Microcarrier / CEF / Media SYSTEM to make Viral Vaccines

描述(申请人提供)：这项研究计划是开发一种 微载体-生物反应器细胞培养系统的高效生长 在受控生物反应器中从微载体中回收受感染的完整细胞 文化。原代鸡胚胎成纤维细胞(CEF)广泛应用于病毒 疫苗生产(用于禽类和人类疫苗)，但目前尚不清楚 利用微载体-生物反应器系统的许可制造协议 为了这个目的。重点将放在整个马雷克细胞的生产上 禽类应用疾病疫苗。发展起来的系统技术 这些研究的过程可以取代大规模的工业过程，其中 (1)原代CEF细胞用于滚筒培养瓶中；(2)胚胎培养，可育 鸡蛋起到了微型生物反应器的作用，例如在制造人类 流感疫苗或(3)全细胞疫苗和/或免疫疗法。 具体目标一、二和六。优化细胞培养条件 使用原CEF电池、二次CEF电池和 在一升生物反应器培养中，高密度永生化CEF样细胞。 具体目标III.优化为期三天的HVT生产的培养条件 在生物反应器培养中使用1升规模的二次CEF细胞进行培养。 具体目标IV和V制定快速采集感染人类疱疹病毒的方案 细胞，并将这些细胞从微载体中分离出来，并实现 马雷克的高效价疫苗生产。 建议的商业应用： 生产了许多动物重要疫苗(如马立克氏病)和人类病毒疫苗 使用鸡胚细胞，无论是在滚瓶系统中还是在鸡胚化中 鸡蛋。这两个过程都不使用微载体-生物反应器系统。大尺度 在苗族载体-生物反应器系统中生产马立克疫苗将导致 与旧技术相比，生物反应器具有显著的经济优势。 产品质量和一致性也应该提高。和制造成本 减少了。本文提出的系统将利用(1)一次或二次CEF 细胞，或永生化的细胞系。(2)特殊介质配方；(3)牛血清 并将改进(4)协议以在该系统中生产疫苗。这个 技术应该外推到其他全细胞人类疫苗。 建议的商业应用： 目前人类流感疫苗供应短缺，突显出 开发替代胚胎卵子的技术的重要性 制作方法。从(1)疾控中心对特定流感的估计得出的准备时间 病毒变种可能袭击美国，以(2)疫苗的大规模可用 规模非常长，长达数月之久。如果发生流感大流行 这一长达数月的提前期可能会导致高死亡率，这让人想起 1917-18年大流行。流感病毒疫苗能否大规模生产 生物反应器，具有快速周转时间(几个月)，高死亡率 大流行或许是可以避免的。提出的这项研究有可能解决 这个问题。

DESCRIPTION (Provided by Applicant): This research plan is to develop a Microcarrier-Bioreactor Cell Culture System for the growth and efficient recovery of infected, whole cells from microcarriers in controlled, bioreactor cultures. Primary chick embryo fibroblast (CEF) cells are widely used in viral vaccine production (for both avian and human vaccines), but there are no known licensed-manufacturing protocols which utilize a Microcarrier-Bioreactor System for this purpose. Focus will be on the production of a whole cell Marek's Disease vaccine for avian applications. The System technology developed in the course of these studies could replace large-scale industrial processes in which (1) primary CEF cells are utilized in roller bottles, (2) embryonated, fertile eggs function as tiny bioreactors, such as in the manufacturing of human influenza vaccine or (3) whole cell vaccines and/or immunotherapies. Specific Aims I, II and VI. To optimize cell culture conditions for a one to two day HVT-production run using primary CEF cells, secondary CEF cells and immortalized CEF-like cells at high density, in a one-liter bioreactor culture. Specific Aim III. To optimize culture conditions for a three-day HVT-production run using secondary CEF cells at the one-liter scale in bioreactor culture. Specific Aim IV & V. To develop protocols for rapid harvesting of HVT-infected cells, and separation of those cells from microcarriers, and to achieve high-titer Marek's vaccine production. PROPOSED COMMERCIAL APPLICATION: Many animal vital vaccines (e.g. Marek's) and human viral vaccines are produced using chick embryo cells, either in roller bottle systems or in embryonated chicken eggs. Neither process uses microcarrier - bioreactor systems. Large scale production of Marek's vaccine in a Miaocarrier-Bioreactor System, will lead to substantial economic advantages when bioreactors are compared to old technology. Product quality and consistency should also be improved. and manufacturing costs reduced. The System herein proposed will utilize (1) primary or secondary CEF cells, or immortalized cell lines. (2) special media formulations, (3) bovine sera and will evolve the (4) protocols to produce vaccines in this System. The technology should extrapolate to other whole-cell human vaccines. PROPOSED COMMERCIAL APPLICATION: The current short supply of human influenza vaccine serves to underline the importance for developing technology that is an alternative to embryonated egg production methods. The lead time from (1) a CDC estimate of the specific influenza virus variant likely to strike the USA, to (2) the availability of the vaccine on a large scale is exceedingly long, many months. In the event of an influenza pandemic this many-month lead-time could result in a high fatality rate reminiscent of the 1917-18 pandemic. If influenza virus vaccine can be produced in very large-scale bioreactors, with a fast turn around time (a couple months), the high fatality rate of a pandemic might be averted. The research proposed has the potential to address this issue.