中国新药杂志

抗EpCAM+CD3双特异性抗体细胞生物学活性报告基因测定方法的建立和验证

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张红梅 ¹^,² , 张峰 ² , 刘春雨 ² , 于传飞 ² , 段茂芹 ² , 曹俊霞 ² , 黄璟 ²^,³ , 王开芹 ² , 郭潇 ² , 王兰 ²

中国新药杂志 | 重大新药创制专项巡礼 2021,30(1): 25-34

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中国新药杂志 |重大新药创制专项巡礼 2021 , 30 (1) : 25 -34

抗EpCAM+CD3双特异性抗体细胞生物学活性报告基因测定方法的建立和验证

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张红梅¹^,², 张峰², 刘春雨², 于传飞², 段茂芹², 曹俊霞², 黄璟²^,³, 王开芹², 郭潇², 王兰²

作者信息

¹烟台大学药学院，烟台　264005

²中国食品药品检定研究院单克隆抗体产品室，卫生部生物技术产品检定方法及标准化重点实验室，北京　102629

³沈阳药科大学，生命科学与生物制药学院，沈阳　110016

通讯作者:

王兰，女，研究员，主要从事抗体类生物治疗药物质量控制研究。联系电话：（010）53852159，E-mail：wanglan@nifdc.org.cn。

作者简介:

张红梅，女，硕士研究生，主要从事药物分析研究。E-mail：1129920097@qq.com。

张峰，男，主任技师，主要从事抗体类生物治疗药物质量控制研究。联系电话：（010）53852178，E-mail：zhangfeng5122@163.com。

Development and validation of a cell-based reporter gene assay for bioactivity determination of anti-EpCAM+CD3 bispecific antibody

Hong-mei ZHANG¹^,², Feng ZHANG², Chun-yu LIU², Chuan-fei YU², Mao-qin DUAN², Jun-xia CAO², Jing HUANG²^,³, Kai-qin WANG², Xiao GUO², Lan WANG²

Affiliations

¹School of Pharmacy, Yantai University, Yantai 264005, China

²Division of Monoclonal Antibody, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing 102629, China

³School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China

出版时间: 2021-01-15

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摘要

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目的：

建立基于细胞的抗EpCAM+CD3双特异性抗体（bispecific antibody，BsAb）生物学活性报告基因测定方法（reporter gene assay，RGA）。

方法：

构建稳定表达萤光素酶（luciferase，Luc）的HcT116-Luc细胞为靶细胞，以HuT78细胞为效应细胞，通过对样品浓度、孵育时间、细胞接种时间、效靶比和细胞接种密度进行优化建立报告基因法，根据人用药品注册技术要求国际协调会（International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use，ICH）Q2指导原则和《中华人民共和国药典》（2015）指导原则，对RGA法的特异性、准确性、精密度、线性、代次稳定性和耐用性进行验证。

结果：

建立RGA方法经优化后的检测条件：效靶比为10∶1；细胞接种密度为1.1×10⁴细胞·孔^-1；BsAb样品以3 000 ng·mL^-1为起始浓度1∶3.5向下稀释9个浓度梯度（共10个检测浓度）；系列稀释样品与细胞孵育时间为72 h。孵育结束后测定Luc强度并以4-参数模式拟合剂量-效应曲线，计算样品相对生物学活性。建立RGA法的特异性、耐用性均符合应用要求。采用50%，70%，80%，100%，120%，130%，150%和200%共8个效价浓度进行准确性、精密度和线性验证，8个效价浓度的生物学活性回收率在95.48%~107.37%，变异系数（coefficient of variation，CV）在4.80%~9.97%，均<10%，实测效价与理论效价线性回归分析相关系数r²>0.99。第14~33代次HcT116-Luc细胞Luc表达情况稳定。

结论：

基于传代细胞建立的抗EpCAM+CD3 BsAb报告基因生物学活性测定方法操作简便，其特异性、精密度、准确性、耐用性方面符合应用要求，可作为此类双特异性抗体活性评价的方法，用于其质量控制中。

关键词

双特异性抗体 / 抗EpCAM+CD3 / 生物学活性 / 报告基因法

Abstract

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Objective:

To develop a cell-based reporter gene assay (RGA) for bioactivity determination of anti-EpCAM+CD3 bispecific antibody (BsAb).

Methods:

A HcT116-Luc cell line stably expressing Luciferase (Luc) was generated as the target cell, and a HuT78 cell line served as effector cell. The RGA was established by optimizing the initial concentration of BsAb, incubation time, cell seeding time, the ratio of effector cell to target cell, and cell seeding density. The specificity, accuracy, precision, linearity, subculture stability and robustness of RGA were verified according to the guideline of International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q2 and Pharmacopoeia of the People's Republic of China (ChP, 2015).

Results:

The parameters of RGA were developed and confirmed after optimization as follows: The ratio of effector cell to target cell was 10∶1; After mixing effector cell and target cell, the mixed cells were seeded in 96-well plate with 1.1×10⁴ cells·well^-1, which was cell seeding density; The initial concentration of BsAb, 3 000 ng·mL^-1, was serially diluted by 1∶3.5 for 9 gradients, and there were 10 detected concentrations in all; The serially diluted samples were incubated for 72 h with the seeding cells. After the incubation, the Luc intensity was measured, and the dose-response curve was fitted in a 4-parameter mode to calculate the relative bioactivity of sample. The accuracy, precision and linearity verification were performed by 8 potency concentrations, which were 50%, 70%, 80%, 100%, 120%, 130%, 150% and 200% potency concentrations. The recovery rate range of relative bioactivities of 8 potency concentrations was 95.48%~107.37%, and the coefficient of variation (CV) ranged 4.80%~9.97%, which was <10%. The measured potency to expected potency was evaluated by linear regressing model analysis (r²>0.99). The Luciferase was stably expressed in HcT116-Luc cell between 14 and 33 passage.

Conclusion:

Based on passage cells, the RGA has been developed as a simple and convenient procedure for determination of bioactivity of anti-EpCAM+CD3 BsAb, and its specificity, precision, accuracy and robustness met the application requirements.

Key words

bispecific antibody / anti-EpCAM+CD3 / bioactivity / reporter gene assay

引用本文

张红梅, 张峰, 刘春雨, 于传飞, 段茂芹, 曹俊霞, 黄璟, 王开芹, 郭潇, 王兰. 抗EpCAM+CD3双特异性抗体细胞生物学活性报告基因测定方法的建立和验证. 中国新药杂志, 2021 , 30 (1) : 25 -34 .

Hong-mei ZHANG, Feng ZHANG, Chun-yu LIU, Chuan-fei YU, Mao-qin DUAN, Jun-xia CAO, Jing HUANG, Kai-qin WANG, Xiao GUO, Lan WANG. Development and validation of a cell-based reporter gene assay for bioactivity determination of anti-EpCAM+CD3 bispecific antibody[J]. Chinese Journal of New Drugs, 2021 , 30 (1) : 25 -34 .

基金

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国家“重大新药创制”科技重大专项资助项目(2018ZX09101001-003)

参考文献

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2021年第30卷第1期

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首发时间：2026-10-29
出版时间：2021-01-15

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录用日期：2020-05-27

基金

国家“重大新药创制”科技重大专项资助项目(2018ZX09101001-003)

作者信息

¹烟台大学药学院，烟台　264005

²中国食品药品检定研究院单克隆抗体产品室，卫生部生物技术产品检定方法及标准化重点实验室，北京　102629

³沈阳药科大学，生命科学与生物制药学院，沈阳　110016

通讯作者:

王兰，女，研究员，主要从事抗体类生物治疗药物质量控制研究。联系电话：（010）53852159，E-mail：wanglan@nifdc.org.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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