Microcarrier culture system for human influenza vaccine

描述(申请人提供)：拟议研究的目标是开发一种优化的微载体-生物反应器细胞培养系统，使快速、高效、大规模、低成本生产人类流感疫苗成为可能。大多数人类流感病毒疫苗是在胚胎鸡蛋中生产的。联邦政府和其他世界组织正在寻找替代细胞培养方法来生产流感疫苗，因为鸡蛋衍生疫苗存在固有的困难。在正常情况下，在(A)确定可能在每个流感季节出现的特定病毒株与(B)获得针对这些病毒株的适当疫苗之间有很长的准备时间。微载体生物反应器细胞培养技术有可能降低生产成本，并将生产9300万剂单价疫苗所需的时间减少到几个月。这种迅速实现大规模的潜力在流感大流行的情况下将是无价的，并可能对抗大流行或生物恐怖行为。为了实现研究目标，五个具体目标将优化影响Vero和MDCK细胞病毒产量的微载体-生物反应器培养工艺参数，并开发适用于经济商业使用的方案。我们将(I)向流感疫苗的大型生产商许可制造协议，并(Ii)销售用于这些协议的Hillex微载体。 明确目标1.确定和整合关键的细胞培养和病毒生长参数，以便在Hillex微载体悬浮培养的Vero细胞中最佳生产人类流感病毒。 具体目的二.扩大病毒生长参数的范围(如具体目标一所确定的)，以包括另外六种流感病毒株，并比较在Hillex微载体悬浮培养中培养的胚胎和Vero细胞之间直接“面对面”竞争中的病毒产量。 具体目的三.评估在1升规模的微载体-生物反应器培养的Vero细胞中产生人类流感病毒。 具体目标四：开发一种将Vero细胞从工作种子安瓶扩大和放大到5升培养的过程，并证明传代细胞将在更大范围内支持高浓度流感病毒。 特定目的V：确定和整合在Hillex微载体-生物反应器培养的MDCK细胞中优化生产人流感病毒(A/武汉/395/95)的关键细胞培养参数。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop an optimized microcarrier-bioreactor cell culture system that enables the rapid, efficient, large scale, low cost production of human influenza vaccine. Most human influenza virus vaccine is produced in embryonated eggs. The Federal government and other worldwide organizations are seeking alternative cell-culture approaches to influenza vaccine production since there are inherent difficulties with the egg-derived vaccines. Under normal conditions there are long lead times between (a) the identification of particular virus strains likely in each flu season and (b) the availability of appropriate vaccines against these virus strains. Microcarrier-bioreactor cell-culture technology has the potential to lower production costs and reduce the time required to produce 93 million doses of monovalent vaccine to a few months. This potential to rapidly achieve large scale, would be invaluable in case of an influenza pandemic and possibly could counter a pandemic or bioterrorist acts. To achieve the research goals, five specific aims will optimize microcarrier-bioreactor culture process parameters that affect viral yield with Vero and MDCK cells and develop protocols applicable to economic commercial use. We will (i) license manufacturing protocols to large producers of flu vaccine and (ii) sell Hillex microcarriers for those protocols. Specific Aim I. To identify and integrate critical cell culture and viral growth parameters for optimal production of human influenza virus in Vero cells grown in Hillex microcarrier-suspension culture. Specific Aim II. To extend the range of viral growth parameters (as identified in Specific Aim I) to include six additional strains of influenza virus and to compare the virus production in direct "head-to-head" competition between embryonated eggs and Vero cells grown in Hillex microcarrier-suspension culture. Specific Aim III. To assess production of human influenza virus in Vero cells grown in a microcarrier-bioreactor culture at the one liter scale. Specific Aim IV: To develop a process to expand and scale-up Vero cells from a working seed ampoule to a five liter culture and to demonstrate that the passaged cells will support high influenza virus concentrations at a larger scale. Specific Aim V: To identify and integrate critical cell culture parameters for optimal production of human influenza virus (A/Wuhan/395/95) in MDCK cells grown in Hillex microcarrier-bioreactor culture.