Optimization of Sonication Process for High Newcastle Disease Virus （NDV） Titer

Cell disruption focuses on obtaining a desired bioproduct within a cell, and it is the cell wall that must be disrupted to allow access to the contents of the cell. In animal cells, the plasma membrane is the only barrier separating cell contents from the environment. Sound waves from sonication, a mechanical technique for cell disintegration, have been used to disrupt as well as to aggregate cells as a step towards purification of a desired bioproduct. In the present study, an improved sonication process for the high yield of Newcastle disease virus （NDV） propagated in tissue culture was described. DF-I cell was cultured in 25cm^2 T flask. When cells were about 80% confluent, a lentogenic strain of NDV （F strain） was used to infect the cell monolayer. With evident cytopathic effect, cells were subjected to cycles of freeze-thaw before sonicating with varying combinations of amplitude, temperature and time. Cells were sonicated using a water bath Sonicator, Jac Ultrasonic 1505 JEIO TECH 4 KHz. From ANOVA analysis, a significant interaction between sonication time and amplitude was observed. This also corresponds to the highest F value observed.

基于实验设计法针对抗VEGFR2-MICA融合蛋白进行发酵工艺优化（英文）

中国仓鼠卵巢细胞(CHO)是目前生产治疗性抗体的主要表达系统,该细胞的培养和抗体表达与培养条件密切相关。本实验室构建出了一种靶向VEGFR2单抗融合MICA的融合蛋白,具有良好的抗肿瘤活性,但是抗体产量较低,限制了该抗体的进一步研发和应用。为了探究适合该抗体的最佳发酵体系,本研究通过Minitab16.0正交法设计多种流加培养方式,探索了4种培养基、流加物以及培养条件对发酵细胞密度和抗体产量的影响,并优化出最佳的发酵方式,即以1×106cells/mL接种密度将细胞接种于商业培养基CDM4PerM Ab,每2 d加入12%(V/V)的流加物Boost2+Boost5。为保持抗体的稳定性和质量一致性,将此发酵方式扩大至3 L发酵罐以提高抗体的批次产量。最终抗体产量可达54.45 mg/L,并且Western blot实验结果验证了融合抗体装配的正确性。MTT实验结果表明JZB01能有效抑制HUVEC、MDA-MB-231和K562的细胞增殖,具有显著的抗血管生成作用以及比母体单抗更有效的抗肿瘤活性。综上,本研究开发出了一种有利于抗体生产的发酵工艺,能够满足实验室抗体研发需求。本实验室将进一步扩大该工艺适用的发酵规模,以满足抗体的临床前研究以及后续工业化生产。