亚洲色综合导航,欧美性爱H版A片,激情导航,亚洲干综合,日本外道魂在线视频,avtt天堂网中文

The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets.

持續(xù)的 COVID-19 大流行引起了全球?qū)魅静〉年P(guān)注，吸引了大量資源用于開發(fā)大流行防范計(jì)劃和疫苗平臺(tái)——這些技術(shù)具有強(qiáng)大的制造工藝，可以快速轉(zhuǎn)向針對(duì)新出現(xiàn)的疾病。新城疫病毒 (NDV) 已被研究作為人類和獸用疫苗的病毒載體，但其生產(chǎn)嚴(yán)重依賴雞胚，很少有研究在細(xì)胞培養(yǎng)中產(chǎn)生 NDV。在這里，NDV 是在懸浮的 Vero 細(xì)胞中產(chǎn)生的，分析測(cè)定 (TCID 50和 ddPCR) 用于量化感染性和總病毒滴度。NDV-GFP 和 NDV-FLS（SARS-CoV-2 全長(zhǎng)刺突蛋白）構(gòu)建體適用于使用無(wú)血清培養(yǎng)基在 Vero 和 HEK293 懸浮培養(yǎng)物中復(fù)制，同時(shí)微調(diào)參數(shù)，如 MOI、溫度和胰蛋白酶濃度. 搖瓶用Vero細(xì)胞生產(chǎn)導(dǎo)致1.07×10的感染滴度8 TCID 50 / mL的NDV-GFP和1.33×10 8 TCID 50為NDV-FLS /毫升。在 1 L 批次生物反應(yīng)器中的生產(chǎn)也導(dǎo)致培養(yǎng)物上清液的高滴度，NDV-GFP達(dá)到 2.37 × 10 8 TCID 50 /mL 和 3.16 × 10 7 TCID 50/mL 用于 NDV-FLS。這顯示了細(xì)胞培養(yǎng)中有效的 NDV 生產(chǎn)，為可應(yīng)用于不同目標(biāo)的可擴(kuò)展載體疫苗制造過程奠定了基礎(chǔ)。

Furthermore, process intensification could increase the quantity of infective particles harvested, using technologies such as fed-batch or perfusion [48]. The lower infectious titers observed at later time points in bioreactors and shake flasks suggest that NDV could be a good candidate for production in perfusion mode, as the viruses could be continuously harvested before suffering a loss in infectivity due to temperature and shear stress in the bioreactor.

Therefore, we have successfully developed the upstream process and analytical methods for suspension Vero cell-based production of NDV, using the constructs NDV-GFP and NDV-FLS as models. Future steps include establishing a scalable purification protocol and testing different bioreactor production modes, such as fed-batch and perfusion, to move towards a complete process based on continuous manufacturing.

此外，使用分批補(bǔ)料或灌注等技術(shù)，過程強(qiáng)化可以增加收獲的感染性顆粒的數(shù)量 [ 48 ]。在生物反應(yīng)器和搖瓶中的稍后時(shí)間點(diǎn)觀察到的較低感染滴度表明 NDV 可能是在灌注模式下生產(chǎn)的良好候選者，因?yàn)榭梢栽谟捎谏锓磻?yīng)器中的溫度和剪切應(yīng)力而喪失傳染性之前連續(xù)收獲病毒.

因此，我們使用構(gòu)建體 NDV-GFP 和 NDV-FLS 作為模型，成功開發(fā)了基于懸浮 Vero 細(xì)胞生產(chǎn) NDV 的上游工藝和分析方法。未來的步驟包括建立可擴(kuò)展的純化方案和測(cè)試不同的生物反應(yīng)器生產(chǎn)模式，如補(bǔ)料分批和灌注，以實(shí)現(xiàn)基于連續(xù)制造的完整工藝。

關(guān)鍵詞：新冠病毒,Vero懸浮培養(yǎng),病毒疫苗生物工藝,生物反應(yīng)器生產(chǎn),疫苗生產(chǎn)平臺(tái),COVID-19,SARS-CoV-2,Newcastle Disease Virus,Vero suspension culture,viral vaccine bioprocess, bioreactor production, vaccine production platform,COVID-19, SARS-CoV-2