药学学报

Drug name	Sponsor	Approval data or status
Recombinant human tumor necrosis factor receptor-immunoglobulin fusion protein for injection	CP Guojian Pharm Celgen Biopharma Zhejiang Hisuan pharmaceutical Co., Ltd	2005 2011 2012
Iodine-~(131)-labeled tumor necrosis therapy chimeric antibody	Shanghai Medipharm Biotech Pharmaceutical	2006
Iodine[¹³¹I]metuximab injection	Chengdu huasuan group inc., Ltd	2006
Nimotuzumab injection	Biotech pharma Co., Ltd	2008
Recombinant humanized anti-CD25 monoclonal antibody injection	CP Guojian Pharm	2011
Conbercept ophthalmic injection	Kanghong Pharmaceutical	2013
Toripalimab injection	TopAlliance Bioscience Inc.	2018
Sintilimab injection	Innovent Biologics, Inc	2018
Rituximab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd/ Innovent Biologics, Inc	2019 Under review
Camrelizumab	Shanghai hengrui pharmaceutical co., Ltd	2019
Adalimumab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd Innovent Biologics, Inc Zhejiang Hisuan pharmaceutical Co., Ltd Sinoasis Pharmaceuticals, Inc	Under review
Recombinant anti-VEGF humanized monoclonal antibody injection	Qilu Pharmaceutical	Under review
Bevacizumab biosimilar	Innovent Biologics, Inc	Under review
Recombinant anti-HER2 humanized monoclonal a ntibody for injection	Sansheng Guojian Pharmaceutical (Shanghai) Co., Ltd	Under review
Tirelizumab injection	BeiGene, Ltd	Under review
Trastuzumab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd	Under review

Drug name	Sponsor	Approval data or status
Recombinant human tumor necrosis factor receptor-immunoglobulin fusion protein for injection	CP Guojian Pharm Celgen Biopharma Zhejiang Hisuan pharmaceutical Co., Ltd	2005 2011 2012
Iodine-~(131)-labeled tumor necrosis therapy chimeric antibody	Shanghai Medipharm Biotech Pharmaceutical	2006
Iodine[¹³¹I]metuximab injection	Chengdu huasuan group inc., Ltd	2006
Nimotuzumab injection	Biotech pharma Co., Ltd	2008
Recombinant humanized anti-CD25 monoclonal antibody injection	CP Guojian Pharm	2011
Conbercept ophthalmic injection	Kanghong Pharmaceutical	2013
Toripalimab injection	TopAlliance Bioscience Inc.	2018
Sintilimab injection	Innovent Biologics, Inc	2018
Rituximab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd/ Innovent Biologics, Inc	2019 Under review
Camrelizumab	Shanghai hengrui pharmaceutical co., Ltd	2019
Adalimumab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd Innovent Biologics, Inc Zhejiang Hisuan pharmaceutical Co., Ltd Sinoasis Pharmaceuticals, Inc	Under review
Recombinant anti-VEGF humanized monoclonal antibody injection	Qilu Pharmaceutical	Under review
Bevacizumab biosimilar	Innovent Biologics, Inc	Under review
Recombinant anti-HER2 humanized monoclonal a ntibody for injection	Sansheng Guojian Pharmaceutical (Shanghai) Co., Ltd	Under review
Tirelizumab injection	BeiGene, Ltd	Under review
Trastuzumab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd	Under review

Variant type	Example	Potential impact	Quality control strategy (test assay)
Size related variants	Aggregates	Increased immunogenicity	Extend characterization (SEC-HPLC, AUC) or in release test (SEC-HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-CE)
	Fragments	Maybe has no potency
Charge related variants	Acid variant (sialylation, deamidation, C-terminal lysine cleavage, high mannose, thiosulfate, glycation, etc.)	Modification in N/C terminal region have no substantial effects on antibody structure, stability and function; modification in CDR regions have affect on potency and stability	Extend characterization (isoelectric focusing, capillary isoelectric focusing, ion exchange chromatography (IEX, LC-MS) or routine release test (IEX, HIC)
	Main species (cyclization of N-terminal Gln to pycoGlu, removal of the heavy chain lysine, glycosylation of Asn297)
	Basic variant (C-terminal lysine, N-terminal Glu, isomerization of Asp, succinimide, methionine oxidation, amidation, aglycosylation, fragments, aggregation)
Sequence related variants	N-Terminal leads sequence	Low risk of safety and effect	Extend characterization (LC-MS) or routine release test (peptide mapping)
Amino acid mis-incorporation
	N-Terminal modification (acetylation, formylation and pyroglutamylation)	Increased charge heterogeneity without impacting safety and efficacy	Extend characterization (LC-MS, reverse phase high-performance liquid chromatography) or in release test (IEX)
	C-Terminal modification (clipping of C-terminal Lys, prolineamidation)
Cysteine-related variants	Free cysteine	Decreased antibody thermal stability and trigger formation of covalent aggregates	Extend characterization (LC-MS, Ellman kit)
	Disulfide isoforms (IgG2A/B, IgG half antibody)	May impact potency and specificity of antibody	Extend characterization (LC-MS)
	Trisulfide bond	Low risk of safety and effect
	Thioether, D-cysteine, cysteinylation
Glycosylation	N-Glycolylneuraminic acid, α-1, 3 Gal	Increased immunogenicity	Extend characterization (LC-MS) and/or routine release test (hydrophilic interaction chromatography, HIC)
	Afucosylation	Increased ADCC effect
	Galatosylation	Increased CDC effect
	High mannose	Decreased half-life time in vivo
	Glycation	Generation of acidic species
Other post-translational modification variants	Asndeamidation, Asp isomerization, Met/Trp oxidation in CDR region	Decreased affinity and immunogenicity

Variant type	Example	Potential impact	Quality control strategy (test assay)
Size related variants	Aggregates	Increased immunogenicity	Extend characterization (SEC-HPLC, AUC) or in release test (SEC-HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-CE)
	Fragments	Maybe has no potency
Charge related variants	Acid variant (sialylation, deamidation, C-terminal lysine cleavage, high mannose, thiosulfate, glycation, etc.)	Modification in N/C terminal region have no substantial effects on antibody structure, stability and function; modification in CDR regions have affect on potency and stability	Extend characterization (isoelectric focusing, capillary isoelectric focusing, ion exchange chromatography (IEX, LC-MS) or routine release test (IEX, HIC)
	Main species (cyclization of N-terminal Gln to pycoGlu, removal of the heavy chain lysine, glycosylation of Asn297)
	Basic variant (C-terminal lysine, N-terminal Glu, isomerization of Asp, succinimide, methionine oxidation, amidation, aglycosylation, fragments, aggregation)
Sequence related variants	N-Terminal leads sequence	Low risk of safety and effect	Extend characterization (LC-MS) or routine release test (peptide mapping)
Amino acid mis-incorporation
	N-Terminal modification (acetylation, formylation and pyroglutamylation)	Increased charge heterogeneity without impacting safety and efficacy	Extend characterization (LC-MS, reverse phase high-performance liquid chromatography) or in release test (IEX)
	C-Terminal modification (clipping of C-terminal Lys, prolineamidation)
Cysteine-related variants	Free cysteine	Decreased antibody thermal stability and trigger formation of covalent aggregates	Extend characterization (LC-MS, Ellman kit)
	Disulfide isoforms (IgG2A/B, IgG half antibody)	May impact potency and specificity of antibody	Extend characterization (LC-MS)
	Trisulfide bond	Low risk of safety and effect
	Thioether, D-cysteine, cysteinylation
Glycosylation	N-Glycolylneuraminic acid, α-1, 3 Gal	Increased immunogenicity	Extend characterization (LC-MS) and/or routine release test (hydrophilic interaction chromatography, HIC)
	Afucosylation	Increased ADCC effect
	Galatosylation	Increased CDC effect
	High mannose	Decreased half-life time in vivo
	Glycation	Generation of acidic species
Other post-translational modification variants	Asndeamidation, Asp isomerization, Met/Trp oxidation in CDR region	Decreased affinity and immunogenicity

Drug name	Sponsor	Approved indication in China	Dose/price (RMB)
Nivolumab	Bristol-Myers Squibb	Advanced non-small cell lung cancer	100 mg/9 260
Pembrolizumab	Merck & Co Inc	Metastatic malignant melanoma	100 mg/17 918
Toripalimab	Shanghai Junshi Bioscience	Melanoma	240 mg/7 200
Sintilimab	Innovent Biologics Co. Ltd	Refractory classic Hodgkin's lymphoma	100 mg/7 839
Camrelizumab	Jiangsu Hengrui Medicine	Advanced solid tumor	100 mg/7 838
Rituximab	Roche	Non-hodgkin lymphoma	100 mg/2 418
Rituximab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd	Non-hodgkin lymphoma; follicular lymphoma; diffuse large B-cell lymphma	100 mg/1 648

Drug name	Sponsor	Approved indication in China	Dose/price (RMB)
Nivolumab	Bristol-Myers Squibb	Advanced non-small cell lung cancer	100 mg/9 260
Pembrolizumab	Merck & Co Inc	Metastatic malignant melanoma	100 mg/17 918
Toripalimab	Shanghai Junshi Bioscience	Melanoma	240 mg/7 200
Sintilimab	Innovent Biologics Co. Ltd	Refractory classic Hodgkin's lymphoma	100 mg/7 839
Camrelizumab	Jiangsu Hengrui Medicine	Advanced solid tumor	100 mg/7 838
Rituximab	Roche	Non-hodgkin lymphoma	100 mg/2 418
Rituximab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd	Non-hodgkin lymphoma; follicular lymphoma; diffuse large B-cell lymphma	100 mg/1 648

关于我国单抗药物上市阶段药学评价的思考

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刘伯宁 , 徐刚领 , 罗建辉 ^*

药学学报 | 新药论坛 2019,54(11): 2126-2134

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药学学报 |新药论坛 2019 , 54 (11) : 2126 -2134

关于我国单抗药物上市阶段药学评价的思考

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刘伯宁, 徐刚领, 罗建辉^*

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:

*罗建辉, Tel: 86-10-68586655, E-mail: luojh@cde.org.cn

Chemistry, manufacturing and controls regulatory considerations for marketing authorization application of therapeutic antibody in China

Bo-ning LIU, Gang-ling XU, Jian-hui LUO^*

Affiliations

Center for Drug Evaluation, National Medical Products Administration, Beijing 100022, China

出版时间: 2019-11-12 doi: 10.16438/j.0513-4870.2019-0534

文章导航

摘要

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申报注册上市是药物生命周期的重要节点，标志着具有临床价值“候选物”成为可以上市销售的“药品”。目前，我国自主研发的抗体药物上市产品仅12个。注册生产阶段的单抗药物研发与评价，对于工业界和监管界而言均缺乏经验。与此同时，近年来单抗生物类似药产品已开始集中报产，国外单抗进口注册进程不断加快，未来单抗药物上市阶段的药学评价将为我国生物制品注册上市审评的重要工作。本文结合笔者从事单抗药物研发与评价的实践经验，重点对单抗药物注册生产阶段的药学研究内容、评价要点及现存问题展开讨论，以期促进工业界规范开展药学研究，加速此类产品注册上市进程。

关键词

抗体药物 / 上市申请 / 药学评价 / 生产现场检查 / 单克隆性 / 质量源于设计 / 工艺变更

Abstract

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The marketing authorization application is a milestone of drug life cycle, which indicates a candidate has potential to become a commercial drug. As of now, there are only 12 domestic therapeutic antibodies approved in China. The chemistry, manufacturing and controls (CMC) development and evaluation of monoclonal antibody were more challenging for both industry and authority agency. As the result of domestic biopharmaceutical industry development and implement of priority review system, the marketing authorization application of domestic antibody biosimilar and imported antibodies had dramatic increased in recent years. Thus, the CMC evaluation of monoclonal antibody become the important task of biological product's marketing authorization registration management. In the article, the CMC regulatory considerations for marketing authorization application based on author's review experience was proposed, in order to accelerate development and registration of commercial antibody in China.

Key words

monoclonal antibody / marketing authorization application / chemistry, manufacturing and controls review / pre license inspection / clonality / quality by design / manufacturing process change

引用本文

刘伯宁, 徐刚领, 罗建辉. 关于我国单抗药物上市阶段药学评价的思考. 药学学报, 2019 , 54 (11) : 2126 -2134 . DOI: 10.16438/j.0513-4870.2019-0534

Bo-ning LIU, Gang-ling XU, Jian-hui LUO. Chemistry, manufacturing and controls regulatory considerations for marketing authorization application of therapeutic antibody in China[J]. Acta Pharmaceutica Sinica, 2019 , 54 (11) : 2126 -2134 . DOI: 10.16438/j.0513-4870.2019-0534

正文

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药物申报注册上市标志着具有临床应用价值的“候选药”有望成为可上市销售的“药品”。充分的药学研发与评价是保证产品上市后工艺稳定、质量可控的前提。近年来, 随着我国抗体药物研发能力的提高, 以及相关产业化技术(细胞系构建^[1]、细胞大规模培养^[2]、表征分析与质控技术^[3]等)的突破, 越来越多的自主研发抗体药物开始注册申报上市(表 1)。与此同时, 国内对于临床急需药物优先审评、有条件接受境外临床试验数据等政策的出台, 也使得国外已上市抗体在国内进口注册的时间差不断缩小^[4]。可以预计, 单抗药物上市阶段的药学评价将是我国生物制品注册上市审评的重要工作。本文结合笔者从事单抗药物研发与评价的实践经验, 重点对单抗药物申报上市阶段的药学研究内容、评价要点及现存问题展开讨论, 以期促进工业界规范开展药学研究, 充分准备研究资料, 加速此类产品注册上市进程。

1 抗体药物上市阶段药学评价的现状

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1.1 我国抗体药物上市注册申请分析

长期以来, 国内外抗体药物产业发展存在显著差距。国际上欧美市场每年上市的单抗药物约5~7个品种^[5], 而在我国自主研发的上市单抗药物总计仅12个(表 1)。近年来, 随着我国抗体药物产业的迅速发展, 已经有近300个抗体药物进入临床研究阶段, 产品类型涵盖抗体生物类似药、新靶点(序列)抗体药物、抗体药物偶联物、单域抗体、双功能抗体(复方抗体)^[6]和抗体样融合蛋白等多种结构类别。自2018年开始, 我国自主研发的多个抗PD-1单抗(特瑞普利单抗、信迪利单抗、卡瑞利珠单抗、替雷利珠单抗等)和抗体生物类似药(利妥昔单抗、阿达木单抗、贝伐珠单抗、曲妥珠单抗等)集中申报注册生产(表 1)。结合已进入临床阶段的候选药物产品线分析, 可以预计未来数年国内抗体药物申报生产品种将进入一个高峰^[4]。

此外, 对于进口注册抗体药物而言, 目前国外已上市的94个抗体药物中完成进口注册的产品尚不足1/4。随着我国临床急需药物优先审评制度^[7]、接受药品境外临床试验数据指导原则等政策公布, 未来国外单抗药物进口注册进程将显著加快。目前, 已有48个抗体药物纳入优先审评程序, 其中进口注册产品将近半数(22个)^[4]。因此, 未来国内外单抗药物的上市阶段评价将为生物制品注册管理的重要工作。

1.2 注册临床与申报上市阶段的药学评价的不同要求

单抗药物的药学研究随临床试验进程“分阶段”、“渐进式”的开展, 药学评价同样遵从药物研发规律。在申请临床阶段药学评价应重点关注受试者的“安全性”, 而注册生产阶段则应全面保证药品的工艺稳健性与质量可控性, 符合监管方对于药物内涵的定义。如: “安全、有效和质量可控”或“安全(safety)、纯度(purity)和效力(potency)”^{[8, 9]}。

单抗药物在注册生产阶段其药学研究内容应基本完成, 与申报临床阶段相比, 药学评价在细胞基质、工艺表征和验证、质量研究和控制等方面均提出符合商业化生产阶段特点的更高要求, 如:工程细胞系在申报临床阶段可以仅构建主细胞库, 特殊情况下甚至是未经单克隆化的细胞池(cell pool), 但在申报上市阶段应规范建立三级细胞库, 生产细胞一般应符合“单克隆化”要求; 工艺开发方面, 申报临床阶段可直接采用平台化的中试工艺, 临床期间进行产品特异化的工艺开发。注册上市阶段生产工艺应完成商业化生产工艺的性能确认与工艺验证^[10]; 质量研究方面, 申报临床阶段对于产品相关变体(分子大小、电荷异构、翻译后修饰等)可采用报告含量的方式进行初步控制, 注册生产阶段则需明确产品相关的杂质或物质, 并将限度要求纳入质量控制; 同时, 应建立反映临床作用机制的生物测活方法并纳入放行标准。稳定性试验考察结果, 临床试验阶段只要能够满足临床试验用药的质量控制即可, 上市阶段则需要提供全面的稳定性考察数据支持上市产品的有效期和储存、运输条件; 病毒去除/灭活工艺, 在临床试验申请时重在有效工艺步骤的初步验证, 注册生产上市时应完成全面的验证。此外, 对于抗体药物生产过程中所接触到高风险的一次性耗材与惯用包材, 在产品注册生产时也应完成产品特异性相容研究。

1.3 加快审评对药学研究与评价带来挑战

目前, 为了尽早满足临床用药亟需, 各国的监管部门都已出台了相关加快审评政策。如美国食品药品监督管理局(Food and Drug Adminstration, FDA)采用加速批准(accelerated approval pathway)、快速通道(fast track designation)、突破性疗法(breakthrough therapy designation)和优先审评(priority review designation)等方式, 对具有显著临床优势或存在未满足临床需求的药物进行加快审评; 欧盟药监局(European Medicines Agency)也于2016年建立了重点药品快速审评程序(priority medicines)。从历史经验看, 一般纳入突破性疗法的单抗药物(nivolumab、blinatumomab、elotuzumab、pembrolizumab、daratumumab等)上市日程至少可提前两年以上; 在我国, 特殊审评程序与优先审评制度也大幅缩短了单抗药物的研发与审评周期^[7]。但是, 上述加快审评制度对抗体药物的药学研发与评价提出了新的挑战, 如:商业化规模工艺验证批次有限, 缺乏效期末的实时稳定性数据、产品上市后频繁的工艺变更与技术转移等^{[11, 12]}。在审评实践中, 近年我国纳入优先审评的抗PD-1单抗或生物类似药等产品也存在着现有厂房多产品共线、注册生产工艺产能过小和上市后即申请工艺变更等情况。

2 抗体药物上市阶段药学研究内容与审评要点

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2.1 临床批件中要求的常见药学研究内容

申报临床试验阶段药学评价的重点在于满足临床用药的安全性, 其他药学研究通常可以“临床试验通知书”内容的形式要求在临床期间完成。常见的遗留问题包括:关注重组生产细胞单克隆性、持续开展本品工艺开发与验证, 建议对产品相关物质或杂质进行深入研究等。

2.1.1 关注重组生产细胞的单克隆性

按照ICH Q5D等相关指导原则的技术要求, 作为抗体药物生产的细胞基质应该来源于一个祖细胞(a single cell progenitor)或一个稳定的、可连续传代的单克隆细胞系(monoclonal cell line)。虽然, 工业界也有观点认为“单克隆性” (clonality)仅仅是保证终产品质量的手段之一, 不应过分强调或要求生产细胞的单克隆性^[13]。但是, 考虑到细胞基质作为抗体药物质量控制的源头和根本保障, 其单克隆性能更好地保证药物在整个生命周期内的工艺与质量一致性。目前, 国内外监管界普遍要求, 生产细胞系构建过程中采用至少两轮有限稀释(0.5 cell/well)或其他方法(ClonePix^®、FACS^®)等来确保生产细胞系的“单克隆性”^[14]。

在审评实践中, 若在申报临床阶段生产细胞单克隆性存疑, 临床期间可通过荧光原位杂交技术(fluorescence in situ hybridization)分析整合位点和亚克隆表型来进一步确认。单抗药物注册生产时, 若不能保证生产细胞系的单克隆性, 应增加额外的质控策略(质谱检测序列变体、糖型控制等)或重新构建细胞库^{[15, 16]}。某单抗上市阶段通过对生产终末期细胞检定, 发现其主细胞库来源于两个不同的克隆亚群。虽然富集不同克隆亚群的产品证实暂不影响产品质量, 但监管方仍要求该品种上市后一定时间内应完成工程细胞的单克隆化, 并开展相关研究证实新细胞库产品与原细胞库产品质量可比。

2.1.2 应持续进行本品工艺开发与验证

抗体药物模块化生产工艺相对成熟, 单抗药物申报临床阶段一般惯用平台化生产工艺^[3]。临床期间应结合自身特点进行产品特异性工艺开发。笔者在单抗生物类似药的工艺开发中发现, 不同亚型的IgG单抗所耐受的pH阈值显著不同(IgG₁ < aglyco-IgG₁ < IgG₂ < IgG₄), nivolumab等IgG₄亚型单抗应对亲和层析洗脱液、低pH值病毒灭活等工序进行产品特异性工艺开发, 才能保证中间体聚体含量控制在验收标准限度范围内^[17]; 审评实践中, 进口注册单抗在临床期间普遍采用“规模缩小模型” (scale-down model)进行系统化工艺特性研究(process characterization), 进而明确生产工艺中的关键操作参数、控制范围及中间体验收标准。

注册生产阶段的工艺验证除完成连续3批拟商业化工艺的性能确认外, 还应结合层析柱使用寿命、纯化工艺病毒灭活/去除能力等进行全面验证, 如:根据未处理原液中逆转录病毒颗粒数, 计算整个纯化工艺对逆转录病毒的累积去除效果(一般 < 1×10^-6个/dose)。审评实践中, 进口注册单抗药物在注册生产时积累多达10批以上的拟商业化工艺验证数据, 通过“批分析”加强对产品工艺的理解与控制。

2.1.3 建议对产品相关杂质/物质进行深入研究

临床期间在保证安全性的前提下, 抗体药物通常可仅对主成分进行限度控制。随着临床期间对产品质量的深入研究和对产品质量属性的逐步认知, 注册生产阶段应将产品相关杂质或物质进行充分研究及控制。对于分子构建中引入的“定点突变”也应完成功能验证, 如:融合蛋白etanercept由于二硫键错配可以在疏水色谱(hydrophobic interaction chromatography, HIC)上出现不同峰, 注册生产阶段对不具生物活性的相关变体含量进行限定^[18]; IgG₂型单抗panitumumab在非还原毛细管电泳(capillary electrophoresis, CE)图谱上出现非单一主峰, 注册生产阶段完成了相关二硫键配变体(IgG₂A、IgG₂A/B、IgG₂B)的鉴定与测活^[19]; IgG4型单抗pembrolizumab为防止半抗体形成, 在铰链区引入定点突变(S228P), 注册上市阶段通过体外、体内研究进行功能验证^[20]。

抗体药物结构复杂且存在较多翻译后修饰。审评实践中, 已经发现存在多种形式的分子变体。因此, 注册生产阶段应尽可能采用先进、互补的分析方法, 对抗体药物进行表征分析。近年来, FDA对生物制品上市申请进行回溯性分析发现, 绝大多数抗体(79/80)均使用了以质谱为代表的先进手段进行氨基酸序列与二硫键确证、翻译后修饰及产品相关杂质鉴定等研究^[21]。笔者在抗体药物偶联药开发过程中, 也曾采用液质联用技术(liquid chromatography-mass spectrometry, LC-MS)对药物偶联比例、修饰位点进行精确分析^[22]。

此外, 任何单一的分析方法均有其局限性, 采用原理互补的分析手段可更好地表征产品质量特性, 如:分子排阻高效液相色谱(size-exclusion chromatography high performance liquid chromatography, SEC-HPLC)可精确定量分析共价和部分非共价聚体含量, 常用于抗体药物放行标准中的纯度检测。但该方法不能检出样品中的非共价可逆性聚体。因此, 纯度研究可采用分析型超速离心(analytical ultracentrifugation, AUC)等技术进行互补分析, 或采用SEC-HPLC进行方法学验证; 制剂不溶性微粒放行检测一般采用光阻法(light obscuration)测定, 近年来兴起的微流成像技术(microflow imaging), 可同时定量和定性表征不同粒径颗粒数分布、区分蛋白和非蛋白颗粒, 能够满足FDA对于蛋白制剂2~10 μm颗粒分析的技术要求^[23]。审评实践中, 某进口品种在制剂中含由蛋白和胶塞浸出物形成的聚合物, 在货架期标准中通过采用该技术对可见异物进行补充控制。结合已上市单抗药物的质量研究, 现将常见分子变体对临床免疫原性、安全性、有效性的影响, 以及质控策略总结如下(表 2)^[24-29]。

2.2 临床期间发生的生产工艺变更

抗体药物临床常见的变更内容包括:引入新的细胞库(或更换宿主细胞)、发酵工艺优化、纯化介质更新、开发适合临床的制剂处方与剂型、商业化工艺放大与地点转移等。工艺变更的可比性研究是桥接不同研究阶段试验结果、确保产品质量一致性的基础。因此, 临床期间工艺变更也是注册上市阶段药学评价的重点内容。

对于不同临床阶段的工艺变更, 其研究程度、评价要求应与临床试验进度相适应(clinic phase-appropriate)。对于临床试验早期阶段的工艺变更, 应关注毒理批次与拟用于临床试验批次的连续性, 一般应至少进行工艺变更前后各一批头对头质量分析, 并确保临床用药的安全性; 临床试验后期阶段发生的工艺变更, 特别是Ⅲ期临床试验前, 通常需要对工艺变更前后各3批样品进行全面的可比性研究; 产品上市后的工艺变更还应结合历史批次数据进行比对分析。因此, 申请人应结合产品开发进度与变更风险等级合理安排变更时间节点, 提前规划变更研究方案。通常多数工艺变更建议发生在临床试验的早期(Ⅱa前), 在关键性验证临床试验(Ⅲ期)开始后应尽可能避免发生重大工艺变更^{[30, 31]}。

工艺变更的药学可比性研究应包括工艺参数、工艺性能、放行检测和质量表征及稳定性研究等。工艺变更前应充分考虑变更因素及影响程度、设计制定完整的“可比性研究方案”, 结合产品质量历史数据预设合理验收标准, 通常限度要求应严于放行标准, 如: palivizumab临床期间中试生产规模为200 L, 商业化生产需放大规模(500 L、10 000 L)至不同产地, 药学可比性研究通过对变更后的产品质量分析, 支持工程细胞的遗传稳定性时间超出历史生产代次^[32]; 审评实践中, 国内某企业临床期间进行多项工艺变更。经药学审评, 建议申请人进行生产终末期细胞检定、开展工程细胞稳定性研究, 以支持培养基改变及发酵规模的放大; 建议对纯化工艺病毒去除/灭活能力进行再评估, 以支持纯化工序的改变。

生产工艺变更后, 若药学评价存在明显质量差异或不足以证明其具有可比性, 应进一步结合非临床及临床试验(药代、免疫原性、药效学等)数据进行综合分析。如: moxetumomabpasudotox生产细胞、发酵与纯化工艺、制剂处方等改变; siltuximab细胞系改变(SP2/0到CHO); canakinumab剂型的改变(冻干制剂到预充针); 曲妥珠给药装置的改变(手动注射剂到自动装置), 上述变更均通过药代或生物等效性试验证明产品质量可比或桥接临床试验结果^{[31, 33]}。

2.3 继续完善《注册质量标准》与《制造与检定规程》

拟定《注册质量标准》与《制造与检定规程》是生物制品进行全程质量控制的基本要求, 也是为后期进行生产现场检查和上市后监管提供技术文件依据。单抗药物注册生产时, 应结合现行工艺及质控策略, 按照《生物制品生产工艺信息登记模板》及药典中相关品种体例, 对原液、制剂的检定方法与生产工艺进行详细描述。

检定方法描述应包括基本原理和范围、仪器设备、操作流程和参数、试剂(来源、级别、贮存条件等)、供试品和内控品制备、系统适用性、结果分析、验收标准和典型图谱(如适用性)等; 质量标准的限度范围应结合临床和商业化生产工艺批次的统计分析结果、生产工艺性能能力、分析方法的变异性, 以及原液和成品长期放置过程中可能的降解情形、质量标准的收紧的情况(如有)合理拟定。

工艺规程应重点明确关键工艺参数及控制范围, 如:培养工序中温度、pH值、溶氧、培养结束(或废弃)标准、最大培养时间(限传代次)、内外源因子控制(微生物负荷、支原体、外源病毒)等参数; 纯化工序中说明所用色谱填料来源、层析柱高度、载量、使用寿命和吸收峰条件等; 制剂工序中说明浓缩液与稀释液配比或辅料混合顺序、过滤后膜的完整性测试等信息。目前, 国内尚未引入“返工” (re-process)的概念, 上述情况应严格限定于除菌过滤工序。审评实践中, 国外单抗药物商业化生产中普遍存在多批原液混合灌装制剂的情况。上述“混批”工艺可提高生产和市场供应的灵活性, 但同时也对终产品的质量控制提出了挑战。因此, 应在工艺规程中对“混批”工序进行严格限定, 除“混批”前后原液与制剂应符合放行标准外, “混批”工序的工艺参数(搅拌速度、搅拌时间)、产品均质性(蛋白浓度等)应进行充分验证, 确保“混批”产品的质量一致性及数据可溯源性。此外, 由于我国自主研发单抗现存生产规模与产品市场容量存在较大差距, 若申请人采用多产地、多线生产等供应策略, 应在拟定规程中对不同产地的生产线(或工艺)进行分别描述。

3 国内企业注册上市阶段药学研究的现存问题

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笔者在审评实践中通过比对国内外申报资料发现, 国内研发者由于经验不足或临床进程时间所限, 以及业界的竞争压力和市场抢占考虑等, 在产品注册生产申请时经常出现以下问题:产品特异性工艺开发及质量研究不足, 工艺验证不充分, 研发型企业缺乏生产现场检查准备经验, 产品上市后工艺变更策略过于激进等。

3.1 产品特异性的工艺开发与质量研究不充分

目前, 国外已经开始应用“质量源于设计” (quality by design, QbD)理念指导产品工艺开发与质量研究, 其中罗氏公司多个基于此理念研发的抗体药物(obinutuzumab、atezolizumab等)已经被FDA批准上市^[34-36]。QbD理念首先结合临床作用机制和相关先进经验确定产品的关键质量属性(critical quality attribute)^[37]。采用规模缩小模型对生产工艺进行充分特性研究, 依据对产品质量的影响程度确定关键控制参数及其范围。并在此基础上引入“设计空间” (design space definition)概念(即:设计空间内的工艺参数变化不属于工艺变更范畴)^[38]。如:采用Spintube^[39]或小型生物反应器^[40]作为规模缩小模型, 对细胞培养工艺参数充分优化后在商业化生产工艺规模进行放大。而国内企业目前在临床试验期间普遍缺乏工艺特性研究, 工艺验证停留在商业化工艺的“性能确认”层面。缺乏对于生产工艺的完整认识和控制, 往往会导致工艺放大后稳健性不足。审评实践中, 国内某单抗药物在采用商业化规模工艺验证时发现, 发酵规模放大后未处理原液中溶氧不足, 导致二硫键无法正确组装、中间体非还原电泳纯度检查项目不合格。

在质量研究方面, 国内自主研发的抗体药物与进口注册品种间也存在着显著的差距。由于单抗药物质控策略与检测方法相对成熟, 国内企业对产品相关杂质的研究不充分, 直接参考相关品种检查项目建立产品放行标准, 审评实践中, 国外某进口单抗药物在其表征研究中对含量大于0.1%杂质、稳定性研究(加速、强制降解等)中形成的杂质成分均进行了鉴定与测活。放行标准中同时采用SEC/IEX/HIC等色谱手段对主成分和多种变体形式进行限度控制, 尤其是HIC法可对影响生物活性的氧化峰实现控制; 国内同类品种受杂质富集手段和分析方法所限, 仅基于历史批次放行质量数据、稳定性(货架期末)研究结果来设立标准限度, 也未将可表征工艺稳定性的糖型修饰纳入质量控制; 此外, 目前国内企业已上市的所有单抗药物, 其宿主蛋白残留测定方法基本上采用外购商业化试剂盒检定, 工艺特异性分析方法开发尚未完成。另外, 国内企业在方法验证上也存在不全面不规范的问题, 如用于过程中控制的检测方法未进行确认或验证; 色谱类方法验证时仅考察主峰, 而未对杂质峰的专属性、准确性、精密性、范围和线性等进行验证; 定量限和检测限仅是采用信噪比的方式制定, 没有充分依据; 未考察方法的稳定性指示作用等。审评实践中, 某国内品种生物学活性测定方法由于优化验证不充分, 生产现场动态核查3批中1批成品企业放行检验不符合质量标准, 复验后合格。此外, 某国内品种的非还原CE方法前期未关注用十二烷基苯硫酸钠(SDS)缓冲液稀释样品到加入碘乙酰胺的时间间隔会影响检测结果, 导致稳定性考察数据波动, 给稳定性趋势分析带来了一定的不确定性。

强制降解实验是在较长期或加速稳定性更为剧烈的条件下进行, 如:高温(35 ℃)、极端pH值、氧化(过氧化氢)和糖化(葡萄糖)等。该方法在进口注册抗体的产品相关杂质制备、降解途径分析、工艺可比性研究等方面应用广泛^[41]。如:国外某进口注册单抗强制降解实验数据提示, 序列中存在多个脱酰胺降解位点。因此其拟定的注册标准中采用反相法对相关杂质进行控制。但是, 目前国内研发的抗体药物基本上均未开展强制降解实验研究。

以上工艺特性和质量研究不足又导致了对工艺理解和产品认知欠缺, 带来后续工艺验证的盲从性。如国内企业上市申报阶段普遍采用3批进行工艺验证, 没有经过对工艺和产品、控制策略全面的风险评估确定合理的验证批次, 而国外企业根据风险等级确定进行3批甚至10批的工艺验证, 为保障工艺重现性和商品化产品质量提供充分依据。

3.2 生产现场检查所暴露的问题

国外的药品上市前现场检查(pre-approved inspection, PAI)主要基于系统风险(system-based)和风险管理(risk-management)评估, 核查过程中可结合共线生产的其他产品进行检查, 一般不要求上市产品的注册检验。整个PAI过程中重点对“六大系统” (质量系统、厂房与设备系统、原材料系统、生产系统、包装与标签系统、实验室控制系统)和“三大元素” (操作规程、人员培训与记录)现场检查^[42]。

在我国, 药审中心对注册生产申请的抗体药物完成技术评价后, 由审核查验中心组织专职检查人员、产品专家和工艺专家共同进行生产现场检查, 同时抽取动态检查批次的原液与制剂产品交中国食品药品检定研究院进行注册检验与标准复核。整个PAI过程中, 通常围绕着生产工艺与申报工艺一致性、生产系统、质量管理系统、实验室控制系统、文件管理和物料系统等开展检查。由于目前生产注册的抗体药物多为研发型企业开发, 申请人对于上市药品的生产质量管理体系缺乏实践经验。结合生产现场检查实践, 建议申请人重点关注以下方面: ①加强产品共线生产的风险评估, 有效防止共线生产的产品间交叉污染; ②加强生产工艺的微生物限度控制(环境、原材料、工艺过程等), 尤其是制剂生产工序的无菌保障; ③对于在发酵培养、缓冲液配制和原液贮存等使用的一次性耗材, 应结合工艺实际进行风险评估后开展相容性研究, 提供浸出物/提取物研究报告^{[43, 44]}。

3.3 产品上市后工艺变更策略过于激进

一般单抗药物上市后, 为了扩大产能、降低成本还会发生较为频繁的工艺变更(平均15次)。如: infliximab上市后已经发生了50次风险等级不同的工艺变更。根据工艺变更风险等级的不同, 美国FDA分别按照prior approval supplement (PAS)、CBE-0/CBE-30和年度报告(annual report)等进行管理^[45]。审评实践中, 经常遇到国内研发型申请人, 在没有充分设计考虑和全面比较研究的前提下, 匆忙进行变更并提交初步的研究结果支持上市阶段的同期变更。这样带来的风险高及可控性差, 建议申请人结合目前上市单抗工艺变更的实际内容(优化培养基、扩大规模、新增生产线等), 参照《生物制品上市后变更研究的技术指导原则》及ICH Q5E进行风险评估后开展充分的可比性研究, 必要时按照补充申请进行注册申报。

根据国内外上市抗体的工艺变更经验, 上游培养工艺的变更(培养基改变、培养规模放大)容易造成产品的电荷异质性、糖基化修饰等质量属性的改变; 下游纯化工艺的变更(碟片离心机代替切向流过滤)则多影响改变工艺相关杂质残留及去除效果。对于纯化工艺变更后病毒灭活是否需要重新验证, 应结合病毒灭活工艺关键参数及样品性质是否发生改变进行判定^[46]。审评实践中, 国内某融合蛋白药物上市后, 通过培养基优化及补料工艺改进, 其细胞密度与发酵产率提高多倍。药学审评中重点关注细胞密度提高后, 纯化工艺对工艺相关杂质(宿主蛋白、宿主DNA等)的去除能力, 以及培养基条件的改变对本品糖型修饰、生物活性的影响。

特别需要指出的是, 近年部分单抗生物类似药为加快产品上市进程, 采用较小的工艺规模(500 L)进行注册申报。产品上市后为应对市场供应短缺, 短时间内提出工艺变更扩大产能。此种注册申报策略不符合上市产品工艺变更一般规律。一方面, 生物类似药的商业化生产工艺应在临床试验早期进行锁定; 另一方面, 产品上市后短时内发生变更, 缺乏商业化产品质量数据的批分析, 较难预设基于自身产品生命周期的可比性验收标准。审评实践中, 某单抗生物类似药上市后不足半年, 为提高产能申请多项工艺变更事项。药学审评中, 除关注细胞限传代次提高后生产终末期细胞检定、纯化工序改变后的病毒去除/灭活再验证外, 由于缺乏足够批次的商业化产品质量分析数据, 其工艺变更前后的质量可比性评价面临很大挑战^[47]。

4 结语

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目前, 我国抗体生物类似药及PD-1单抗上市已极大程度地降低了原研药在国内的售价, 显著提高了上述药物在国内的用药可及性(表 3)。但是, 与国外产业发展现状相比仍然差距明显。2018年全球市场销售额前10的药物中单抗药物占据8种。如infliximab自1998年产品上市以来, 已经累计生产了近1.5亿支制剂, 满足了世界上将近300万患者的临床需求。在长期商业化生产过程中, 企业建立贯穿产品生产至临床应用全过程的质量管理体系, 其中涉及原料采购(16~24周)、细胞复苏与发酵(16~20周)、纯化和原液放行(8~9周)、冻干与制剂检定(5~7周)、包装与最终放行(8~9周)、供应链运输(4~6周)等环节, 总计共250个检测项目。此外, infliximab上市后欧美监管方先后批准了本品14次工艺变更, 最大程度地满足了产品的商业化供应^[48]。与国外已上市“重磅炸弹”级单抗药物相比, 一方面, 我国上市的单抗药物仍然存在产能规模受限、市场容量尚在培育等明显差距。更为重要的是, 研发型企业对于上市产品的生产质量管理理念不够重视、管理经验不足; 另一方面, 监管方对于产品上市后监管能力也有待加强。在此情况下, 国内药品审评机构如何借鉴欧美发达国家对于上市药物的监管经验, 结合国内行业发展现状与临床用药需求展开技术评价, 将是近期生物制品注册上市审评的一个重要课题。

基金

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国家“重大新药创新”资助项目(2015ZX09501008)

参考文献

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文献

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2019年第54卷第11期

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doi: 10.16438/j.0513-4870.2019-0534

接收时间：2019-07-08
首发时间：2026-01-26
出版时间：2019-11-12

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出版历史

收稿日期：2019-07-08
修回日期：2019-08-07

基金

国家“重大新药创新”资助项目(2015ZX09501008)

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:

*罗建辉, Tel: 86-10-68586655, E-mail: luojh@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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TxT

Drug name	Sponsor	Approval data or status
Recombinant human tumor necrosis factor receptor-immunoglobulin fusion protein for injection	CP Guojian Pharm Celgen Biopharma Zhejiang Hisuan pharmaceutical Co., Ltd	2005 2011 2012
Iodine-~(131)-labeled tumor necrosis therapy chimeric antibody	Shanghai Medipharm Biotech Pharmaceutical	2006
Iodine[¹³¹I]metuximab injection	Chengdu huasuan group inc., Ltd	2006
Nimotuzumab injection	Biotech pharma Co., Ltd	2008
Recombinant humanized anti-CD25 monoclonal antibody injection	CP Guojian Pharm	2011
Conbercept ophthalmic injection	Kanghong Pharmaceutical	2013
Toripalimab injection	TopAlliance Bioscience Inc.	2018
Sintilimab injection	Innovent Biologics, Inc	2018
Rituximab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd/ Innovent Biologics, Inc	2019 Under review
Camrelizumab	Shanghai hengrui pharmaceutical co., Ltd	2019
Adalimumab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd Innovent Biologics, Inc Zhejiang Hisuan pharmaceutical Co., Ltd Sinoasis Pharmaceuticals, Inc	Under review
Recombinant anti-VEGF humanized monoclonal antibody injection	Qilu Pharmaceutical	Under review
Bevacizumab biosimilar	Innovent Biologics, Inc	Under review
Recombinant anti-HER2 humanized monoclonal a ntibody for injection	Sansheng Guojian Pharmaceutical (Shanghai) Co., Ltd	Under review
Tirelizumab injection	BeiGene, Ltd	Under review
Trastuzumab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd	Under review

Drug name

Sponsor

Approval data or status

Recombinant human tumor necrosis factor receptor-immunoglobulin fusion protein for injection

CP Guojian
Pharm Celgen Biopharma
Zhejiang Hisuan pharmaceutical Co., Ltd

2005
2011
2012

Iodine-~(131)-labeled tumor necrosis therapy chimeric antibody

Shanghai Medipharm Biotech Pharmaceutical

2006

Iodine[¹³¹I]metuximab injection

Chengdu huasuan group inc., Ltd

2006

Nimotuzumab injection

Biotech pharma Co., Ltd

2008

Recombinant humanized anti-CD25 monoclonal antibody injection

CP Guojian Pharm

2011

Conbercept ophthalmic injection

Kanghong Pharmaceutical

2013

Toripalimab injection

TopAlliance Bioscience Inc.

2018

Sintilimab injection

Innovent Biologics, Inc

2018

Rituximab biosimilar

Shanghai Henlius Biopharmaceutical Co., Ltd/
Innovent Biologics, Inc

2019
Under review

Camrelizumab

Shanghai hengrui pharmaceutical co., Ltd

2019

Adalimumab biosimilar

Shanghai Henlius Biopharmaceutical Co., Ltd Innovent Biologics, Inc
Zhejiang Hisuan pharmaceutical Co., Ltd
Sinoasis Pharmaceuticals, Inc

Under review

Recombinant anti-VEGF humanized monoclonal antibody injection

Qilu Pharmaceutical

Under review

Bevacizumab biosimilar

Innovent Biologics, Inc

Under review

Recombinant anti-HER2 humanized monoclonal a ntibody for injection

Sansheng Guojian Pharmaceutical (Shanghai) Co., Ltd

Under review

Tirelizumab injection

BeiGene, Ltd

Under review

Trastuzumab biosimilar

Shanghai Henlius Biopharmaceutical Co., Ltd

Under review

Variant type	Example	Potential impact	Quality control strategy (test assay)
Size related variants	Aggregates	Increased immunogenicity	Extend characterization (SEC-HPLC, AUC) or in release test (SEC-HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-CE)
	Fragments	Maybe has no potency
Charge related variants	Acid variant (sialylation, deamidation, C-terminal lysine cleavage, high mannose, thiosulfate, glycation, etc.)	Modification in N/C terminal region have no substantial effects on antibody structure, stability and function; modification in CDR regions have affect on potency and stability	Extend characterization (isoelectric focusing, capillary isoelectric focusing, ion exchange chromatography (IEX, LC-MS) or routine release test (IEX, HIC)
	Main species (cyclization of N-terminal Gln to pycoGlu, removal of the heavy chain lysine, glycosylation of Asn297)
	Basic variant (C-terminal lysine, N-terminal Glu, isomerization of Asp, succinimide, methionine oxidation, amidation, aglycosylation, fragments, aggregation)
Sequence related variants	N-Terminal leads sequence	Low risk of safety and effect	Extend characterization (LC-MS) or routine release test (peptide mapping)
Amino acid mis-incorporation
	N-Terminal modification (acetylation, formylation and pyroglutamylation)	Increased charge heterogeneity without impacting safety and efficacy	Extend characterization (LC-MS, reverse phase high-performance liquid chromatography) or in release test (IEX)
	C-Terminal modification (clipping of C-terminal Lys, prolineamidation)
Cysteine-related variants	Free cysteine	Decreased antibody thermal stability and trigger formation of covalent aggregates	Extend characterization (LC-MS, Ellman kit)
	Disulfide isoforms (IgG2A/B, IgG half antibody)	May impact potency and specificity of antibody	Extend characterization (LC-MS)
	Trisulfide bond	Low risk of safety and effect
	Thioether, D-cysteine, cysteinylation
Glycosylation	N-Glycolylneuraminic acid, α-1, 3 Gal	Increased immunogenicity	Extend characterization (LC-MS) and/or routine release test (hydrophilic interaction chromatography, HIC)
	Afucosylation	Increased ADCC effect
	Galatosylation	Increased CDC effect
	High mannose	Decreased half-life time in vivo
	Glycation	Generation of acidic species
Other post-translational modification variants	Asndeamidation, Asp isomerization, Met/Trp oxidation in CDR region	Decreased affinity and immunogenicity

Variant type

Example

Potential impact

Quality control strategy (test assay)

Size related variants

Aggregates

Increased immunogenicity

Extend characterization (SEC-HPLC, AUC) or in release test (SEC-HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-CE)

Fragments

Maybe has no potency

Charge related variants

Acid variant (sialylation, deamidation, C-terminal lysine cleavage, high mannose, thiosulfate, glycation, etc.)

Modification in N/C terminal region have no substantial effects on antibody structure, stability and function; modification in CDR regions have affect on potency and stability

Extend characterization (isoelectric focusing, capillary isoelectric focusing, ion exchange chromatography (IEX, LC-MS) or routine release test (IEX, HIC)

Main species (cyclization of N-terminal Gln to pycoGlu, removal of the heavy chain lysine, glycosylation of Asn297)

Basic variant (C-terminal lysine, N-terminal Glu, isomerization of Asp, succinimide, methionine oxidation, amidation, aglycosylation, fragments, aggregation)

Sequence related variants

N-Terminal leads sequence

Low risk of safety and effect

Extend characterization (LC-MS) or routine release test (peptide mapping)

Amino acid mis-incorporation

N-Terminal modification (acetylation, formylation and pyroglutamylation)

Increased charge heterogeneity without impacting safety and efficacy

Extend characterization (LC-MS, reverse phase high-performance liquid chromatography) or in release test (IEX)

C-Terminal modification (clipping of C-terminal Lys, prolineamidation)

Cysteine-related variants

Free cysteine

Decreased antibody thermal stability and trigger formation of covalent aggregates

Extend characterization (LC-MS, Ellman kit)

Disulfide isoforms (IgG2A/B, IgG half antibody)

May impact potency and specificity of antibody

Extend characterization (LC-MS)

Trisulfide bond

Low risk of safety and effect

Thioether, D-cysteine, cysteinylation

Glycosylation

N-Glycolylneuraminic acid, α-1, 3 Gal

Increased immunogenicity

Extend characterization (LC-MS) and/or routine release test (hydrophilic interaction chromatography, HIC)

Afucosylation

Increased ADCC effect

Galatosylation

Increased CDC effect

High mannose

Decreased half-life time in vivo

Glycation

Generation of acidic species

Other post-translational modification variants

Asndeamidation, Asp isomerization, Met/Trp oxidation in CDR region

Decreased affinity and immunogenicity

Drug name	Sponsor	Approved indication in China	Dose/price (RMB)
Nivolumab	Bristol-Myers Squibb	Advanced non-small cell lung cancer	100 mg/9 260
Pembrolizumab	Merck & Co Inc	Metastatic malignant melanoma	100 mg/17 918
Toripalimab	Shanghai Junshi Bioscience	Melanoma	240 mg/7 200
Sintilimab	Innovent Biologics Co. Ltd	Refractory classic Hodgkin's lymphoma	100 mg/7 839
Camrelizumab	Jiangsu Hengrui Medicine	Advanced solid tumor	100 mg/7 838
Rituximab	Roche	Non-hodgkin lymphoma	100 mg/2 418
Rituximab biosimilar	Shanghai Henlius Biopharmaceutical Co., Ltd	Non-hodgkin lymphoma; follicular lymphoma; diffuse large B-cell lymphma	100 mg/1 648

Drug name

Sponsor

Approved indication in China

Dose/price (RMB)

Nivolumab

Bristol-Myers Squibb

Advanced non-small cell lung cancer

100 mg/9 260

Pembrolizumab

Merck & Co Inc

Metastatic malignant melanoma

100 mg/17 918

Toripalimab

Shanghai Junshi Bioscience

Melanoma

240 mg/7 200

Sintilimab

Innovent Biologics Co. Ltd

Refractory classic Hodgkin's lymphoma

100 mg/7 839

Camrelizumab

Jiangsu Hengrui Medicine

Advanced solid tumor

100 mg/7 838

Rituximab

Roche

Non-hodgkin lymphoma

100 mg/2 418

Rituximab biosimilar

Shanghai Henlius Biopharmaceutical Co., Ltd

Non-hodgkin lymphoma; follicular lymphoma; diffuse large B-cell lymphma

100 mg/1 648