High-Expression, Rapid Production of Influenza Vaccines in Cell-Based Systems

在细胞系统中高表达、快速生产流感疫苗

• 批准号: 8321463
• 负责人: Pamuk Bilsel
• 金额: $ 29.77万
• 依托单位: CDI BIOSCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321463
• 关键词: Agglutinins; Apoptosis; Binding; Biomass; Bioreactors; Cell Culture Techniques; Cell Density; Cell Line; Cell Survival; Cell division; Cell membrane; Cells; Chemicals; Chickens; Chinese Hamster Ovary Cell; Culture Media; Disease Outbreaks; Engineering; Environment; Feeds; Fluorescein; Hour; Influenza A Virus, H1N1 Subtype; Isopropyl Thiogalactoside; Label; Legal patent; Length; Mammalian Cell; Measures; Methods; Monitor; Production; Protein Secretion; Proteins; Protocols documentation; Reaction Time; Risk; Staging; Surface; System; Technology; Time; Vaccine Production; Vaccines; Viral; Virus; Virus Diseases; Virus Receptors; base; bioprocess; cell growth; cellular engineering; cost; egg; expression vector; falls; feeding; feeding schedule; flasks; flexibility; influenza outbreak; influenza virus vaccine; influenzavirus; innovation; meetings; new technology; pandemic disease; receptor; receptor expression; scale up

DESCRIPTION (provided by applicant): We propose to develop an innovative high-expression CHO cell based platform for rapid Influenza vaccine production, which will have the ability to complete a 12 week vaccine production cycle, leading to a drastic reduction in response time to Influenza outbreak. This system will have advantages over the traditional approach to vaccine production in chicken eggs in that it can more feasibly meet the rapid "surge capacity" needs that would be required during a pandemic outbreak. Traditional methods of generating Influenza vaccines in embryonated chicken eggs fall short of the needs during a pandemic because of lengthy times required for scale-up, high costs, limited quantities of specialized eggs and potential risks of contamination. Cell-culture based vaccines are potentially a more reliable, flexible, and easily scalable method of producing vaccines; however, cell lines available for efficient large-scale vaccine production are limited. The approach is to integrate two novel technologies for creating a highly efficient, rapid vaccine production system within mammalian cells. The first technology, developed by FluGen, increases the number of surface bound influenza virus receptors for expanding the amount of virus that can infect the cell. The second technology, developed by CDI Bioscience, is incorporated into a high-expression mammalian host cell line (PACE") that, when activated, will be capable of more than doubling the expression of the surface receptors of the FluGen technology. We will achieve the objectives of the project by pursuing three specific aims over a three year period: (Aim 1) Develop a PACE" cell line that stably expresses FluGen surface receptors on cell membrane; Three to four high-expressing PACE" clonal lines stably expressing FluGen surface receptors on cell membrane will be developed. These clonal cell lines will be considered suitable for further characterization if they (a) show over 2-fold FluGen surface receptors expression upon RP shift; and (b) produce higher viral titers when infected with H1N1 (A/PR/8/34 strain) virus, as compared to the PACE" cells that are not engineered with the FluGen surface receptors. (Aim 2) Develop bioprocess protocols for influenza virus production in FluGen surface receptors expressing PACE" cells in shake flasks; We will determine the best CD medium for cell growth and culture viability, and the optimal VCD for induction of RP shift(R). We will then determine the best medium/feed/supplements combination that achieves: (a) at least 2-fold higher expression of FluGen surface receptors upon induction of RP shift(R). (2) Virus titers of at least 107-108 pfu/ml. (Aim 3) Develop scalable bioprocess protocols for influenza virus production in bench top and industrial bioreactors (50-100L); Adapted cells and optimal conditions from Aim 2 will be used in small-scale bench top bioreactors to determine the best cell growth and induction conditions in order to achieve the highest viral titer in an industrial environment. Subsequently, we will develop scale up strategy for optimal influenza virus production in 50-100L bioreactor. Narrative Traditional methods of generating Influenza vaccines in embryonated chicken eggs fall short of the needs during a pandemic outbreak. This project combines two novel technologies for creating a highly efficient, rapid vaccine production system within mammalian cells, allowing completion of a 12 week vaccine production cycle. Achieving the objectives of this project will form a major milestone towards leading to a drastic reduction in response time to Influenza outbreak.

描述（由申请人提供）：我们建议开发一种创新的基于高表达CHO细胞的快速流感疫苗生产平台，该平台将有能力完成12周的疫苗生产周期，从而大大缩短对流感爆发的反应时间。该系统将比传统的鸡蛋疫苗生产方法具有优势，因为它可以更可行地满足大流行爆发期间所需的快速“激增能力”需求。在有胚胎的鸡蛋中生产流感疫苗的传统方法无法满足大流行期间的需求，因为扩大规模需要很长时间、成本高、专用鸡蛋数量有限以及潜在的污染风险。基于细胞培养的疫苗可能是一种更可靠、更灵活、更容易扩展的疫苗生产方法；然而，可用于高效大规模疫苗生产的细胞系有限。该方法是将两种新技术结合起来，在哺乳动物细胞内创建一种高效、快速的疫苗生产系统。第一项技术是由FluGen开发的，它增加了表面结合的流感病毒受体的数量，以扩大可以感染细胞的病毒数量。第二项技术是由CDI Bioscience开发的，它被整合到高表达的哺乳动物宿主细胞系（PACE）中，当被激活时，将能够使FluGen技术的表面受体的表达增加一倍以上。我们将通过在三年时间内实现三个具体目标来实现该项目的目标：（目标1）开发一种能够在细胞膜上稳定表达FluGen表面受体的PACE细胞系；将在细胞膜上稳定表达FluGen表面受体的3 ~ 4株高表达PACE“克隆系”。如果这些克隆细胞系(a)在RP移位时显示超过2倍的FluGen表面受体表达，则认为它们适合进一步表征；(b)当感染H1N1 （A/PR/8/34株）病毒时，与未使用FluGen表面受体进行工程改造的PACE细胞相比，产生更高的病毒滴度。（目标2）开发在摇瓶中表达PACE的FluGen表面受体细胞中生产流感病毒的生物工艺方案；我们将确定细胞生长和培养活力的最佳CD培养基，以及诱导RP移位(R)的最佳VCD。然后，我们将确定最佳培养基/饲料/添加物组合，以实现：(a)诱导RP移位(R)后FluGen表面受体的表达至少提高2倍。(2)病毒滴度≥107- 108pfu /ml。（目标3）在台式和工业生物反应器（50-100L）中制定可扩展的流感病毒生产生物工艺方案；Aim 2中的适应细胞和最佳条件将用于小型台式生物反应器，以确定最佳细胞生长和诱导条件，以便在工业环境中实现最高的病毒滴度。随后，我们将制定在50-100L生物反应器中最佳流感病毒生产的扩大策略。在大流行爆发期间，在有胚胎的鸡蛋中生产流感疫苗的传统方法无法满足需求。该项目结合了两种新技术，用于在哺乳动物细胞内创建高效、快速的疫苗生产系统，从而可以完成12周的疫苗生产周期。实现这一项目的目标将成为大幅缩短流感爆发应对时间的一个重要里程碑。