重组HSV-Ⅱ病毒疫苗的微载体悬浮培养生产工艺研究

doi:10.13523/j.cb.20140310

中国生物工程杂志

2014, Vol. 34

Issue (3): 68-78 DOI: 10.13523/j.cb.20140310

技术与方法

重组HSV-Ⅱ病毒疫苗的微载体悬浮培养生产工艺研究

林美玲, 唐寅, 张明, 易小萍, 黄明志

华东理工大学生物反应器工程国家重点实验室上海 200237

Bioprocess Optimization for Recombinant Herpes Simplex Virus type Ⅱ Vaccine Production Based Cell Microcarrier Suspention Culture

LIN Mei-ling, TANG Yin, ZHANG Ming, YI Xiao-ping, HUANG Ming-zhi

The State Laboratory Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

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摘要： 经过重组减毒的Ⅱ型溶瘤单纯疱疹病毒（rHSV-Ⅱ）在体外具有良好的溶瘤效果，并对小鼠体内B16R黑色素瘤内注射具有较高的疗效。重组HSV-Ⅱ病毒作为高效的新型溶瘤病毒疫苗有望用于肿瘤的临床治疗。Vero细胞是广泛应用于病毒疫苗生产的细胞基质，该细胞系对多种病毒敏感且产量高，其微载体培养安全，开发重组HSV-Ⅱ溶瘤病毒肿瘤疫苗的Vero细胞微载体反应器悬浮培养生产工艺具有重要的意义。以自主开发的低血清培养基为基础，对Vero细胞的反应器微载体球转球转移放大工艺及在位消化放大培养工艺进行了研究和比较，以新老微载体比3：1的比例成功实现了微载体球转球转移放大，建立了可实现至少四次/级连续放大的球转球转移放大培养工艺，可用于工业化生产过程。在此基础上，初步建立了以Vero细胞为基质的重组HSV-Ⅱ病毒疫苗反应器微载体无血清悬浮培养生产工艺，最大病毒滴度可达到6.62 lgTCID50/ml以上。为以Vero细胞为基质的无血清病毒疫苗规模化培养提供了一种简便、高效的工艺方法。

关键词： Vero细胞; 生物反应器; 球转球转移放大; 重组HSV-Ⅱ

Abstract: The attenuated recombinant herpes simplex virus type Ⅱ(rHSV-Ⅱ) have a good oncolytic effect in vitro, and showed higher efficacy against B16R melanoma in mice. As an efficient new oncolytic virus, rHSV-Ⅱ is expected for the clinical treatment of cancer. African Green Monkey Kidney (Vero) Cells have been widely used for human vaccine production since they were sensitive and high production hosts, and safety in micro-carrier culture. With the self-development chemically defined serum-free medium added 1% NBCS, the bead-to-dead transfer of vero cells in bioreactor was developed. The optimal condition of transfer were about 30 cells/MC inoculated, cultured for 60 h when the cells density reached about the same density with the initial inoculated, the static time/stiring time was 42 min/3min. It can be achieved at least four times successive transfer for bioreactor amplification by the 1:4 (V:V), cells density reach up to 6.0×10⁶ cells/ml. Under these conditions, the bioreactor culture process of rHSV-Ⅱ in microcarrier suspension culture has been developed, maximum virus titer was reached up to 6.62 lgTCID50/ml. A simple, efficient process method is provided for large-scale production of anti-tumor vaccine of rHSV-Ⅱ.

Key words: Vero cells Bioreactor Bead to bead transfer rHSV-Ⅱ

收稿日期: 2013-12-26 出版日期: 2014-03-25

ZTFLH:

Q813.1+1

通讯作者: 易小萍, 黄明志 E-mail: xpyi@ecust.edu.cn;huangmz@ecust.edu.cn

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引用本文:

林美玲, 唐寅, 张明, 易小萍, 黄明志. 重组HSV-Ⅱ病毒疫苗的微载体悬浮培养生产工艺研究[J]. 中国生物工程杂志, 2014, 34(3): 68-78.

LIN Mei-ling, TANG Yin, ZHANG Ming, YI Xiao-ping, HUANG Ming-zhi. Bioprocess Optimization for Recombinant Herpes Simplex Virus type Ⅱ Vaccine Production Based Cell Microcarrier Suspention Culture. China Biotechnology, 2014, 34(3): 68-78.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140310 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I3/68

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