From single-cell cloning to high-yield influenza virus production – implementing advanced technologies in vaccine process development

IF 3.9 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Engineering in Life Sciences Pub Date : 2024-02-18 DOI:10.1002/elsc.202300245
Tilia Zinnecker, Najd Badri, Diogo Araujo, Kristin Thiele, Udo Reichl, Yvonne Genzel
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Abstract

Innovations in viral vaccine manufacturing are crucial for pandemic preparedness and to meet ever-rising global demands. For influenza, however, production still mainly relies on technologies established decades ago. Although modern production shifts from egg-based towards cell culture technologies, the full potential has not yet been fully exploited. Here, we evaluate whether implementation of state-of-the-art technologies for cell culture-based recombinant protein production are capable to challenge outdated approaches in viral vaccine process development. For this, a fully automated single-cell cloning strategy was established to generate monoclonal suspension Madin-Darby canine kidney (MDCK) cells. Among selected cell clones, we could observe distinct metabolic and growth characteristics, with C59 reaching a maximum viable cell concentration of 17.3 × 106 cells/mL and low doubling times in batch mode. Screening for virus production using a panel of human vaccine-relevant influenza A and B viruses in an ambr15 system revealed high titers with yields competing or even outperforming available MDCK cell lines. With C113, we achieved cell-specific virus yields of up to 25,000 virions/cell, making this cell clone highly attractive for vaccine production. Finally, we confirmed process performance at a 50-fold higher working volume. In summary, we present a scalable and powerful approach for accelerated development of high-yield influenza virus production in chemically defined medium starting from a single cell.

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从单细胞克隆到高产流感病毒生产--在疫苗工艺开发中采用先进技术
病毒疫苗生产的创新对于防范大流行病和满足不断增长的全球需求至关重要。然而,流感疫苗的生产仍主要依赖几十年前的技术。虽然现代生产已从以鸡蛋为基础转向细胞培养技术,但其潜力尚未得到充分挖掘。在此,我们评估了基于细胞培养的重组蛋白生产的最新技术是否能够挑战病毒疫苗工艺开发中的过时方法。为此,我们建立了一种全自动单细胞克隆策略,以生成单克隆悬浮麦丁-达比犬肾(MDCK)细胞。在选定的细胞克隆中,我们可以观察到不同的代谢和生长特性,其中 C59 的最大存活细胞浓度为 17.3 × 106 cells/mL,批处理模式下的倍增时间较短。在ambr15系统中使用一组与人类疫苗相关的甲型和乙型流感病毒进行病毒生产筛选,结果显示病毒滴度很高,产量可与现有的MDCK细胞系相媲美,甚至更胜一筹。利用 C113,我们实现了高达 25,000 病毒/细胞的细胞特异性病毒产量,使这一细胞克隆在疫苗生产中极具吸引力。最后,我们确认了在工作容量提高 50 倍的情况下的工艺性能。总之,我们提出了一种可扩展的强大方法,可以从单个细胞开始,在化学定义培养基中加速开发高产流感病毒的生产。
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来源期刊
Engineering in Life Sciences
Engineering in Life Sciences 工程技术-生物工程与应用微生物
CiteScore
6.40
自引率
3.70%
发文量
81
审稿时长
3 months
期刊介绍: Engineering in Life Sciences (ELS) focuses on engineering principles and innovations in life sciences and biotechnology. Life sciences and biotechnology covered in ELS encompass the use of biomolecules (e.g. proteins/enzymes), cells (microbial, plant and mammalian origins) and biomaterials for biosynthesis, biotransformation, cell-based treatment and bio-based solutions in industrial and pharmaceutical biotechnologies as well as in biomedicine. ELS especially aims to promote interdisciplinary collaborations among biologists, biotechnologists and engineers for quantitative understanding and holistic engineering (design-built-test) of biological parts and processes in the different application areas.
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