中国新药杂志

建立CAR-T细胞治疗产品质量控制检测方法及其定量参考品的数字PCR方法研究

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豆敏华 ¹ , 朱冰美 ² , 宗伟英 ² , 吴晓双 ² , 杨志行 ¹

中国新药杂志 | 实验研究 2021,30(24): 2306-2314

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中国新药杂志 |实验研究 2021 , 30 (24) : 2306 -2314

建立CAR-T细胞治疗产品质量控制检测方法及其定量参考品的数字PCR方法研究

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豆敏华¹, 朱冰美², 宗伟英², 吴晓双², 杨志行¹

作者信息

¹中国科学院上海生命科学研究院湖州营养与健康产业创新中心，湖州　313000

²湖州申科生物技术有限公司，湖州　313000

通讯作者:

杨志行，男，高级工程师，研究方向：生物技术药物质量安全控制技术和产品研究。E-mail：zhixingy@nhc.ac.cn。

作者简介:

豆敏华，男，硕士，研究方向：生物技术药物质量安全控制技术和产品研究。E-mail：minhuad@nhc.ac.cn。

Research on the development of methods in CAR-T cell therapy and its quantitative reference using digital PCR

Min-hua DOU¹, Bing-mei ZHU², Wei-ying ZONG², Xiao-shuang WU², Zhi-xing YANG¹

Affiliations

¹Huzhou R&D Center for Nutrition and Health Shanghai Institutes for Biological Sciences Chinese Academy of Sciences, Huzhou 313000, China

²Huzhou Shenke Biotechnology Co., Ltd., Huzhou 313000, China

出版时间: 2021-12-30

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摘要

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目的：

本研究建立嵌合抗原受体T（CAR-T）细胞治疗产品质量控制中猿猴病毒40大T抗原（SV40LTA），腺病毒早期区域1A蛋白（E1A）和质粒残留水平的荧光定量聚合酶链式反应（PCR）检测方法，同时采用微流控芯片式数字PCR技术对质粒定量参考品进行赋值。

方法：

通过建立多重荧光定量PCR的方法，在一个反应中同时检测SV40LTA和E1A的残留水平。针对目前慢病毒等包装技术存在多种质粒组合系统（如四质粒和五质粒），对质粒的残留检测标准难以统一，本研究从同源性很高的复制子区域入手，设计和筛选引物探针，能同时检测多种不同质粒的总体残留水平。建立数字PCR技术分别对SV40LTA和E1A的质粒定量参考品拷贝数浓度标定方法进行方法学验证和赋值。

结果：

SV40LTA和E1A双重定量PCR方法的特异性好，最低定量限分别达到2.80×10和2.98×10 copies·μL^-1。质粒残留检测法的最低定量限可达到2.80×10 copies·μL^-1。通过采用目前快速发展的数字PCR技术进行赋值，经典吸光度法计算质粒定量参考品SV40LTA和E1A的拷贝数浓度为2.80×10⁸和2.98×10⁸ copies·μL^-1；经数字PCR标定分别为1.06×10⁸和1.21×10⁸ copies·μL^-1。

结论：

本研究建立的SV40LTA，E1A和质粒检测方法灵敏度较高、特异性强，能满足CAR-T细胞治疗产品的质量控制要求。经典的吸光度法定值的结果大于数字PCR方法，大约有2倍左右的差别。

关键词

嵌合抗原受体T / 细胞治疗 / 猿猴病毒40大T抗原 / 腺病毒早期区域1A蛋白 / 质粒残留 / 数字聚合酶链式反应

Abstract

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Objective:

To establish real-time PCR methods for the detection of residual SV40LTA&E1A and plasmids in the quality control of CAR-T cell therapy products, as well as the assay of reference plasmid calibration by microfluidic digital PCR technology.

Methods:

SV40LTA&E1A were detected in one tube at the same time by multiplex TaqMan probe PCR. In view of the fact that it was hard to unify the standard of residue detection because multiple plasmid systems (four or five plasmids) existed in the current lentiviral packaging technologies, primers were designed from the significant sequence homology of replicon of those plasmids. Meanwhile, digital PCR method was validated.

Results:

Good specificity and quantitative limits of 2.80×10 copies·μL^-1 (SV40LTA) and 2.98×10 copies·μL^-1 (E1A) were achieved in the SV40LTA&E1A PCR method. The quantitative limit was 2.80×10 copies·μL^-1 for plasmids detection method. The quantitative calculation results of plasmid copy number concentration of SV40LTA and E1A were 2.80×10⁸ copies·μL^-1 and 2.98×10⁸ copies·μL^-1, corresponding to 1.06×10⁸ copies·μL^-1 (SV40LTA) and 1.21×10⁸ copies·μL^-1 (E1A) by digital PCR method.

Conclusion:

The SV40LTA&E1A and residual plasmid PCR detection methods have good specificity, sensitivity and accuracy. The calibration results of classical absorption method are about twice times larger than those by digital PCR method.

Key words

chimeric antigen receptor T-cell / cell therapy / SV40 large T antigen / E1A / residual plasmid / digital PCR

引用本文

豆敏华, 朱冰美, 宗伟英, 吴晓双, 杨志行. 建立CAR-T细胞治疗产品质量控制检测方法及其定量参考品的数字PCR方法研究. 中国新药杂志, 2021 , 30 (24) : 2306 -2314 .

Min-hua DOU, Bing-mei ZHU, Wei-ying ZONG, Xiao-shuang WU, Zhi-xing YANG. Research on the development of methods in CAR-T cell therapy and its quantitative reference using digital PCR[J]. Chinese Journal of New Drugs, 2021 , 30 (24) : 2306 -2314 .

基金

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湖州市科技局公益性技术应用研究资助项目(2018GZ29)

参考文献

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参考文献引证文献

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2021年第30卷第24期

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首发时间：2026-10-28
出版时间：2021-12-30

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录用日期：2021-07-06

基金

湖州市科技局公益性技术应用研究资助项目(2018GZ29)

作者信息

¹中国科学院上海生命科学研究院湖州营养与健康产业创新中心，湖州　313000

²湖州申科生物技术有限公司，湖州　313000

通讯作者:

杨志行，男，高级工程师，研究方向：生物技术药物质量安全控制技术和产品研究。E-mail：zhixingy@nhc.ac.cn。

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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