Guideline on the Development and Manufacture of Oligonucleotides 寡核苷酸药物开发和生产指南

Executive summary 执行摘要

This guideline addresses specific aspects regarding the manufacturing process, characterisation, specifications and analytical control for synthetic oligonucleotides which are not covered in the Guideline on the Chemistry of Active Substances (EMA/454576/2016) or Chemistry of Active Substances for Veterinary Medicinal Products (EMA/CVMP/QWP/707366/2017). It also contains requirements and considerations related to conjugation, to active substance in solution, to medicinal product development, to oligonucleotide generics development, to oligonucleotide personalized medicine approaches and to clinical trial applications (human products only).

本指南针对合成寡核苷酸的制造过程、表征、规格和分析控制提出了具体要求，这些内容未在《活性物质化学指南》(EMA/454576/2016)或《兽药活性物质化学指南》(EMA/CVMP/QWP/707366/2017)中涵盖。该指南还涉及与偶联、溶液形式的活性物质、药物开发、寡核苷酸仿制药开发、个性化寡核苷酸药物方法以及临床试验申请（仅限人用产品）相关的要求和考虑。

1.Introduction 引言

This guideline has been prepared in accordance with the structure agreed for the quality part of the  dossier for human medicinal products (Format ICH-CTD). The subheadings have been included for the sake of clarity.

本指南按照人用药品质量部分档案的结构（格式 ICH-CTD）编写。为了清晰起见，包含了各个小标题。

2. Scope 范围

The purpose of this guideline is to set out the type of information required for the development, manufacture and control of synthetic oligonucleotides (existing or new chemical entities) used in a medicinal product, in the context of obtaining a marketing authorisation. There is also a chapter on the requirements for clinical trial applications.

本指南的目的是明确在获得药品上市许可时，关于合成寡核苷酸（包括现有和新化学实体）的开发、制造和控制所需的信息类型。此外，指南中还包含了一章，详细说明了临床试验申请的要求。

Synthetic oligonucleotides are fully or partially excluded from the scope of ICH Q3A/B (VICH 73 GL10/GL11), ICH Q6A/B (VICH GL39/GL40) and ICH M7 (EMA/CVMP/SWP/377245/2016). This guideline addresses those specific aspects regarding the manufacturing process, characterisation, specifications and analytical control for synthetic oligonucleotides which are not covered in the Guideline on the Chemistry of Active Substances (EMA/454576/2016) and Chemistry of Active Substances for Veterinary Medicinal Products (EMA/CVMP/QWP/707366/2017), and is to be considered complementary to the latter guidelines. mRNA entities are out of scope of this guideline.

合成寡核苷酸完全或部分排除在 ICH Q3A/B（VICH GL10/GL11）、ICH Q6A/B（VICH GL39/GL40）和 ICH M7（EMA/CVMP/SWP/377245/2016）的适用范围之外。本指南补充了活性物质化学指南（EMA/454576/2016）和兽医药品活性物质化学指南（EMA/CVMP/QWP/707366/2017）中未涉及的关于制造过程、表征、规格和分析控制的具体方面。mRNA 实体不在本指南的范围内。

Whilst veterinary products are outside the scope of ICH guidelines there are no corresponding VICH guidelines on certain topics. Nevertheless, the principles outlined in these ICH guidelines may also be relevant to veterinary products to facilitate flexibility and to allow the applicant the option of using different approaches to product development.

尽管兽医产品不在 ICH 指南的适用范围内，但在某些领域没有相应的 VICH 指南。然而，ICH 指南中提出的原则也可能适用于兽医产品，以提供灵活性并允许申请人在产品开发中选择不同的方法。

For the veterinary products, the reference to GMP Eudralex Volume 4 will eventually be superseded by respective Implementing Acts, when they come into force.

对于兽医产品，当相关实施法生效时，将不再引用 GMP Eudralex 第 4 卷。

3. Legal basis and relevant guidelines 法律基础及相关指南 /

This guideline should be read in conjunction with the introduction and general principles of Annex I to Directive 2001/83/EC as amended for human medicinal products and Annex II of Regulation (EU) 2019/6 as amended for veterinary medicinal products, and all other relevant EU and (V)ICH guidelines. These include, but are not limited to:

本指南应结合《指令 2001/83/EC》附件 I 中修订版关于人类药品的介绍和一般原则，以及《法规（EU）2019/6》附件 II 中修订版关于兽医药品的介绍和一般原则，同时参考所有其他相关的 EU 和(V)ICH 指南一同阅读。这些指南包括但不限于

•            Guideline on the Chemistry of Active Substances EMA/454576/2016 and Chemistry of Active Substances for Veterinary Medicinal Products (EMA/CVMP/QWP/707366/2017)

•            EU GMP guide Part II: Basic Requirements for Active Substances used as Starting Materials

•            EU GMP guide Part II, Q&A 12 on GMP requirements to be applied for the formulation of  biological active substances with excipients, when described in the active substance section of a registration dossier

•            ICH Q1 A-F Stability testing of new drug substances and drug products – Scientific guidelines (veterinary VICH GL3-5, GL45, GL51 and GL58)

•            ICH Q2 Guideline on validation of analytical procedures (veterinary VICH GL1 and GL2)

•            ICH Q3A Impurities in new drug substances CPMP/ICH/2737/99 (veterinary VICH GL10)

•            ICH Q3B Impurities in new drug products CPMP/ICH/2738/99 (veterinary VICH GL11)

•            ICH Q3C Guideline for residual solvents EMA/CHMP/ICH/82260/2006 (veterinary VICH GL18)

•            ICH Q3D Elemental impurities EMA/CHMP/ICH/353369/2013 (veterinary Reflection paper EMA/CVMP/QWP/153641/2018)

•            ICH Q6A Specifications: Test Procedure and Acceptance Criteria for New Drug Substances and New Drug Products – Chemical Substances CPMP/ICH/367/96 (veterinary VICH GL39)

•            ICH Q8 Pharmaceutical development – scientific guideline EMA/CHMP/ICH/167068/2004

•            ICH Q9 Quality risk management EMA/CHMP/ICH/24235/2006

•            ICH Q11 Guideline on development and manufacture of drug substances (chemical entities and biotechnological/ biological entities) EMA/CHMP/ICH/425213/2011

•            ICH Q13 Continuous manufacturing of drug substances and drug products EMA/CHMP/ICH/427817/2021

•            ICH M7 Guideline on assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk EMA/CHMP/ICH/83812/2013 (veterinary EMA/CVMP/SWP/377245/2016)

•            Investigation of Chiral Active Substances 3CC29a for human products, EMEA/CVMP/128/95 for the veterinary products

•            Reflection paper on statistical methodology for the comparative assessment of quality attributes in drug development - EMA/CHMP/138502/2017

•            CHMP SWP reflection paper on the assessment of the genotoxic potential of antisense oligodeoxynucleotides (EMEA/CHMP/SWP/199726/2004)

•            Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container (EMA/CHMP/CVMP/QWP/850374/2015)

•            Guideline on the requirements to the chemical and pharmaceutical quality documentation concerning investigational medicinal products in clinical trials - EMA/CHMP/QWP/545525/2017

4. Active Substance 原料药

4.1. General Information 基本信息 3.2.S.1

4.1.1. Nomenclature 药品名称3.2.S.1.1

No additional requirements.

无额外要求

4.1.2. Structure 结构 3.2.S.1.2

Letter codes may be used for the primary structure of the active substance, the used codes should  include the nucleobases, the sugars (or morpholinos), and any substitutions thereof, and should reflect the nature of the linkages between sequential nucleosides (e.g. phosphodiester, phosphorothioate diester, thiophosphoramidate, phosphorodiamidate linkages). A legend should accompany the structure. For oligonucleotide chains, the 5`-terminus and the 3`-terminus of the chain should be clearly indicated. For small interfering RNA (siRNA), the structure of sense- and antisense strands should be provided, and the place of hybridisation of the complementary nucleotides of the sense and antisense strands should be indicated, as well as any un-hybridised overhangs in any of the strands. Counter-ions should be indicated. Molecular formula and molecular mass of the active substance, and for siRNA also the sense- and antisense strands are to be provided. If relevant, the secondary and tertiary structure (e.g. in case of hairpin loops or aptamers) should be visualised.

可以使用字母代码表示活性物质的一级结构，使用的代码应包括核碱基、糖（或吗啉代）及其任何取代基，并应反映连续核苷之间的连接性质（例如磷酸二酯、硫代磷酸二酯、硫代磷酰胺、磷酰二胺连接）。结构图应附有图例说明。对于寡核苷酸链，应明确标示链的 5'端和 3'端。对于小干扰 RNA（siRNA），应提供正义链和反义链的结构，标明正义链和反义链互补核苷酸的杂交位置以及任何链上未杂交的突出端。应标明抗衡离子。需提供活性物质的分子式和分子质量，对于 siRNA，还应提供正义链和反义链的分子式和分子质量。如有需要，还应展示二级和三级结构（例如发夹环或适配体的情况下）。

Full chemical structure of side chains and linkers is expected.

需要提供侧链和连接子的完整化学结构。

4.1.3. General Properties 基本性质3.2.S.1.3

Relevant general properties of the oligonucleotide in question should be listed.

应列出相关寡核苷酸的一般性质。

In most cases, for active substances presented as powder, appearance, water content, pH of a solution of the oligonucleotide, molar absorptivity, and solubility in different media would be expected. Hygroscopicity needs to be indicated, e.g. with moisture sorption isotherms, or reference could be made to 3.2.S.3.1 where such information could be provided in more detail. Polymorphic form is generally not applicable, since most oligonucleotide powders are amorphous. Melting point is relevant for certain oligonucleotides e.g. siRNAs, aptamers.

对于以粉末形式存在的活性物质，通常需要提供外观、水分含量、寡核苷酸溶液的 pH 值、摩尔吸光度以及在不同介质中的溶解度。需标明吸湿性，例如通过水分吸附等温线，或者在 3.2.S.3.1 中更详细地提供此类信息。由于大多数寡核苷酸粉末为无定形状态，多晶形式通常不适用。熔点对于某些寡核苷酸（如 siRNA、适配体）是相关的。

For active substances in solution, the composition of the formulation and the pH are expected.

对于溶液形式的活性物质，需要提供其成分和 pH 值。

The counter ion needs to be indicated, if relevant, and whether it is present in a stoichiometric or non stoichiometric ratio.

如有必要，应明确说明所使用的抗衡离子及其与药物成分的比例关系（化学计量比或非化学计量比）。

The stereochemistry of the nucleosides and of the phosphorothioate diester linkages needs to be discussed in this section (alternatively reference can be made to 3.2.S.3.1). If there are stereocenters without absolute configuration, i.e. if the active substance is a mixture of diastereoisomers, this should be mentioned. The control strategy to ensure consistency of stereochemistry (stereochemical purity of starting materials, coupling reagent and conditions, etc) needs to be explained.

本部分需详细阐述核苷和硫代磷酸二酯键的立体化学特性（也可参考 3.2.S.3.1）。若药物成分中存在无绝对构型的立体中心，即为非对映异构体的混合物，则需进行明确说明。同时，还需阐述确保药物成分立体化学一致性的具体控制策略，包括原料、偶联试剂和反应条件等方面的考虑。

4.2. Manufacture 生产 3.2.S.2

4.2.1. Manufacturer(s) 生产商 3.2.S.2.1

No additional requirements.

无额外需求。

4.2.2. Description of Manufacturing Process and Process Controls 生产工艺和工艺控制 3.2.S.2.2

The chemical synthesis of a therapeutic oligonucleotide is typically carried out on a functionalised solid support using an automated synthesiser. In addition to solid-phase synthesis, alternative synthesis  methods (e.g., liquid synthesis, enzymatic synthesis) could be used. However, at the time the guideline was written these methods were considered too premature to be included.

治疗性寡核苷酸通常通过自动化合成仪在功能化的固体载体上进行化学合成。除了固相合成法外，还可以采用液相合成和酶促合成等其他方法，但由于这些方法尚不成熟，本指南暂未涉及。

Irrespective of the manufacturing strategy, the dossier should describe relevant process characteristics and equipment in adequate detail.

无论选择何种生产方式，申报资料都应详细说明具体的工艺参数和使用的设备。

Schematic representation of the manufacturing process 生产工艺流程图

For the graphical presentations of the synthetic process(es) for oligonucleotides, it is considered acceptable to replace certain chemical structures with-letter codes in the reaction schemes to improve legibility. Letter codes should be accompanied with a legend.

为了提高可读性，可以在表示寡核苷酸合成过程的示意图中使用字母代码代替部分化学结构。这些字母代码需附带相应的说明。

Sequential procedural narrative 连续操作描述

The sequential procedural narrative should describe each step in the manufacturing process. During oligonucleotide synthesis the same standardised steps are typically repeated cyclically, e.g. in Solid Phase Oligonucleotide Synthesis (SPOS) the oligonuclelotide sequence is built up on a solid support by repeated cycles of deprotection, coupling, oxidation/sulfurisation and capping steps. These standardised steps with their associated Proven Acceptable Ranges (PARs) need not be described in detail each time they are used, provided clear descriptions of the used general conditions (e.g. equivalents, reagents, solvents, reaction times, …) are given. The final cleavage and deprotection step should be described in detail, including any use of reagents, in case of which a discussion of their mutagenic potential should also be included in 3.2.S.3.2. Amounts can be described as weights/volumes or equivalents.

应逐一详细描述寡核苷酸生产过程中的各个步骤。在固相寡核苷酸合成过程中，通常会重复一系列标准化操作，如脱保护、偶联、氧化/硫化和封端等。对于这些标准化步骤，只需明确说明通用的反应条件（如反应物用量、溶剂、反应时间等），无需每次重复详细描述。合成过程的最终环节，即裂解和脱保护步骤，应详细说明所用试剂及其潜在的致突变性（详见 3.2.S.3.2）。物料的计量可以使用重量、体积或当量表示。

Splitting or combining of sub-batches/multiple cycles may be performed at different stages during manufacturing, e.g. based on equipment capacity or operational efficiency in SPOS. The quality criteria applied in the decision on splitting or pooling of sub-batches should be provided, along with an adequate justification for the selected approach. Moreover, material traceability from the synthesis steps through the final drug substance is expected and S.2.2 should contain an unambiguous definition of the commercial batch size (range). When continuous manufacturing approaches are intended, the requirements of ICH Q13 on the description of the manufacturing process should be considered.

生产过程中可根据设备能力或操作效率等因素，对中间产品进行拆分或合并处理。应明确说明用于决定是否拆分或合并的质量控制标准，并合理地解释选择的依据。此外，需确保从合成起始阶段到最终产品的可追溯性，并在 S.2.2 中明确定义商业化产品的批次规模。对于连续生产模式，应遵循 ICH Q13 的相关规定对生产过程进行详细描述。

Synthetic oligonucleotides are generally purified using chromatographic techniques, often starting from a relatively complex crude intermediate. It is recognised that the crude mixture typically contains pre- and post-, and often co-eluting, structurally related impurities. Detailed information on fraction collection, preparation of mock pools and all applied acceptance criteria during the purification steps should be provided. In the case that re-purification steps of side fractions is part of the established purification procedure, this should be addressed in the dossier as well. Appropriate measures to prevent cross-contamination due to the successive purification of different oligonucleotides using the same column should be in place, as required by GMP.

合成寡核苷酸通常需要经过色谱纯化，起始原料通常是较为复杂的粗品混合物。由于混合物中含有结构相似的杂质，如结构相关的前体和后体，并且经常共同洗脱，因此需详细说明馏分收集、模拟混合物的制备以及纯化过程中所采用的质量控制标准。如果需要对部分馏分进行二次纯化，也应在申报资料中有所体现。为避免使用同一色谱柱纯化不同寡核苷酸时产生交叉污染，应采取符合 GMP 要求的预防措施。

Annealing (for siRNA) 退火

The annealing process is performed to assemble the two complementary single strands (sense and antisense) into the drug substance duplex. Annealing conditions (e.g. buffer composition, time, temperature) should be specified, and volumetric ratio of the single strands should be optimised in order to minimise the unhybridised excess for any single strand. If any small-scale experiments are performed at batch level in view of optimisation of the volumetric ratio of the single strands, the approach should be explained, as well as the in-process control (IPC) non-denaturing method(s) used for measurement of duplex purity, with the applied limit for the residual single strand. The volumetric ratio used in the manufacturing process should be recorded, as well as the residual single strand excess after annealing and the duplex purity, measured as IPC.

退火是指将互补的正义链和反义链结合形成双链药物分子的过程。需明确退火条件（如缓冲液组成、时间、温度）并优化单链的比例，以尽量减少未结合的单链残留。如需通过小规模实验优化单链比例，应详细说明实验方法，并明确用于检测双链纯度的中控非变性方法及残留单链的允许限度。生产过程中使用的单链比例、退火后的单链残留量和双链纯度等均应作为中控参数详细记录。

Concentration step 浓缩步骤

If any concentration step (e.g. evaporation under vacuum, ultrafiltration) is in place, this needs to be described including the relevant process parameters and IPCs.

如生产过程中涉及浓缩步骤（如真空蒸发、超滤等），需详细说明操作参数和过程控制要点。

Lyophilisation 冻干

Lyophilisation of synthetic oligonucleotides is considered common practice. Lyophilisation process parameters should be described.

冻干是处理合成寡核苷酸的常规方法。应描述冻干工艺中的参数设置。

Oligonucleotide active substance in solution 溶液中的寡核苷酸原料药

See 4.9.

参见 4.9

Reprocessing, recovery and rework 再加工、回收和返工

The terms should be used and understood as defined in EU GMP Part II.

这些术语的定义和使用应遵循 EU GMP 第二部分的规定。

4.2.3. Control of Materials 物料控制 3.2.S.2.3

Active Substance (AS) Starting Material(s) 原料药起始物料

The considerations for selection and justification of starting materials outlined in ICH Q11 and its associated Q&A can be applied to synthetic oligonucleotides. The name and address of all starting material manufacturers should be provided. The addition of manufacturers for the starting materials needs to be approved by a variation according to European legislation. Information, in the form of flowcharts, indicating the synthetic process(es) of all starting materials including details of reagents, solvents and catalysts used, should be provided, followed by a criticality assessment of which starting material impurities may have an impact on the impurity profile of the oligonucleotide.

起始物料的选择和论证应遵循 ICH Q11 及其相关问答的规定。需提供所有起始物料制造商的名称和地址。增加新的起始物料制造商必须根据欧盟法规获得变更批准。应以流程图的形式详细说明各起始物料的合成过程，包括所用试剂、溶剂和催化剂等信息，并评估起始物料杂质对最终产品杂质谱的影响。

Starting materials from human or animal origin should, if possible, be avoided. If used, Ph. Eur. chapter 5.2.8 on ‘Minimizing the Risk of Transmitting Animal Spongiform Encephalopathy Agents via Medicinal Products,’ and the ‘Note for Guidance on Minimizing the Risk of Transmitting Animal Spongiform Encephalopathy Agents via Human and Veterinary Medicinal Products’ (EMA/410/01) should be considered, and transmissible spongiform encephalopathy (TSE) safety should be addressed.

应尽量避免使用动物或人源性起始原料。如必须使用，则需遵守欧盟药典 5.2.8 章《最小化通过药品传递动物海绵状脑病（TSE）病原体的风险》和 EMA/410/01 《最小化通过人用和兽药传递动物海绵状脑病（TSE）病原体的指导意见》的相关规定，并处理传染性海绵状脑病（TSE）的安全性问题。

Nucleoside phosphoramidites 亚磷酰胺

Protected nucleoside phosphoramidites (with protection of 5'-hydroxy group and heterocyclic base as relevant) are generally acceptable as starting materials in the manufacturing process of synthetic oligonucleotides. Nevertheless, a justification on the designation of starting materials needs to be provided.

通常将受保护的亚磷酰胺（5'-羟基和碱基部分受保护）作为合成寡核苷酸的起始原料，但需提供选择该原料的具体依据。

For more complex nucleotide derivatives carrying modifications in the phosphate, sugar or base moiety, more detailed information regarding their manufacture (e.g., precursors and used reagents) and impurity profile is required than for established building blocks such as 5’-dimethoxytrityl (5’-DMT) protected 2’-deoxyribose 2-cyanoethyl-N,N-diisopropylaminophosphoramidites.

对于结构更为复杂的核苷酸衍生物（磷酸基团、糖基或碱基部分带有修饰基团），除了常规质量控制指标外，还需详细说明其合成工艺（如前体物质、反应试剂等）和杂质情况。相比之下，对于常用的 5'-二甲氧基三苯甲基保护的 2'-脱氧核糖 2-氰乙基-N,N-二异丙基氨基磷酰胺等原料，只需进行常规质量控制即可。

Quality attributes for nucleoside phosphoramidites used as starting materials for synthetic oligonucleotide generally include: appearance, identification, assay, impurities, purity, water content and residual solvents. Identity, assay, and impurity profile are typically controlled using liquid chromatography with ultraviolet (UV) and/or mass spectrometry (MS) detection. In addition,³¹P nuclear magnetic resonance (NMR) spectroscopy may be used to assess the starting material purity. The critical impurities of the nucleoside phosphoramidites which can react like the parent compound during coupling and may accumulate in the final drug substance (e.g. 3’-DMT-5’-amidite isomer) should be adequately controlled and limited in the starting material specifications. The impurity profiles of the starting materials and their potential impact on the quality of the final drug substance should be investigated during manufacturing process development. This should include a fate and purge assessment of the impurities that may be formed downstream in the manufacturing process.

用于合成寡核苷酸的亚磷酰胺的质量属性一般包括：外观、鉴别、含量测定、杂质、纯度、水分和残留溶剂。通常通过液相色谱结合紫外（UV）和/或质谱（MS）检测来控制其鉴别、含量和杂质特征。此外，³¹P 核磁共振（NMR）光谱也可以用于评估起始材料的纯度。对于在合成过程中可能像母体化合物一样反应并可能在最终药物中积累的关键杂质（如 3’-DMT-5’-氨基化物异构体），需要在起始物料标准中加以控制和限制。在生产工艺开发阶段，需要研究起始物料的杂质谱及其对最终原料药质量的影响，包括对可能在生产过程中下游形成的杂质的去向和清除评估。

The solid support preloaded with the first nucleotide of the oligonucleotide sequence (through a linker) is also considered starting material. However, the unloaded solid support itself is not considered a starting material as it is not incorporated as a significant structural fragment into the structure of the drug substance.

连接有起始核苷酸的固体载体也可视为一种起始原料，但未连接核苷酸的固体载体则不在此列，因为它不作为药物结构中的重要组成部分。

For nucleoside loaded solid supports, quality attributes related to the purity and potential impurities of the loaded nucleoside are recommended. For the solid support itself, reference is made to section ‘Other materials used in the manufacturing process’.

对于核苷酸负载型固体载体，需关注负载核苷酸的纯度和杂质情况，而固体载体本身的相关信息可参考“生产过程用辅料”章节。

Non-nucleotide structural moieties 非核苷酸结构部分

Conjugation and other derivatisations of oligonucleotides are commonly used. In these cases, sometimes complex structures are added to the oligonucleotide sequence. The classification of these materials will be handled on a case-by-case basis and early interaction (scientific advice) with the regulatory agencies is recommended. However, also for starting materials of non-nucleotide structural moieties (e.g. poly(ethylene glycol) (PEG)-chains, N-acetyl galactosamine (GalNAc) moieties, fatty acids), compliance with the requirements as laid down in ICH Q11 and its associated Questions and answers is expected and its selection as starting material should be justified. For instance, sufficient subsequent chemical transformation steps after the starting material should be performed under good manufacturing practices (GMP).

寡核苷酸的偶联和其他衍生化方法是常见的。在这些过程中，可能会向寡核苷酸序列中添加复杂的结构。对于这些结构单元的分类需具体情况具体分析，建议提前与监管机构沟通（科学建议）。然而，即使是非核苷酸结构成分的起始材料（如聚乙烯醇（PEG）链、N-乙酰半乳糖胺（GalNAc）成分、脂肪酸），也需要遵循 ICH Q11 及其相关问答中的要求，并提供选择该原料的合理性说明。例如，在起始物料的基础上应进行充分的化学转化步骤，并遵循 GMP 规范。

Other materials used in the manufacturing process 生产过程中的其他物料

A list of all other reagents, such as solid support, solvents and chromatographic materials used in the manufacturing process of an oligonucleotide should be provided. Adequate specifications for all materials should be laid down considering their role in the process but covering as a minimum identity as well as purity and/or assay where applicable. For some specific reagents (e.g. acids used in the detritylation step) certain impurities have to be controlled to minimise the likeliness of the generation of product-related impurities. For materials used in the coupling steps residual moisture may be a critical attribute.

需列出寡核苷酸生产过程中所使用的全部辅助材料，包括固体载体、溶剂和色谱填料等。应根据这些材料在生产过程中的作用制定相应的质量控制标准，但至少应包括鉴别以及适用情况下的纯度或含量。对于某些特殊试剂（如脱保护步骤中的酸类），需严格控制可能导致产品相关杂质的杂质含量。偶联反应中使用的材料应严格控制水分含量。

The solid support is a key component of the SPOS process, controlled pore glass (CPG) and polystyrene resins are most commonly used. Typical quality attributes of the resin include: appearance, identification, mesh size and loading. For CPG also the pore size and for polystyrene cross-linking and swelling volume are typical quality attributes.

固体载体是固相合成法的关键材料，常用的载体包括控制孔径玻璃 (CPG) 和聚苯乙烯树脂。树脂的质量控制指标主要包括外观、成分、网孔大小和负载量等。对于 CPG，还需检查孔径；对于聚苯乙烯，则需关注交联度和膨胀体积。

4.2.4. Control of Critical Steps and Intermediates 关键步骤和中间体的控制 3.2.S.2.4

The criticality of the manufacturing steps for oligonucleotides made by solid phase synthesis should be evaluated during development according to the principles described in ICH Q9-Q11. In-process controls should be defined. The control of critical steps can be achieved by a combination of analytical tests and process controls. During SPOS critical steps could include, e.g., DMT deprotection, coupling, oxidation/ sulfurisation reaction or capping monitoring, cleavage, concentration and drying, annealing (if relevant) and lyophilisation (if relevant) steps.

应根据 ICH Q9-Q11 指南对寡核苷酸固相合成过程中的关键步骤进行评估，并明确相应的过程控制点。通过结合分析检测和工艺控制手段对关键步骤进行有效管控。固相合成过程中的关键步骤可能包括脱保护、偶联、氧化/硫化、封端、裂解、浓缩、干燥、退火（如果适用）和冻干（如果适用）等。

During oligonucleotide purification by preparative chromatography, individually collected fractions are usually combined into a pool of fractions. The pooling strategy should be defined and acceptance criteria for the purity of individual fractions and the main pool should be stated. These criteria for purity usually include overall purity and criteria for individual impurities. In case secondary purification is proposed in the manufacturing process, adequate requirements for side-fractions that are allowed to undergo such purification, and the conditions thereof, should be defined. It should be stated which fractions are discarded. Filtration and lyophilisation steps should also be adequately controlled.

在通过制备色谱进行寡核苷酸纯化时，通常会将单独收集的分馏液合并成一个分馏池。需要定义分馏液合并的策略，并说明各分馏液和主池的纯度标准。这些纯度标准通常包括总体纯度以及个别杂质的标准。如果制造过程中需要进行二次纯化，则应明确允许进行该纯化的副分馏液的要求及其条件，并说明哪些分馏液会被丢弃。过滤和冷冻干燥步骤也需进行适当控制。

Intermediates as defined in the manufacturing process are tested before use in the next stage of the manufacturing process. The methods used for IPC and/or intermediate testing should be described and confirmation of analytical method validation provided where applicable.

生产过程中的中间产品需在用于下道工序前进行检验。应详细说明检验方法，并提供相关分析方法的验证数据。

The EU GMP Part II definition of intermediate should be considered, i.e., “A material produced during steps of the processing of an API that undergoes further molecular change or purification before it becomes an API. Intermediates may or may not be isolated.” For intermediates, justified specifications should be presented. Only intermediates complying with specifications may finally be pooled.

中间体的定义应参考 EU GMP Part II，即“在 API 加工步骤中生产的材料，需经过进一步的分子变化或纯化后成为 API。中间体可能会或可能不会被分离。”对于中间产品应设定合理的质量标准，仅符合标准的中间产品方可合并使用。

For double stranded oligonucleotides, specifications for the single strand intermediates prior to annealing, consisting of at least identity, purity and impurities, a description of the analytical methods used, and relevant analytical method validation data should be provided. Results from forced degradation studies on the single strands should be provided to demonstrate the stability indicating nature of the analytical methods. The holding time– and storage conditions for single strand intermediates should be supported by stability data.

对于双链寡核苷酸，需提供退火前单链中间体的质量控制标准，包括鉴别、纯度、杂质检测方法及其验证数据。此外，还需进行强制降解试验以验证检测方法的稳定性指示功能。单链中间体的储存条件和存储时间应有稳定性数据支撑。

4.2.5. Process Validation and/or Evaluation 工艺验证/评价 3.2.S.2.5

No additional requirements apply for synthetic oligonucleotides than for other synthetic substances (i.e. process validation data would normally not be expected in the dossier), except for oligonucleotide active substance in solution: see 4.9. Re-use of preparative columns should be appropriately validated.

合成寡核苷酸与其他合成药物相比，没有额外的要求（例如，通常不需要在申报文件中提供工艺验证数据），唯一的例外是溶液中的寡核苷酸原料药：参见 4.9。制备色谱柱的重用应经过适当的验证。

4.2.6. Manufacturing Process Development 生产工艺开发 3.2.S.2.6

In order to support the development of a manufacturing process that produces an active substance of suitable quality, a risk-based approach as outlined in ICH Q8 Pharmaceutical development, ICH Q9 Quality risk management, and ICH Q11 Guideline on development and manufacture of drug substances (chemical entities and biotechnological/biological entities) is expected to be applied throughout the process development.

为确保生产出符合质量要求的活性药物成分，应在整个研发过程中遵循 ICH Q8《药物开发》、Q9《风险管理》 和 Q11《原料药开发和生产指导原则（化学实体、生物技术/生物实体）》等指南提出的风险评估原则。

Risk assessments should be performed to determine the criticality of individual quality attributes on the overall quality of the active substance and resulting finished product. These risk assessments should support the overall control strategy.

需要对各个质量属性对原料药整体质量和最终产品的影响进行风险评估，以帮助制定全面的控制策略。

Process risk assessments and process characterisation studies should be carried out to identify the impact of process parameters on the quality attributes, including an assessment of the potential impact of process limits and failures on quality and/or process consistency, and the identification of appropriate risk mitigation actions where relevant.

通过工艺风险评估和工艺特性研究，明确工艺参数对产品质量的影响，包括评估工艺偏差对产品质量和生产一致性的潜在风险，并采取相应的风险控制措施。

The process risk assessments, together with the knowledge gained from process characterisation and development studies, should be used to establish the final set of process controls for active substance manufacturing. This includes the classification of the IPCs and tests as critical or non-critical.

工艺风险评估以及从工艺表征和开发研究中获得的知识应用于建立最终的原料药制造工艺控制方案。这包括将 IPC 和测试分类为关键或非关键。

It is acknowledged that there are general aspects of oligonucleotide synthesis where prior knowledge and manufacturing experience may be extrapolated between different processes. Also, the starting materials and their properties are mostly well-known. If justified, manufacturers may make reference to prior knowledge for general aspects of the manufacturing process (e.g. choice of solid support or coupling reagents). However, it is expected that substance specific aspects such as reaction times, temperatures and molar equivalents are addressed in the development section. If in-house knowledge from other products is referred to, the data and source should be identified as appropriate and differentiated from product-specific data. A discussion of how this data is to be used should be integrated with the relevant product-specific data to provide an overall understanding of product development and control. The use of prior knowledge should always be explained and justified in the dossier, with a focus on the context and relevance of the prior knowledge to the current assessment. If prior knowledge from scientific papers is quoted, copies of the paper should be provided as appropriate.

寡核苷酸合成领域存在一些通用的经验和知识，可用于指导不同生产工艺。此外，起始原料及其性质也相对成熟稳定。因此，在合理的情况下，企业可参考以往的生产经验（如固体载体或偶联试剂的选择）。然而，对于具体的工艺参数，如反应时间、温度和物料配比等，仍需进行详细的研发和论证。如引用其他产品的内部数据，应明确数据来源并区分与本产品的差异性，同时需结合具体情况说明如何参考历史数据。如引用科学文献，需提供相关文献的复印件。

4.3. Characterisation 特性鉴定 3.2.S.3

4.3.1. Elucidation of Structure and other Characteristics 结构和理化性质 3.2.S.3.1

The structure of the oligonucleotide should be confirmed by analytical data, this includes the primary, secondary, and tertiary structure where relevant. Mass spectrometry is a suitable analytical tool for the structure elucidation of oligonucleotides. Variants of the MS technique can be used to determine the molecular mass of an oligonucleotide and to confirm its nucleotide sequence. Typical representative spectra and interpretation of the fragmentation data, including assignments and tables with theoretical and observed mass values, should be provided.

需通过分析数据确认寡核苷酸的一级、二级和三级结构。质谱是解析寡核苷酸结构的理想工具。质谱技术的不同变体可以用于确定寡核苷酸的分子质量并确认其核苷酸序列。应提供典型的质谱图谱，并对谱图中的碎片离子进行详细解析，包括理论与实测质量值的比对。

Elemental analysis (e.g. by combustion analysis, or inductively coupled plasma optical emission spectroscopy (ICP-OES) for phosphorous and sodium content) may be used in view of structure confirmation.

元素分析（如燃烧分析法或电感耦合等离子体发射光谱法测定磷和钠含量）也可用于结构确认。

NMR experiments are recommended to be part of the characterisation studies, to elucidate the nucleobase, sugar and backbone compositions, identity, and connectivity of nucleotides. One- and two dimensional techniques should be used to assign the structure by means of ¹H, ¹³C and ³¹P NMR data where relevant. ³¹P NMR typically provides information on primary and secondary structure of phosphorothioate diester and/or phosphodiester backbones. In case of fluorination (e.g. 2-fluoro substitution of the riboses), also ¹⁹F NMR can be used.

建议在表征研究中进行核磁共振 (NMR) 实验，以明确核碱基、糖和骨架的组成、身份和核苷酸的连接性。通过一维和二维 NMR 技术结合 ¹H、¹³C 和³¹P 核磁共振谱图可确定化合物的结构信息。其中，³¹P NMR 可用于分析磷酸二酯或硫代磷酸二酯主链的结构特点。对于含氟修饰的寡核苷酸，可进一步采用¹⁹F NMR 进行分析。

Nucleoside stereochemistry, including stereochemical purity of the phosphoramidite starting materials originating from their nucleoside precursors, and potential anomerisation propensity during chain elongation of the oligonucleotide synthesis, needs to be discussed.

需关注核苷的立体化学信息，包括磷酰胺原料的立体纯度及其在寡核苷酸合成链延伸过程中可能发生的异构化现象。

For oligonucleotides with phosphorothioate diester internucleotide linkages, the phosphorus atoms of the phosphorothioate diester internucleotide linkages are chiral. The stereochemistry of the internucleotide linkages is determined during the coupling reaction, and delivers a mixture of 2n diastereomers (with n the number of phoshorothioate linkages). If separation of these 2n diastereomers is not practically possible, an estimation of the diastereoisomeric distribution (ratio of isomers), and the reproducibility thereof, is expected.

对于具有磷硫代二酯核苷间键的寡核苷酸，这些核苷间键的磷原子是手性的。核苷间键的立体化学在偶联反应过程中决定，并产生 2ⁿ 对映异构体的混合物（n 为磷硫代键的数量）。如果无法实际分离这些 2ⁿ对映异构体，则需要估计对映异构体的分布（即异构体的比例）及其再现性。

Phosphodiester internucleotide linkages are non-chiral.

磷酸二酯链间连接是非手性的。

The impact of the stereochemistry on the biological/pharmacological activity should be discussed (see Investigation of Chiral Active Substances 3CC29a for human products or EMEA/CVMP/128/95 for veterinary products).

需要讨论立体化学对生物和药理活性的影响（参见《人用药物中手性活性物质的研究 3CC29a》或《兽医用药物 EMEA/CVMP/128/95》）。

Ultraviolet, circular dichroism (CD) and infrared (IR) spectroscopy are part of the standard characterisation program. Additional information on the secondary structure can be gathered from these techniques.

紫外光谱、圆二色（CD）光谱和红外（IR）光谱是标准表征程序的一部分。这些技术可以提供关于二级结构的附加信息。

Usually, no biological assay is required for the characterisation or routine release of antisense oligonucleotides or siRNA, which exert their function by annealing onto a complementary strand.

通常无需进行生物学活性检测即可表征或释放反义寡核苷酸或 siRNA，这类药物通过与互补链结合发挥作用。

Aptamers exert their biological activity by selectively binding to a target molecule. The biological activity of aptamers depends heavily on their 3-dimensional structure. Therefore, characterisation of this attribute (e.g. binding to biological target) is expected. Also, the secondary structure (the presence of guanine residues could lead to the formation of G-quadruplexes) should be investigated and results provided.

适配体通过选择性结合目标分子发挥其生物活性，其生物活性主要取决于其三维结构。因此，需要对其三维结构（例如，与生物目标的结合）进行表征。此外，还应研究其二级结构（例如，鸟嘌呤残基的存在可能形成 G-四联体）并提供相关结果。

In case of conjugated molecules, characterisation tests on the conjugated and non-conjugated moieties are expected. Specificity of conjugation needs to be demonstrated, and the secondary structure of conjugated versus unconjugated oligonucleotide should be compared unless otherwise justified.

对于偶联型寡核苷酸，需分别对偶联部分和寡核苷酸主体进行表征，并验证偶联的特异性。此外，除有特别理由，还需比较偶联前后寡核苷酸的二级结构差异。

In case of siRNA, characterisation tests on the sense strand, the antisense strand and the duplex are expected.

对于 siRNA，需分别对正义链、反义链和双链结构进行表征。

Evidence of chemical structure 化学结构证据

The information will normally include such evidence as信息通常包括以下证据:

List of characterisation techniques used for oligonucleotides (example table): 用于寡核苷酸的表征技术列表（示例表）

* for siRNA duplexes 适用于 siRNA 双链

** for aptamers or for oligonucleotides conjugated to moieties with 3-dimensional structure (e.g. antibody oligonucleotide conjugates) 适用于适配体或与三维结构部分偶联的寡核苷酸（例如抗体寡核苷酸偶联物）

Physico-chemical characteristics 理化特性

Physicochemical characterisation of the drug substance could include solubility and hygroscopicity studies, determination of the isoelectric point (pI) and thermogravimetric studies as e.g. differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The morphology may be examined by powder X-ray diffraction (PXRD) and/or DSC.

药物物质的理化性质研究包括溶解度、吸湿性、等电点测定以及热分析（如差示扫描量热法 DSC 和热重分析 TGA）等。此外，可通过粉末 X 射线衍射 (PXRD) 或 DSC 等方法研究药物的晶型结构。

4.3.2. Impurities 杂质3.2.S.3.2