中国新药杂志

CHO宿主细胞蛋白残留量检测方法适用性验证

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张峰 ¹ , 秦俭 ² , 郭莎 ¹ , 王兰 ¹

中国新药杂志 | 生物医药前沿 2021,30(21): 1967-1974

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中国新药杂志 |生物医药前沿 2021 , 30 (21) : 1967 -1974

CHO宿主细胞蛋白残留量检测方法适用性验证

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张峰¹, 秦俭², 郭莎¹, 王兰¹

作者信息

¹中国食品药品检定研究院，北京　102629

²依科赛生物科技（太仓）有限公司，太仓　215434

通讯作者:

王兰，女，研究员，研究方向：治疗性单克隆抗体产品的质量研究。联系电话：（010）53852159，E-mail：iamouran@163.com。

作者简介:

张峰，男，主任技师，研究方向：治疗性单克隆抗体产品的质量研究。E-mail：zhangfeng5122@163.com。

Verification of applicability of the method for detecting CHO host cell protein residues

Feng ZHANG¹, Jian QIN², Sha GUO¹, Lan WANG¹

Affiliations

¹National Institute for Food and Drug Control, Beijing 102629, China

²ExCell Biotech (Taicang) Co., Ltd., Taicang 215434, China

出版时间: 2021-11-15

文章导航

摘要

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目的：

对CHO宿主细胞蛋白（host cell protein，HCP）残留量检测方法应用于候选治疗性单克隆抗体（以下简称单抗）产品生产过程中质控和放行检测的适用性进行验证。

方法：

采用荧光标记的二维差异凝胶电泳（2-dimensional difference gel electrophoresis，2D-DIGE）测定羊抗HCP多克隆抗体对空发酵宿主细胞上清中HCP蛋白的覆盖率；采用统计学方法根据40次检测结果确定检出限（limit of detection，LOD）和定量限（limit of quantitation，LOQ）；依照人用药品注册技术要求国际协调会（ICH）Q2要求验证方法特异性、精密性/中间精密性、准确性、线性和耐受性；依照《美国药典》〈1132〉要求验证方法用于过程中质控的适用性、关键HCP清除步骤和各步骤样品测定稀释度范围。

结果：

2D-DIGE测定羊抗HCP多抗对CHO HCP的覆盖率为56%，应用该抗体建立的CHO HCP检测方法的LOD和LOQ分别为2.47和7.41 ng·mL^-1。经验证，方法特异性、精密性/中间精密性、准确性、线性和耐受性均符合要求。系列稀释单抗纯化过程中间产物，采用选定方法进行检测，确定各生产步骤产物HCP测定的稀释范围，计算各纯化步骤的HCP清除率，确定蛋白A亲和层析为HCP的主要清除步骤。根据上述结果，确定所选定的CHO HCP残留量测定方法可做为蛋白A纯化的过程中控制项目和原液的放行项目。

结论：

所选定CHO HCP检测方法可应用于候选治疗性单抗的过程中控制和放行。

关键词

宿主细胞蛋白残留量 / 检测 / 验证 / 覆盖率 / 单克隆抗体

Abstract

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Objective:

To validate the applicability of a residual CHO host cell protein (HCP) test method for the in-process candidate therapeutic monoclonal antibody and its release control.

Methods:

The coverage ratio of goat anti-CHO HCP polyclonal antibody to the HCPs from supernatant of fermented mock CHO cells was determined by fluorescent-labeled two-dimensional differential gel electrophoresis (2D-DIGE). The limit of detection (LOD) and limit of quantitation (LOQ) were determined statistically from results of 40 tests. The specificity, precision/intermediate precision, accuracy, linearity and robustness were validated according to ICH Q2. To verify the suitability of the method for in-process quality control, key HCP removal steps, and sample dilution ranges for each step were determined according to USP 〈1132〉.

Results:

The coverage ratio of goat anti-CHO HCP polyclonal antibody was 56%. The LOD and LOQ was 2.47 and 7.41 ng·mL^-1, respectively. The validation results showed that the specificity, repeatability/intermediate precision, accuracy, linearity and robustness of the selected CHO HCP test method all met the application requirements. With the detection results of serially diluted purification intermediates, the dilution factor range of intermediates and HCP clearance ratio of each purification step was confirmed, and protein A affinity chromatography was identified as the main clearance step for HCP. Deduced with the above results, the selected CHO HCP test method can be used as the in-process control method for protein A chromatography and release test for drug substance.

Conclusion:

The selected CHO HCP test method can be used in the process control and drug substance release of candidate therapeutic mAbs.

Key words

residual host cell protein / detection / validation / coverage rate / monoclonal antibody

引用本文

张峰, 秦俭, 郭莎, 王兰. CHO宿主细胞蛋白残留量检测方法适用性验证. 中国新药杂志, 2021 , 30 (21) : 1967 -1974 .

Feng ZHANG, Jian QIN, Sha GUO, Lan WANG. Verification of applicability of the method for detecting CHO host cell protein residues[J]. Chinese Journal of New Drugs, 2021 , 30 (21) : 1967 -1974 .

基金

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国家“重大新药创制”科技重大专项资助项目(2018ZX0901001-003)
创新生物技术药评价及标准化研究
子课题3：免疫检查点单抗和其他创新生物技术药的质量评价方法和标准研究

参考文献

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2021年第30卷第21期

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首发时间：2026-10-30
出版时间：2021-11-15

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录用日期：2021-07-28

基金

国家“重大新药创制”科技重大专项资助项目(2018ZX0901001-003)

创新生物技术药评价及标准化研究

子课题3：免疫检查点单抗和其他创新生物技术药的质量评价方法和标准研究

作者信息

¹中国食品药品检定研究院，北京　102629

²依科赛生物科技（太仓）有限公司，太仓　215434

通讯作者:

王兰，女，研究员，研究方向：治疗性单克隆抗体产品的质量研究。联系电话：（010）53852159，E-mail：iamouran@163.com。

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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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