Kon Theone C, Onu Adrian, Berbecila Laurentiu, Lupulescu Emilia, Ghiorgisor Alina, Kersten Gideon F, Cui Yi-Qing, Amorij Jean-Pierre, Van der Pol Leo
Department of Product Development, Intravacc, Institute for Translational Vaccinology, Bilthoven, The Netherlands.
Laboratory of Biotechnology, Cantacuzino National Research Institute, Bucharest, Romania.
PLoS One. 2016 Mar 9;11(3):e0150700. doi: 10.1371/journal.pone.0150700. eCollection 2016.
The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween®, either Triton™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.
本研究的目的是评估不同的灭活和裂解程序对流感疫苗产品成分、稳定性和回收率的影响，以支持工艺技术的转移。制备了四种裂解型和两种全病毒灭活（WIV）流感疫苗原液，并就放行标准、原液稳定性和血凝素回收率进行了比较。将在25000枚受精鸡蛋上制备的一批流感H3N2 A/乌拉圭病毒澄清收获物平均分配到六个下游工艺中。纯化的主要单元操作是蔗糖梯度区带超速离心。病毒的灭活使用磷酸盐缓冲液中的甲醛或柠檬酸盐缓冲液中的β-丙内酯进行。对于在吐温存在下裂解病毒产物，使用了Triton™ X-100或二乙醚。通过离心和蒸发去除乙醚，而通过疏水相互作用色谱法去除Triton-X100。所有产品均经过无菌过滤，并进行了为期5个月的实时稳定性研究。在所有工艺中，无菌过滤后均检测到主要产品损失；裂解病毒的损失比全病毒灭活疫苗更大。与使用甲醛灭活的工艺相比，β-丙内酯灭活平均回收率更高。特别是乙醚裂解甲醛产品在五个月的时间内回收率低且稳定性最差。
