加工修饰之一。对蛋白质的结构稳定性、血清半衰期、生物活性及溶解度等关键属性都可能具有重要影响，通常是重组蛋白/抗体类生物制品的关键质量属性（CQA）。 酸性单糖唾液酸通常位于修饰蛋白的寡糖链末端，唾液酸化对重组蛋白类药物的药代动力学具有重要影响，可降低蛋白质在体内被清除的速率，还参与细胞识别、信号传导等生物过程。 今天为大家介绍一种增强CHO细胞培养物中Fc融合蛋白的唾液酸化的有效方法。 人类蛋白质糖链的唾液酸通常为α2,6-唾液酸和α2,3-唾液酸。由于编码α2,6- ST的st6gal1基因启动子被甲基化失活，做为重组蛋白类药物生产重要宿主的CHO细胞无法产生α2,6-唾液酸，因此仅能对其表达的蛋白质进行α2,3-唾液酸化修饰。 对CHO细胞来源的重组蛋白唾液酸连接进行双重处理可使其更接近人类的蛋白质，从而减少或避免治疗性蛋白引发的机体的免疫反应。 目前许多研究聚焦于通过上调唾液酸转移酶活性和下调唾液酸酶活性来最大化唾液酸含量。其中最直接的方法是通过调整培养基组分来实现唾液酸水平的提升。 糖皮质激素（GCs）一类类固醇激素。因与糖皮质激素受体（GRs）可特异性结合并激活其功能，常被用来调控细胞代谢、增殖、分泌以及糖蛋白糖基化等生理过程。 今天挑选常见的六种GCs（图1），分别是氢化可的松（Hyd）、皮质酮（Ccs）、地塞米松（Dex）、 倍他米松（Bet）、 泼尼松龙（Prl） 和 泼尼松（Prd），评估GCs作为培养基添加剂在提升CHO细胞培养中重组蛋白质量方面的应用价值。 图1：用作CHO细胞培养物添加剂的GCs的化学结构 1 材料与方法 细胞：CHO DUKX-B11，在ST6Gal1中过表达并表达α2,6-ST的CTLA4-Ig 测试添加剂：Hyd、Ccs、Dex、Bet、Prl和Prd 浓度范围：0、0.5、1、10 和 20 mg/L 培养模式：CHO细胞以3×106个细胞/mL的密度接种至工作体积为20 mL的125 mL锥形瓶中，置于在 37 ℃、100 rpm 、 5% CO2 摇床中培养。从第1天到第8天，每24小时取样一次。监测活细胞密度（VCD）和活率。 2 结果 GCs对CHO细胞培养性能的影响 图2：补充GC培养物的活细胞密度（VCD）分布曲线 除20 mg/L浓度外，添加GC对VCD的影响与对照组相比无显著差异。 图3：补充GC培养物的细胞活力曲线 在所有测试的GC浓度下，CHO细胞培养物的活率均未受影响。 图4：GC对CHO细胞培养中（A） IVCD和（B）滴度的影响 通过ELISA定量检测CTLA4-Ig水平发现，GCs对CTLA4-Ig的产生具有浓度相关性的负面影响（图4B）。20 mg/L Ccs下CTLA4-Ig的产量降低了47%，但10 mg/L Ccs培养的CTLA4-Ig滴度则与对照相当。 这些数据表明，糖皮质激素的使用会导致CTLA4-Ig的生成减少，但在CHO细胞培养体系中，糖皮质激素的使用浓度可达到10 mg/L。 结合此前研究报道指出，糖皮质激素会抑制哺乳动物细胞的葡萄糖转运和谷氨酰胺摄取功能。CHO细胞培养物中添加GC后，细胞生长和重组蛋白产量降低与葡萄糖和谷氨酰胺摄取受抑制有关。 盐酸水解α2,3-和α2,6-唾液酸，而唾液酸酶S仅能水解α2,3-唾液酸。如图5所示，添加10 mg/L浓度的Ccs可显著提升总唾液酸含量。与对照组相比，含量变化幅度为1.16倍。实验数据表明，在CHO细胞培养体系中添加Ccs等某些糖基化辅酶， 能有效提升唾液酸化水平。 CTLA4-Ig中唾液酸的相对丰度   图5：α2,3-唾液酸含量（A）与总唾液酸含量（B）的分析结果 细胞内唾液酸转移酶在将CMP-唾液酸递送至糖链残基的过程中起着关键作用。因此，提高该酶的活性可有效调控重组蛋白的唾液酸化水平。如图6所示，与对照组相比，添加10 mg/L Ccs后，细胞内唾液酸转移酶活性显著增加。 细胞内唾液酸转移酶活性 图6：第6天的相对细胞内唾液酸转移酶活性 CHO 细胞的内源性唾液酸酶，可在细胞培养过程中在培养上清液中积累，导致分泌至培养上清中糖蛋白糖链末端唾液酸被移除。因此，唾液酸酶的下调可提高糖蛋白的唾液酸化程度。如图7所示，在培养第6天，补充有10 mg/L Ccs 的细胞外唾液酸酶活性比对照组降低了25%。 细胞外唾液酸酶活性 图7：第6天细胞外唾液酸酶活性分析 3 结论 本文描述了在表达α2,3和α2,6-ST的CHO细胞培养液中，添加不同种类的GCs对细胞培养性能和Fc-融合蛋白唾液酸化的影响。尽管额外添加GCs可对生产效率产生负面影响，但可提升重组蛋白的唾液酸总量。其中，Ccs对提升Fc融合蛋白总唾液酸化水平最为显著，包括具有类人唾液酸化特征的α2,6-唾液酸。 唾液酸含量的增加主要归因于两个因素：细胞内唾液酸转移酶活性上调与细胞外唾液酸酶活性下调。唾液酸转移酶活性增强后，能更有效地将唾液酸转移到末端半乳糖残基上。此外，在Ccs处理条件下，由于唾液酸酶活性降低，Fc融合蛋白上的唾液酸裂解过程受到抑制。 本文介绍了一种提高CHO细胞培养物中重组蛋白质量的有效策略，通过将Ccs作为细胞培养添加剂，可有效增强细胞内唾液酸转移酶活性，并抑制细胞外唾液酸酶活性，从而提高唾液酸化水平，最终实现提升CHO细胞重组蛋白质量的目标。 参考文献： Utilization of Glucocorticoids as Additives for Enhanced Sialylation of Fc-fusion Protein in CHO Cell Cultures[J]. Biotechnology and Bioprocess Engineering 26: 286-293

President Donald Trump signed an executive order on Wednesday directing an office within the Department of Health and Human Services (HHS) to increase its supply of ingredients for "critical" drugs, part of his latest push to bring more facets of the pharmaceutical supply chain into the US. The order follows closely behind a new initiative unveiled by the FDA last week, called the PreCheck programme, designed to offer more support for groups seeking to build US production facilities.  Under the threat of steep tariffs that could reach 250%, Trump has spent the majority of his second term so far pressuring drug developers to onshore manufacturing — and broadly, big pharma appears to have complied, with about a dozen companies having announced plans to invest billions in US operations.Some of the heftiest financial promises were from European drugmakers, with AstraZeneca and Roche each pledging $50-billion investments. For more, see – Spotlight On: AstraZeneca puts its money where its mouth is.The most recent update came from AbbVie, which said Tuesday that it will invest $195 million to build a new active pharmaceutical ingredient (API) manufacturing facility at its North Chicago campus as part of a broader $10-billion commitment to bolster its US manufacturing.Now it seems the Trump administration is also homing in on the importance of APIs. Under the executive order, the Office of the Assistant Secretary for Preparedness and Response (ASPR) has been directed to first develop a list of 26 drugs deemed "vital to national health and security," and then ensure the Strategic Active Pharmaceutical Ingredients Reserve (SAPIR) is stocked with a 6-month supply of components for these treatments, preferentially using APIs made in the US. In addition, ASPR has been instructed to update a list of 86 essential medicines created in 2022, and draft a plan to secure a half-year supply for these drugs' relevant APIs as well. The office will also create a proposal for a second SAPIR. "Stockpiling APIs, which are lower-cost and have longer shelf lives, strengthens the nation’s ability to ensure access to critical drugs during emergencies," the White House said in a fact sheet for the executive order. "Restoring capacity for domestic production of essential pharmaceutical products is essential to safeguarding national health and security against global supply chain disruptions."The order comes as the Trump administration has raised concerns that the lack of US manufacturing leaves the country vulnerable to foreign supply chain disruptions. According to the fact sheet, only about 10% of APIs for US prescription drugs are manufactured in the country.A recent report from a US security commission found that in 2022, up to 28% of API imports were sourced from China, along with 90% of common medicines such as ibuprofen, hydrocortisone and acetaminophen."The United States is overly and dangerously reliant on foreign sources for our medicines and therapeutics," the National Security Commission on Emerging Biotechnology wrote.