药学学报

Drug administration	Guideline	Year
NMPA	General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products	2005
General Principles of Evaluation of Mammalian Cell Substrate Quality Control Technology for the Production of Recombinant Biological Products	2008
Chinese Pharmacopoeia. Manufacture and Quality Control of Animal Cell Substrate for Biological Products	2015
FDA	Points to Consider in the Characterization of Cell Lines Used to Product Biologicals	1993
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use	1997
EMA	Guideline on Virus Safety Evaluation of Biotechnological Investigational Products	2008

Drug
administration

Guideline

Year

NMPA

General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products

2005

General Principles of Evaluation of Mammalian Cell Substrate Quality Control Technology for the Production of
Recombinant Biological Products

2008

Chinese Pharmacopoeia. Manufacture and Quality Control of Animal Cell Substrate for Biological Products

2015

FDA

Points to Consider in the Characterization of Cell Lines Used to Product Biologicals

1993

Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use

1997

EMA

Guideline on Virus Safety Evaluation of Biotechnological Investigational Products

2008

Drug administration	Guideline	Year
NMPA	General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products	2005
General Principles of Evaluation of Mammalian Cell Substrate Quality Control Technology for the Production of Recombinant Biological Products	2008
Chinese Pharmacopoeia. Manufacture and Quality Control of Animal Cell Substrate for Biological Products	2015
FDA	Points to Consider in the Characterization of Cell Lines Used to Product Biologicals	1993
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use	1997
EMA	Guideline on Virus Safety Evaluation of Biotechnological Investigational Products	2008

Drug
administration

Guideline

Year

NMPA

General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products

2005

General Principles of Evaluation of Mammalian Cell Substrate Quality Control Technology for the Production of
Recombinant Biological Products

2008

Chinese Pharmacopoeia. Manufacture and Quality Control of Animal Cell Substrate for Biological Products

2015

FDA

Points to Consider in the Characterization of Cell Lines Used to Product Biologicals

1993

Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use

1997

EMA

Guideline on Virus Safety Evaluation of Biotechnological Investigational Products

2008

Test item	Application 1	Application 2	Application 3
EOPC	+^a	-^b	+^a
UPB	-^b	+^a	+^a

Test item

Application 1

Application 2

Application 3

EOPC

+^a

-^b

+^a

UPB

-^b

+^a

Test item	Application 1	Application 2	Application 3
EOPC	+^a	-^b	+^a
UPB	-^b	+^a	+^a

Test item

Application 1

Application 2

Application 3

EOPC

+^a

-^b

+^a

UPB

-^b

+^a

Drug administration	Guideline	Year
NMPA	Guideline on Technology and Validation of Virus Removal or Inactivation of Blood Products	2002
General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products	2005
FDA	Points to Consider in the Characterization of Cell Lines Used to Product Biologicals	1993
USP < 1050 > . Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin	2013
USP < 1050.1 > . Design, Evaluation, and Characterization of Viral Clearance Procedures Introduction	2016
EMA	Guideline on Virus Safety Evaluation of Biotechnological Investigational Products	2008
Virus Validation Studies: the Design, Contribution, and Interpretation of Studies Validating the Inactivation and Removal of Viruses	1996
ICH	ICH Q5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Biotechnology Products Derived from Cell Lines of Human or Animal Origin	1999

Drug
administration

Guideline

Year

NMPA

Guideline on Technology and Validation of Virus Removal or Inactivation of Blood Products

2002

General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products

2005

FDA

Points to Consider in the Characterization of Cell Lines Used to Product Biologicals

1993

USP < 1050 > . Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin

2013

USP < 1050.1 > . Design, Evaluation, and Characterization of Viral Clearance Procedures Introduction

2016

EMA

Guideline on Virus Safety Evaluation of Biotechnological Investigational Products

2008

Virus Validation Studies: the Design, Contribution, and Interpretation of Studies Validating the Inactivation and Removal of Viruses

1996

ICH

ICH Q5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Biotechnology Products Derived from Cell Lines of Human or Animal Origin

1999

Drug administration	Guideline	Year
NMPA	Guideline on Technology and Validation of Virus Removal or Inactivation of Blood Products	2002
General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products	2005
FDA	Points to Consider in the Characterization of Cell Lines Used to Product Biologicals	1993
USP < 1050 > . Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin	2013
USP < 1050.1 > . Design, Evaluation, and Characterization of Viral Clearance Procedures Introduction	2016
EMA	Guideline on Virus Safety Evaluation of Biotechnological Investigational Products	2008
Virus Validation Studies: the Design, Contribution, and Interpretation of Studies Validating the Inactivation and Removal of Viruses	1996
ICH	ICH Q5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Biotechnology Products Derived from Cell Lines of Human or Animal Origin	1999

Drug
administration

Guideline

Year

NMPA

Guideline on Technology and Validation of Virus Removal or Inactivation of Blood Products

2002

General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products

2005

FDA

Points to Consider in the Characterization of Cell Lines Used to Product Biologicals

1993

USP < 1050 > . Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin

2013

USP < 1050.1 > . Design, Evaluation, and Characterization of Viral Clearance Procedures Introduction

2016

EMA

Guideline on Virus Safety Evaluation of Biotechnological Investigational Products

2008

Virus Validation Studies: the Design, Contribution, and Interpretation of Studies Validating the Inactivation and Removal of Viruses

1996

ICH

ICH Q5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Biotechnology Products Derived from Cell Lines of Human or Animal Origin

1999

Manufacturing process	Application 1	Application 2	Application 3
Low pH	Two viruses (MuLv and Prv), three lots, one experiment	Two viruses (MuLv、Prv), three lots, one experiment; One GMP lot, virus (MuLv), two independent experiments	One GMP lot, virus (MuLv), two independent experiments
Filtration	Two viruses (MMV and Reo-3), three lots, one experiment	Two viruses (MMV and Reo-3), three lots, one experiment; One GMP lot, viruses (MuLv and MMV), two independent experiments	One GMP lot, viruses (MuLv and MMV), two independent experiments
Anion-exchange chromatography	None	One GMP lot, viruses (MuLv and MMV), two independent experiments	One GMP lot, viruses (MuLv and MMV), two independent experiments
Others	None	None	Optional

Manufacturing process

Application 1

Application 2

Application 3

Low pH

Two viruses (MuLv and Prv), three lots, one experiment

Two viruses (MuLv、Prv), three lots, one experiment; One GMP lot, virus (MuLv), two independent experiments

One GMP lot, virus (MuLv), two
independent experiments

Filtration

Two viruses (MMV and Reo-3), three lots, one experiment

Two viruses (MMV and Reo-3), three lots, one experiment; One GMP lot, viruses (MuLv and MMV), two independent experiments

One GMP lot, viruses (MuLv and MMV), two independent experiments

Anion-exchange chromatography

None

One GMP lot, viruses (MuLv and MMV), two independent experiments

Others

None

Optional

Manufacturing process	Application 1	Application 2	Application 3
Low pH	Two viruses (MuLv and Prv), three lots, one experiment	Two viruses (MuLv、Prv), three lots, one experiment; One GMP lot, virus (MuLv), two independent experiments	One GMP lot, virus (MuLv), two independent experiments
Filtration	Two viruses (MMV and Reo-3), three lots, one experiment	Two viruses (MMV and Reo-3), three lots, one experiment; One GMP lot, viruses (MuLv and MMV), two independent experiments	One GMP lot, viruses (MuLv and MMV), two independent experiments
Anion-exchange chromatography	None	One GMP lot, viruses (MuLv and MMV), two independent experiments	One GMP lot, viruses (MuLv and MMV), two independent experiments
Others	None	None	Optional

Manufacturing process

Application 1

Application 2

Application 3

Low pH

Two viruses (MuLv and Prv), three lots, one experiment

Two viruses (MuLv、Prv), three lots, one experiment; One GMP lot, virus (MuLv), two independent experiments

One GMP lot, virus (MuLv), two
independent experiments

Filtration

Two viruses (MMV and Reo-3), three lots, one experiment

Two viruses (MMV and Reo-3), three lots, one experiment; One GMP lot, viruses (MuLv and MMV), two independent experiments

One GMP lot, viruses (MuLv and MMV), two independent experiments

Anion-exchange chromatography

None

One GMP lot, viruses (MuLv and MMV), two independent experiments

Others

None

Optional

创新单抗药物申报临床试验病毒安全性相关监管指南调研与技术考量

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崔靖 , 宋丽娜 ^*

药学学报 | 新药论坛 2021,56(8): 2308-2314

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药学学报 | 新药论坛 2021, 56(8): 2308-2314

创新单抗药物申报临床试验病毒安全性相关监管指南调研与技术考量

全屏

崔靖, 宋丽娜^*

作者信息

国家药品监督管理局药品审评中心, 北京 100022

通讯作者:

*宋丽娜, Tel: 86-10-85243036, E-mail: songln@cde.org.cn

Guidelines investigation and technical considerations of virus safety during investigational new drug application of innovative monoclonal antibody products

Jing CUI, Li-na SONG^*

Affiliations

Center for Drug Evaluation, National Medical Products Administration (CDE, NMPA), Beijing 100022, China

出版时间: 2021-08-12 doi: 10.16438/j.0513-4870.2021-0195

文章导航

摘要

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随着我国生物制药自主创新能力的不断提高，越来越多的创新型单抗药物申报注册临床试验，同时国外已上市和/或国外开展临床试验的单克隆抗体药物在国内申报注册临床的数量也不断增加。2018年国家药品监督管理局对外发布了《新药Ⅰ期临床试验申请技术指南》(2018年第16号)，该指南对创新药申报临床试验阶段药学研究及申报资料提出了相关要求，极大提高了生物创新药的申报质量。但相比美国药品监管机构和欧盟药品监管机构，我国针对创新药申报临床阶段细化的技术指南尚不完善，如生物技术产品申报临床阶段的病毒安全性评价。本文调研了2018~2020年申请人针对创新单抗药物申报临床阶段的一般性技术问题，发现申请人聚焦的问题包括生产终末细胞和/或细胞收获液检定以及病毒去除或灭活验证。同时，审评中也发现由于申请人对国内外技术指南阶段性要求理解的差异，导致申报资料也存在一定差异。结合我国和美国、欧盟药品监管机构病毒安全性相关技术指南的调研并基于技术考量，本文对生产过程中外源病毒因子检测和病毒去除或灭活验证提出个人建议，旨在确保创新单抗药物在申报临床试验阶段的病毒安全性。

关键词

创新单抗药物 / 申报临床试验 / 病毒安全性 / 生产终末细胞和/或细胞收获液 / 病毒去除或灭活验证 / 技术考量

Abstract

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With the development of antibody manufacturing technology and improvement of new drug research in domestic industry, more innovative monoclonal antibody products submitted investigational new drug (IND) application. At the same time, monoclonal antibody products from abroad which have been approved marketing authorization and/or conducted clinical trials submitted IND applications in China. The National Medical Products Administration (NMPA) issued the "Guideline of Investigational New Drug Application" (No. 16, 2018) which emphasized the chemical, manufacturing, and control (CMC) regulatory, and dossier requirements in IND application, greatly promoted the application quality of innovative biological products. However, compared to the Food and Drug Administration (FDA) and European Medicines Agency (EMA), our particular guidelines are insufficient, such as guideline on virus safety evaluation of biotechnological investigational medicinal products. This review investigated the questions raised by sponsors from 2018 to 2020, including the end of production cell (EOPC) and/or unprocessed bulk (UPB) testing and virus removal or inactivation validation. Meanwhile, sponsors submitted different dossiers due to differences in understanding of stage requirements of guidelines from domestic and abroad. Based on the guidelines of virus safety from NMPA, FDA, and EMA, and the technical considerations, this review puts forward personal suggestions on the adventitious agents testing and virus removal or inactivation validation in manufacturing process, aim to ensure virus safety of innovative monoclonal antibody products in clinical trials.

Key words

innovative monoclonal antibody product / investigational new drug application / virus safety / end of production cell and/or unprocessed bulk / virus removal or inactivation validation / technical consideration

引用本文

崔靖, 宋丽娜. 创新单抗药物申报临床试验病毒安全性相关监管指南调研与技术考量. 药学学报, 2021 , 56 (8) : 2308 -2314 . DOI: 10.16438/j.0513-4870.2021-0195

Jing CUI, Li-na SONG. Guidelines investigation and technical considerations of virus safety during investigational new drug application of innovative monoclonal antibody products[J]. Acta Pharmaceutica Sinica, 2021 , 56 (8) : 2308 -2314 . DOI: 10.16438/j.0513-4870.2021-0195

正文

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近年来, 我国单克隆抗体药物产业发展迅速, 截至2019年2月, 国内企业已有近300个单克隆抗体药物申报临床, 10余个单克隆抗体药物进入报产阶段或获批上市, 与此同时, 国外已上市单克隆抗体药物在国内开展临床试验或申报上市进程不断加快^[1]。创新单抗药物的研发有别于已上市抗体药物或传统生物类似药, 其存在靶点多样化、结构多样化以及作用机制多样化等特点, 且其安全性、有效性需要全面的临床前研究和临床试验研究, 在审评时药学审评重点关注与安全性相关的问题。目前, 一般认为, 对于创新药可能引起的临床安全性的主要风险包括: 产品未知或不纯组分、化学结构含有或潜在毒性成分、稳定性差、杂质未充分确定等^[2]。其中涉及的创新单抗药物的风险主要包括动物源性成分的生物安全风险、细胞库未充分检定、生产或贮存过程中生物学活性改变、病毒去除或灭活验证不充分等。

创新单抗药物一般来源于细胞系表达, 一个主要特点是存在病毒污染风险, 一般情况下病毒污染来自于细胞基质本身和生产过程中引入。为确保重组表达生物制品无潜在病毒污染的安全性风险, 人用药品注册技术国际协调组织(International Conference on Harmonization of Technical Requirements Registration of Pharmaceuticals for Human Use, ICH) 在《来源于人或动物细胞系生物技术产品的病毒安全性评价》 (Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Biotechnology Products Derived from Cell Lines of Human or Animal Origin, Q5A) 中提出采用3种互补方法以控制生物制品潜在病毒污染: ①选择和测试细胞系以及其他原材料, 包括对起始原材料、原材料和辅料等的控制; ②对生产工艺清除病毒的能力进行评估; ③在生产过程中的适当步骤或阶段对产品进行测试, 以确保产品无感染性病毒污染^[3]。作者充分调研了国内外针对病毒安全性的技术指南, 基本理念与ICH Q5A是一致的, 但在创新单抗药物申报临床阶段针对生产终末细胞(end of production cell, EOPC) 和/或细胞收获液(unprocessed bulk, UPB) 检定、病毒去除或灭活验证等研究和评价方面存在一定差异。究其原因, 主要是由于美国药品监管机构(Food and Drug Administration, FDA)、欧盟药品监管机构(European Medicines Agency, EMA) 建立病毒安全性控制的技术指南较早, 体系也相对完善, 除了有针对上市申请的技术要求相关指南, 还细化了针对申报临床试验阶段病毒安全性评价的技术指南, 而我国法规起步稍晚, 目前主要是针对上市申请的指南, 而尚未出台针对临床试验申请阶段病毒安全性的技术指南。本文将结合目前已公开的国内外病毒安全性相关技术指南的技术要求, 展开以上两个问题的调研与分析, 同时结合目前的审评要求和技术考虑提出个人建议, 以供业界借鉴和探讨。

1 EOPC和/或UPB检定

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为确保生产的可持续性以及产品的一致性, 重组单抗药物生产过程中一般需要建立细胞库, 包括原始细胞库(primitive cell bank, PCB)、主细胞库(master cell bank, MCB) 和工作细胞库(work cell bank, WCB)。对于已建立的细胞库, 应参照《中华人民共和国药典》 (Chinese Pharmacopoeia, Ch.P) 通则《生物制品生产检定用动物细胞基质的制备及质量控制》进行检测, 检测项目包括鉴别、微生物污染检测、内外源病毒因子检测、逆转录病毒检测及特异性病毒检测等。虽然在细胞库阶段对MCB和WCB进行了内外源病毒因子检测, 但有些内源性病毒可能在MCB和WCB阶段未被检出, 或者在生产过程中引入偶然的病毒因子污染, 因而对生产终末细胞和/或细胞收获液中的病毒因子进行检测和控制非常重要。

1.1 EOPC和/或UPB检测相关技术指南调研

本文整理了国内外关于EOPC和/或UPB检定检测相关指导原则的情况, 如下表 1。我国目前尚未有专门针对创新单抗药物申报临床阶段病毒检测的技术指南, 目前申请人主要参照《中华人民共和国药典》通则《生物制品生产检定用动物细胞基质制备及质量控制》要求进行细胞库鉴定; 而FDA和EMA生物制药发展较早, 因而在监管体系和监管指南方面较为完善, FDA参照《Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use》 (人用单克隆抗体产品的生产和检测的相关考虑, 即PCT1997) 进行UPB的检定; EMA在2008年发布了《Guideline on Virus Safety Evaluation of Biotechnological Investigational Products》 (生物技术研究产品病毒安全性评价技术指南, 即EMA2008), 主要针对生物技术产品在申报临床试验(investigational new drug application, IND application) 时病毒安全性评价的指南, 指南中对于EOPC和/或UPB检定的要求给予了明确规定。

作者将针对以上技术指南具体要求展开说明。我国目前虽然在《中华人民共和国药典》、《生物组织提取制品和真核细胞表达制品的病毒安全性评价技术审评一般原则》以及《重组制品生产用哺乳动物细胞质量控制技术评价的一般原则》中分别提及了对EOPC或UPB检定的要求^[4-6], 但以上指南主要是针对上市的要求。国内企业早期申报临床试验阶段对于生产过程中病毒检测主要参照了《中华人民共和国药典》相关要求, 对临床申报注册工艺下的EOPC进行了一次全面检定, 而并未考虑对工艺过程中UPB进行病毒检测。FDA主要参考PCT1997, 目前对于创新药申报临床试验阶段的UPB检测主要参考该指南, 该技术指南对UPB、原液和最终产品的检定提出了要求, 明确说明对于每个批次收获液进行生物负荷、支原体、外源病毒因子、特定外源病毒和逆转录病毒的控制(逆转录病毒颗粒定量)^[7]。EMA2008指南强调, 每一批用于临床试验用样品的UPB需参照ICH Q5A进行检测, 待测样品应包括细胞, 在适用的情况下测试应包括体外和基于聚合酶链反应(polymerase chain reaction, PCR) 的外源病毒因子试验和逆转录病毒颗粒评估, 逆转录病毒或逆转录病毒颗粒的定量仅需针对特定开发阶段的前3批UPB进行(或更少, 如制备少于3批UPB)。对于源自中国仓鼠卵巢细胞系(Chinese hamster ovary cell, CHO), 无需进一步测试; 对于基于小鼠骨髓瘤细胞(mouse myeloma cell) 的NS0或Sp2/0细胞系, 应进行一次传染性逆转录病毒检测; 但如果细胞培养发生重大变化(如生产规模), 应重新进行传染性逆转录病毒检测。对于基于任何其他细胞系的产品, 应进行一次传染性逆转录病毒测试和体内测试, 但如果生产发生重大变化(如生产规模), 则应重复进行传染性逆转录病毒测试。如果细胞产品采用的细胞系对鼠细小病毒(mouse minute virus, MMV) 易感, 则应考虑进行MMV测试^[8]。同时指南还强调如果UPB进行了上述检测, 则无需在对体外限传代次细胞进行检测, 这里的体外限传代次细胞即为EOPC。

1.2 EOPC和/或UPB检测申报现状与问题分析

由于目前申请人对国内外指南技术阶段性要求理解的差异, 导致申请人在开展研究和递交申报资料时存在一定差异。根据目前创新单抗药物的申报现状, 将主要差异总结如下表 2。国内申请人在申报IND阶段对于EOPC/UPB检测经历了两个阶段, 第一阶段(申报1) 是国内单抗药物起步时期, 一般按照《中华人民共和国药典》要求对EOPC进行一次全面检定; 第二阶段(申报3) 是国内单抗药物发展时期, 很多国内申请人采用中美双报策略, 因而在开展研究中同时进行EOPC和UPB检测; 而申报2一般是在国外申请IND时常见, 一般针对临床试验用每个批次的UPB进行检测, 而不进行EOPC检测。

1.3 思考与建议

由于我国创新单抗药物发展相比美国、欧盟等国家起步较晚, 目前尚未出台针对申报IND阶段病毒安全性检测的细化相关指导原则, 因此国内申请人在早期申报IND阶段按照上市阶段的要求对EOPC进行检测。作者认为, 申报IND阶段的EOPC一般来自于申报临床注册工艺下的细胞进行连续几次体外传代后冻存得到的细胞, 属于一次全面检定, 后续临床试验用样品的UPB缺少对病毒污染的检查。UPB作为整个生产工艺中病毒污染最易检出的工序, 相比仅进行一次EOPC检定对于病毒污染的控制策略更为有效, 主要基于以下考虑: ①确保每个用于临床试验用样品在收获液阶段均进行了病毒污染检测; ②未对UPB进行操作和处理, 可排除额外操作可能引起的病毒滴度降低或病毒活性被破坏的风险; ③相比EOPC一次全面检定, 可更加严格地控制细胞收获液中可能引入的病毒污染的风险, 可最大程度确保最终用于人体临床试验用样品的安全性。通常情况下, 建议IND阶段尽可能对每批用于临床试验用药物的UPB内外源病毒污染检测和微生物污染进行检测, 一般检测项目包括微生物负荷或无菌、支原体、外源病毒因子(体外法)、特定外源病毒因子检测(如适用) 以及逆转录病毒颗粒定量等。需要关注的是, 逆转录病毒颗粒定量研究可针对特定开发阶段的前3批UPB (或者更少, 如制备少于3批UPB) 进行。临床开发过程中发生细胞培养工艺重大变更(如制备规模、培养基和培养方式等), 应重新评估细胞培养收获液中病毒颗粒数量。

2 病毒去除或灭活验证

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病毒清除验证研究的目的是证明实际生产工艺对病毒去除或灭活的有效性, 并结合细胞培养收获液中病毒定量分析结果对终产品制剂的病毒安全性风险进行评估。虽然在细胞系和生产用其他原材料方面进行了病毒安全性测试, 以及在生产工艺过程中进行了病毒污染的控制, 但是所有定量测试病毒的方法都具有局限性, 没有任何一种方法能够必然确保产品的安全性, 因而在确保最终产品中不存在病毒污染的风险除了进行直接测试病毒, 还需要进行病毒去除/灭活验证。

2.1 病毒去除/灭活验证技术指南调研

本文整理了国内外关于病毒清除验证相关指导原则的情况, 如表 3。我国目前在申报IND阶段开展病毒去除或灭活验证主要参考了《血液制品去除/灭活病毒技术方法及验证指导原则》和《生物组织提取和真核细胞表达制品的病毒安全性评价技术审评的一般原则》相关要求。FDA主要参考PCT1997及《美国药典》 (United States Pharmacopoeia, USP)《Design, Evaluation, and Characterization of Viral Clearance Procedures Introduction》 (简称USP < 1050.1 > ) 在申报临床阶段采用简化的病毒去除/灭活验证。EMA主要参考EMA在1996年对外公开的《Virus Validation Studies: the Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses》 (简称EMA1996)相关技术要求对申报临床的生物技术产品开展病毒去除/灭活验证。ICH Q5A是目前国际通用的针对人或动物细胞系生产的生物技术产品上市阶段病毒安全性评价的指南。

下文将结合以上调研对相关指南中的具体技术要求展开阐述。国内技术指南对于选择指示病毒类型规定较为详细, 明确至少应包括单链和双链的核糖核酸(RNA) 及脱氧核糖核酸(DNA)、脂包膜和非脂包膜、强和弱抵抗力、大和小颗粒等病毒; 从工艺步骤选择上, 该技术指南规定“对于真核细胞表达制品应至少包含一个有效工艺步骤, 并能够有效去除和/或灭活非脂包膜病毒; 从病毒去除或灭活验证有效性判定上, 国内技术指南一般认为病毒感染性滴度减少≥4 lg的处理步骤为有效的病毒去除或灭活工艺步骤, 如果去除效果仅够1 lg甚至低于1 lg, 通常可忽略不计, 不计入工艺的总去除指数内^[7]; 从验证批次方面, 参照《血液制品去除灭活病毒技术方法及验证指导》要求, 一般需提供连续3批病毒灭活前的中间品进行病毒灭活方法效果验证^[9]。

FDA早期发布的技术指南在指示病毒选择上未明确具体的要求, 总体原则是建议选择一种或多种病毒, 应包括已知或怀疑污染细胞系病毒, 也可采用模型病毒(与污染病毒性质类似、可培养至高滴度且易于检测分析)^[10]; 2013年USP 《Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin》 (简称USP < 1050 > ) 指出, 用于病毒清除率评估和表征研究的病毒, 应采用性质与可能污染产品相似的病毒以及具有较强的物理化学耐受特性的代表性病毒, 根据评估和表征研究的目的说明病毒选择的合理性^[11]; 2016年USP < 1050.1 > 中指出, 通常在产品开发的早期临床阶段使用至少两种病毒, 一种是包膜的[通常是逆转录病毒, 如鼠白血病病毒(murine leukemia virus, MuLv)], 另一种是无包膜的(最好是细小病毒, 如MMV), 用于过程评估和过程特性研究的模型病毒应与可能污染产品的病毒性质相似, 但是还应检测具有较强的物理化学耐受特性的其他病毒, 后者对于证明纯化过程能够去除/灭活多种病毒至关重要^[12]; 从验证工艺步骤方面FDA相关技术指南均指出应采用不同作用机制的至少两个正交步骤, 且在USP < 1050.1 > 中指出, 在产品开发的早期临床阶段, 每个病毒至少应评估两个正交的病毒去除或灭活步骤, 有效步骤的可重复性应通过执行两个独立的试验来评估, 或者过程开发历史(或经验) 应能支持可重复性^[13]; 从病毒去除或灭活验证有效性判定上FDA相关技术指南指出, 通常认为单个病毒清除步骤有效需证明病毒滴度降低4 lg或更多, 相反证明1~3 lg的清除步骤被认为是辅助步骤, 但考虑到测定方法的内在变异性, 减少1 lg值不计入总去除指数内。

EMA发布的有关于病毒安全性和病毒清除验证相关的技术指南中对于指示病毒选择、验证步骤选择、去除效果有效性差异等方面的总体原则与FDA基本一致。主要差异是EMA强调了病毒安全性评估还应包括每剂量中病毒颗粒量估算, 同时应结合临床试验的总体风险进行评估, 如适应症、剂量、给药频率、人群暴露量、研究持续时间和患者免疫状态等^{[13, 14]}。

ICH Q5A主要是针对上市产品的病毒安全性评估, 这个阶段需要开展全面的、完整的工艺去除或灭活验证研究, 并评价整体工艺对病毒污染控制的可行性, 对于病毒种类的选择、验证步骤的要求以及最终逆转录病毒颗粒数量的安全性评估均进行了全面的阐述和说明。

2.2 病毒去除或灭活验证申报现状与问题分析

近年来, 随着国外申报和中美双报的生物制品企业越来越多, 作者发现在申报临床试验阶段病毒去除或灭活验证的批次、验证病毒种类以及验证步骤略有差异, 主要差异总结如表 4 (以CHO细胞为例)。我国目前在病毒去除或灭活验证研究方面, 普遍存在两种做法, 申报1 (application 1) 是国内申请人在早期申报临床时普遍采用做法, 提供了3个验证批次、4种理化特性不同的指示病毒、2个步骤的验证策略, 但这种做法未考虑多个工艺步骤对相关或特异性模型病毒的去除或灭活能力, 也未考虑一种病毒验证多个工艺步骤, 以确保工艺步骤对相关或特异性模型病毒具有有效的清除能力的同时对物理化学抗性较强的病毒也具有一定的清除能力。申报2 (application 2) 是近年来国内申请人在采用中美双报策略时的普遍做法, 既可满足目前中国监管要求, 也符合国外监管机构指南要求, 开展了两套不同的验证方案, 相比国内早期申报临床及国外申报临床的病毒去除或灭活验证, 额外增加了申请人的负担。申报3 (application 3) 是国外申请人在申报IND阶段的一般做法, 一般情况下采用了简化的病毒去除或灭活验证方案, 采用符合《药品生产质量管理规范》 (Good Manufacturing Practice, GMP) 的一个临床试验用品评价至少两个以上不同作用原理的正交步骤、每个步骤验证两种病毒(低pH除外)、每种病毒至少验证两个工艺步骤、且每个批次分别独立开展两次试验的验证形式。

2.3 思考与建议

我国监管指南与国际监管指南在病毒去除或灭活验证技术要求的理念上基本一致, 但我国病毒安全性相关监管指南主要针对上市要求, 申报临床阶段如何在控制病毒安全性风险的前提下进行简化研究, 仍需监管机构和工业界不断进行探讨。国外申请人对于病毒去除/验证的策略相比国内申请人虽然减少了验证病毒的种类以及验证批次, 但在验证步骤方面考察了3种不同作用机制的工艺步骤, 且每个步骤基本考察了对相关或特异性模型病毒以及理化抗性较强、易污染CHO细胞且较难灭活的相关模型病毒的去除或灭活效果, 在一定程度上也证明了工艺步骤去除或灭活其他病毒的能力。虽然在验证批次方面未开展3批中间品的病毒去除或灭活验证, 但采用一个临床工艺代表性批次重复两次试验的验证策略, 证明去除/灭活效果的稳健性和重复性更加合理, 且一个批次重复两次试验的做法与目前ICH Q5A的要求也一致。

综上, 作者认为, 在申报临床阶段可基于宿主细胞的种属、病毒种类和载量水平、感染性和致病性等因素综合考虑风险, 采用简化的病毒去除或灭活验证研究。在上市注册时提供全面的、完整的病毒去除或灭活验证研究资料, 并评价整体工艺对病毒去除效果的可行性。一般情况下, 简化的病毒去除或灭活验证研究包含两个方面, 即验证工艺步骤和验证指示病毒种类的简化。在申报临床阶段应根据潜在污染病毒特性、结合产品特性和生产工艺、病毒清除工艺的作用机制和清除能力综合评估, 选择适宜的病毒清除工艺, 原则上应至少采用两种正交、原理不同的两个步骤, 一个有效的工艺步骤应当在两个独立的验证研究中进行重复试验以确保去除步骤的可重复性。指示病毒应至少包括与已知或怀疑污染细胞系的病毒或与该病毒性质相似病毒, 以及具有广泛理化特性且具有高抵抗力的病毒, 鼓励申请人对于相关和特异性模型病毒采用至少两个正交步骤进行验证, 原则上减少4 lg或更高数量级的lg表示所研究工艺步骤对特定测试病毒有明显去除效果。验证批次应采用拟用于临床试验的代表性工艺批次, 验证条件和参数应能反映生产工艺的最差条件, 如早期开发阶段尚未确立生产工艺参数的最差条件, 也可采用经证明的生产工艺设定点代表实际生产工艺进行验证。

3 结语

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单克隆抗体药物作为生物药的细分品种, 其在免疫性疾病、癌症等治疗领域取得的巨大成功使得抗体药物在未来将是生物制药行业发展的主要方向。虽然我国抗体药物发展晚于欧洲和美国等发达国家, 但是近十年来也先后涌现出一大批专门从事单抗药物生产的企业, 同时, 许多传统的制药企业近年来也逐步涉足抗体药物领域, 另外国内以服务抗体药物开发和检测的合同研究和合同生产企业(contract research organization/contract manufacturing organization, CRO/CMO)公司也逐步发展成熟, 因而国内创新单抗药物发展的局面日益壮大。

我国目前针对单克隆抗体药物的药学技术指南主要是侧重上市的要求, 而针对申报临床试验阶段一些细化的技术指南尚待进一步完善。随着近年来国内创新药发展日益迅速, 越来越多的创新型企业由“引进来”逐步向“走出去”发展, 由于申请人对国内外指南技术阶段性要求理解的差异, 导致在开展研究和递交申报资料时存在一定差异。笔者在充分梳理了申报资料差异及业界反馈集中的问题基础上, 从中筛选出比较典型的且与申报临床试验阶段病毒安全性相关的焦点问题, 结合国内外不同监管机构的要求以及查阅相关文献资料基础上, 提出了对以上问题的技术考量, 希望通过这些思考与业界就创新单抗药物申报临床阶段病毒安全性问题进行沟通和探讨, 以期提高我国创新单抗药物研发进度和研发水平, 使更多更好的创新单抗药物尽快进入临床试验, 使得国内创新药物更好地走向国际, 最终惠及民众。

作者贡献: 本文主要由崔靖撰写, 宋丽娜修改。

利益冲突: 所有作者均声明不存在利益冲突。

参考文献

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2021年第56卷第8期

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doi: 10.16438/j.0513-4870.2021-0195

接收时间：2021-02-05
首发时间：2025-12-18
出版时间：2021-08-12

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收稿日期：2021-02-05
修回日期：2021-03-10

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国家药品监督管理局药品审评中心, 北京 100022

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*宋丽娜, Tel: 86-10-85243036, E-mail: songln@cde.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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BibTeX
EndNote
RefWorks
TxT

Drug administration	Guideline	Year
NMPA	General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products	2005
General Principles of Evaluation of Mammalian Cell Substrate Quality Control Technology for the Production of Recombinant Biological Products	2008
Chinese Pharmacopoeia. Manufacture and Quality Control of Animal Cell Substrate for Biological Products	2015
FDA	Points to Consider in the Characterization of Cell Lines Used to Product Biologicals	1993
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use	1997
EMA	Guideline on Virus Safety Evaluation of Biotechnological Investigational Products	2008

Drug
administration

Guideline

Year

NMPA

General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products

2005

General Principles of Evaluation of Mammalian Cell Substrate Quality Control Technology for the Production of
Recombinant Biological Products

2008

Chinese Pharmacopoeia. Manufacture and Quality Control of Animal Cell Substrate for Biological Products

2015

FDA

Points to Consider in the Characterization of Cell Lines Used to Product Biologicals

1993

Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use

1997

EMA

Guideline on Virus Safety Evaluation of Biotechnological Investigational Products

2008

Test item	Application 1	Application 2	Application 3
EOPC	+^a	-^b	+^a
UPB	-^b	+^a	+^a

Test item

Application 1

Application 2

Application 3

EOPC

+^a

-^b

+^a

UPB

-^b

+^a

Drug administration	Guideline	Year
NMPA	Guideline on Technology and Validation of Virus Removal or Inactivation of Blood Products	2002
General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products	2005
FDA	Points to Consider in the Characterization of Cell Lines Used to Product Biologicals	1993
USP < 1050 > . Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin	2013
USP < 1050.1 > . Design, Evaluation, and Characterization of Viral Clearance Procedures Introduction	2016
EMA	Guideline on Virus Safety Evaluation of Biotechnological Investigational Products	2008
Virus Validation Studies: the Design, Contribution, and Interpretation of Studies Validating the Inactivation and Removal of Viruses	1996
ICH	ICH Q5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Biotechnology Products Derived from Cell Lines of Human or Animal Origin	1999

Drug
administration

Guideline

Year

NMPA

Guideline on Technology and Validation of Virus Removal or Inactivation of Blood Products

2002

General Principles of Virus Safety Evaluation of Biological Tissue Extraction and Eukaryotic Cell Expression Biological Products

2005

FDA

Points to Consider in the Characterization of Cell Lines Used to Product Biologicals

1993

USP < 1050 > . Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human of Animal Origin

2013

USP < 1050.1 > . Design, Evaluation, and Characterization of Viral Clearance Procedures Introduction

2016

EMA

Guideline on Virus Safety Evaluation of Biotechnological Investigational Products

2008

Virus Validation Studies: the Design, Contribution, and Interpretation of Studies Validating the Inactivation and Removal of Viruses

1996

ICH

ICH Q5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Biotechnology Products Derived from Cell Lines of Human or Animal Origin

1999

Manufacturing process	Application 1	Application 2	Application 3
Low pH	Two viruses (MuLv and Prv), three lots, one experiment	Two viruses (MuLv、Prv), three lots, one experiment; One GMP lot, virus (MuLv), two independent experiments	One GMP lot, virus (MuLv), two independent experiments
Filtration	Two viruses (MMV and Reo-3), three lots, one experiment	Two viruses (MMV and Reo-3), three lots, one experiment; One GMP lot, viruses (MuLv and MMV), two independent experiments	One GMP lot, viruses (MuLv and MMV), two independent experiments
Anion-exchange chromatography	None	One GMP lot, viruses (MuLv and MMV), two independent experiments	One GMP lot, viruses (MuLv and MMV), two independent experiments
Others	None	None	Optional

Manufacturing process

Application 1

Application 2

Application 3

Low pH

Two viruses (MuLv and Prv), three lots, one experiment

Two viruses (MuLv、Prv), three lots, one experiment; One GMP lot, virus (MuLv), two independent experiments

One GMP lot, virus (MuLv), two
independent experiments

Filtration

Two viruses (MMV and Reo-3), three lots, one experiment

Two viruses (MMV and Reo-3), three lots, one experiment; One GMP lot, viruses (MuLv and MMV), two independent experiments

One GMP lot, viruses (MuLv and MMV), two independent experiments

Anion-exchange chromatography

None

One GMP lot, viruses (MuLv and MMV), two independent experiments

Others

None

Optional