Center for Drug Evaluation, NMPA

　　February, 2024

　　I. Foreword

　　Antibody-drug Conjugate (ADC) is a novel antibody drug in which an antibody or antibody fragment targeting a specific antigen is conjugated to a payload through a linker. Compared with traditional antibody drugs, ADCs have both the potent effects of traditional small molecule drugs and the targeting of antibody drugs to reduce systemic toxicity and more selectively deliver payloads to tumor cells, tumor microenvironment, or other target cells. In recent years, with the rapid development of antibodies, payloads, linkers, conjugation technology, and analytical technology, etc., ADCs have higher uniformity, stability and treatment index, which has greatly promoted the development boom of ADCs.

　　Taking into account the complexity, specificity and development status of ADCs, the guideline is formulated to standardize and guide the development of ADCs.The Guidelines is based on current scientific knowledge and puts forward recommended technical requirements for pharmaceutical study of ADCs in the new drug application stage, so as to provide technical guidance to R&D institutions. Applicants may also conduct studies with other equivalent or more effective techniques and methods based on the actual development of product, but they should comply with the rules of drug development and provide information to prove the scientificity and applicability. With the development of technology, deepening knowledge, and accumulation of experience, the relevant content of these guidelines will be gradually refined and updated.

　　II. Scope of application

　　The guideline mainly applies to ADCs made of antibody/antibody fragments and payloads (such as small molecule cytotoxins) conjugated through a linker. Other conjugated drugs, such as antibody-nuclide conjugates, polypeptide-drug conjugates, antibody-oligonucleotide conjugates, etc., can also refer to these guidelines. Due to the complexity and diversity of ADCs, the requirements covered by established technical guidelines (such as small molecule drugs, antibodies, etc.) will not be detailed in these guidelines; please refer to the corresponding technical requirements.

　　III. General principles

　　Pharmaceutical studies on ADCs shall meet the relevant requirements of the “Drug Administration Law of the People’s Republic of China”, “Drug Registration Regulations”, and the “Pharmacopoeia of the People’s Republic of China” (hereinafter referred to as the “Chinese Pharmacopoeia”), and the manufacturing of clinical materials should meet the relevant requirements of the current “Good Manufacture Practic - Appendix Drugs for Clinical Trials (Trial)”.

　　1. General requirements

　　In terms of product design, although ADC products are usually considered to be large molecule drugs, they have the dual attributes of large molecule drugs and small molecule drugs. The target antigens, antibodies, payloads, linkers, and the selection of conjugation methods are all key factors affecting the safety and efficacy of ADCs. Therefore, the safety and efficacy of ADCs depend on strict selection, multi-faceted optimization and reasonable combination of the above components. Comprehensive consideration is required from various aspects such as targeting, stability in body circulation, and biological activity.

　　In terms of manufacturing process, the manufacturing process of ADCs usually involves multiple manufacturing processes such as naked antatibody manufacturing, small molecule manufacturing, ADC DS, and ADC DP manufacturing. In the process of product development, process design and process development may be carried out based on the concepts of “quality by design” and “risk assessment”. Special attention should be paid to integrate each component (naked antibody, small molecule, ADC DS, and ADC DP) as a whole to comprehensively consider the risks and control of impurities and viruses safety of the entire manufacturing process.

　　In terms of formulation development, compared with antibody drugs, ADC molecules may have higher hydrophobicity, irregular surface charges, poor conformational stability, and high heterogeneity, which brings more complexity and instability to formulation development. In addition, naked antibodies, small molecules and ADC molecules all have different biophysical properties, and the conjugation process may also affect the stability of the protein (such as causing conformational changes, aggregation and degradation, etc.). Therefore, formulation development requires finding a suitable formulation to balance the stability of naked antibodies, small molecules and conjugation, and pay close attention to the aggregation and particle formation of proteins in the drug product, as well as the shedding of payloads.

　　In terms of quality study and control, the molecular structure, mechanism of action, and quality study and control strategies of ADCs are all unique. Different products also have special characteristics in quality study and quality control due to their different design concepts and manufacturing process, which require specific analysis case by case. Due to the complex structure and strong heterogeneity of ADCs, including many kinds of product-related impurities and process-related impurities, in addition to the critical quality attributes of antibody drugs, other critical quality attributes such as small molecules, conjugation processes-related attributes, etc. will be introduced as well. It is necessary to adopt appropriate technical means or a strategy that combines multiple analysis methods to fully characterize critical quality attributes such as structural characteristics, purity and impurities, heterogeneity, biological activity, and payload distribution.

　　2. Considerations at different development stages

　　As an innovative antibody drug, the development and production of ADCs must follow the general rules of drug development, and be gradually improved and continuously optimized on the premise of ensuring general clinical safety. According to the rules of drug development life cycle, development strategies based on science and risk assessment are used at different stages of development and post-marketing processes.

　　During the clinical trial application, factors affecting clinical safety should be focused on to ensure the safety of clinical subjects. For example, the control of adventitious agents should be comprehensively considered from the starting materials (such as cell substrates, other raw materials for manufacturing, etc.), manufacturing in-process control, release testing, etc., to avoid contamination by adventitious agents; the manufacturing process of clinical materials should have preliminary robustness with preliminary control items and relevant criteria for intermediates having been established, and focuses on the control of viral safety, microbial safety, sterility assurance, etc. during the manufacturing process of clinical materials; perform quality research and develop and validate analytical methods for safety evaluation and potency methods on representative samples, establish specification based on platform experiences, product knowledge and multi-batch testing data, and conduct reasonable control of impurities based on product characteristics and manufacturing processes; conduct preliminary stability studies, and the study results should be able to support the development of clinical trials; Select appropriate container closure system according to the products characteristics, and perform risk assessment on compatibility based on stability study data and extractables data provided by suppliers. Non-clinical research is an important reference for evaluating the safety of products for human use. Special attention should be paid to the comparison or bridging analysis of the manufacturing process and quality of clinical samples and non-clinical samples. In principle, the quality of clinical samples should not be inferior than that of non-clinical samples or clinical trial samples that have been studied to demonstrate safety in humans.

　　During clinical trials, based on process development and understanding of product quality attributes, it is necessary to gradually confirm critical process procedures and process parameters, control items and critical quality attributes in the manufacturing process, etc., to establish a stable manufacturing process and a complete quality control system. During R&D, raw materials for manufacturing, manufacturing process, specifications, etc. may change with process development or optimization. The complexity of ADCs’ structures, manufacturing process, and supply chains poses an additional challenge to evaluating the potential impact of changes, and change plans should be assessed and implemented more carefully. The implementation of various change protocols needs to be based on comparability studies appropriate to the R&D stage. Comparability studies should be carried out based on risk assessment principles and with reference to the requirements of relevant guidelines such as ICH Q5E, etc., with sufficiently precise and sensitive analytical methods to reasonably assess the impact of changes on product quality. Particular attention should be paid to whether differences in quality attributes of naked antibodies and small molecules caused by changes in upstream process will adversely affect the critical quality attributes of subsequent conjugation or ADC.

　　In the new drug application stage, after process development and systematic process validation, critical process procedures and critical process parameters are determined, and critical quality attributes are controlled to ensure that the commercial manufacturing process can continuously and stably produce products that meet the target quality. Pharmaceutical study data should be able to support the safety, efficacy and quality controllability of the product. At the same time, a work plan for continuous qualification and optimization of the manufacturing process after marketing should be formulated to continuously ensure product quality.

　　IV. Risk assessment and control

　　ADCs are innovative antibody drugs made by combination of large molecule and small molecule, which face many challenges in product design, manufacturing process, formulation development, quality study and control, as well as stability. Product development involves multiple disciplines such as chemistry and biology, from starting materials and process development to quality control. The molecular structure and manufacturing process are complex and diverse, product heterogeneity is high, and different ADCs may also show large differences. In addition, new antibody forms, payloads, linkers, and new conjugation strategies are also constantly emerging, so that the manufacturing process and control strategy of each ADC has its personalized characteristics. In addition, the manufacturing process of ADCs includes multiple manufacturing steps and there is a high degree of change complexity. Therefore, it is necessary to carry out risk assessment, by scientifically utilizing risk assessment tools, from multiple factors such as molecular design, manufacturing process, quality control, and stability based on product and process characteristics, referring to the quality risk management concept of ICH Q8, ICH Q9 and ICH Q11. According to the risk assessment results, combined with the understanding of products and processes, formulate corresponding risk control strategies. The revision of the risk control strategy should run through the entire life cycle of the product, and it should be continuously updated with the accumulation of new knowledge, production experience and in-depth understanding of product quality attributes.

　　V. Materials for production

　　Materials for production mainly refer to all materials used in the manufacturing process of ADCs, including starting materials, materials used or added during manufacture (such as medium and additives, purification reagents, conjugation reagents, conjugation enzymes, etc.), excipients, and production consumables (such as culture bags, liquid storage bags, pipetting lines, filter membranes, etc.), etc. Materials used in manufacture are closely related to the quality, safety and efficacy of products. A good and standardized quality management system should be established, and the materials should be controlled based on risk assessment principles with reference to relevant requirements such as the Chinese Pharmacopoeia.

　　The cell substrates used in the manufacturing process shall meet the relevant requirements of the “Chinese Pharmacopoeia” general principle “Management and Quality Control of Bacterial and Viral Strains Used in the Manufacturing and Testing of Biological Products” and “Preparation and Quality Control of Animal Cell Substrates for Manufacturing and Testing of Biological Products”; other manufacturing materials used should meet the relevant requirements of the “Chinese Pharmacopoeia” general priciple “Quality Control of Raw Materials and Excipients for the Manufacturing of Biological Products” and ICH Q11. For starting materials with complex chemical structures and manufacturing processes, their quality should be comprehensively analyzed and reasonably controlled based on the manufacturing process of the starting materials.

　　The consumables and containers used in the manufacturing process, such as disposable bioreactors, ultrafiltration membrane packs, filters, pipelines, etc., should have stable physical and chemical properties, and the consumables should have good compatibility with the solutions and intermediate products in direct contact. Risk assessments or corresponding compatibility studies should be conducted based on the material of consumables, usage stages, suppliers’ CoAs, etc. In addition, if a certain proportion of organic solvents is required in the ADC manufacturing process, materials directly in contact during manufacture (such as disposable reaction bags, ultrafiltration membranes, manufacturing equipment, etc.) should be resistant to organic solvents, and leachables must meet relevant requirements. At the same time, attention should be paid to the impact of the leachables on product quality. For ADC products with high hydrophobicity, it is also necessary to pay attention to the non-specific binding of ADC molecules to the filter membrane used for sterilization, and select an appropriate filter membrane.

　　VI. Manufacturing process

　　(I) Manufacturing process development

　　The manufacturing process of ADCs usually includes multiple manufacturing steps such as naked antibody production, small molecules manufacturing, ADC DS manufacturing, and DP manufacturing. In accordance with general rules of drug manufacturing process development, the process should be gradually improved based on the understanding of the Quality Target Product Profile (QTPP) as well as the correlation between manufacturing process and quality. The manufacturing process development should complete the development process from laboratory to commercial scale manufacturing, and gradually determine critical quality attributes, process steps and critical process parameters. Based on risk analysis, establish a full-process control strategy and reasonably set controls in the manufacturing process, especially the quality control of adventitious agent contamination and key intermediate products in the manufacturing process. In the product life cycle, the manufacturing process should be continuously optimized following the advancement and in-depth understanding of process technology. Process optimization should fully evaluate the impact of changes on product safety and efficacy based on change types and development stages, and the corresponding comparability studies should be conducted on process optimization with reference to relevant guidelines to ensure product quality.

　　For light-sensitive small molecules, chemical structure changes will occur under specific light bands or high-intensity irradiation, and this chemical structure change may affect the quality attributes of ADC products (such as biological activity, etc.). Therefore, it is necessary to take appropriate measures during the manufacturing process (such as using yellow light instead of white light in the manufacturing environment, protecting containers of intermediate from light, etc.), and conduct systematic research on the DP filling process, storage conditions, and selection of packaging containers.

　　1. Small molecule portion

　　The manufacturing process development of small molecules (including semi-synthetic sources) should be performed with reference to the requirements of chemical DS mentioned in the "Technical Guidelines for the manufacturing and Structure Elucidation of Chemical Drug substances" and "General Review of Recombinant DNA Protein Products for Human Use" , ICH and other relevant guidelines.

　　According to the chemical characteristics of ADC modification and conjugation reactions, rationally select and design linkers, payloads, and synthesis/process routes of ADC molecules. Refer to ICHQ11 and its Q&A to rationally select starting materials for chemical synthesis, and develop synthesis process and quality control strategies of small molecules. For linkers and payloads containing multiple chiral centers, attention should be paid to whether the chiral centers will affect the biological activity of the ADC product, and chiral impurities should be reasonably controlled. The critical quality attributes of small molecules should be confirmed through risk assessment in terms of their impact on the safety and efficacy of the ADC drug conjugation process and the final product.

　　2. Naked antibody

　　The development of manufacturing process for naked antibodies should be carried out in accordance with the “Chinese Pharmacopoeia”, “Technical Guideline for Quality Control of Human Monoclonal Antibodies”, as well as relevant international technical requirements such as ICH and WHO. The manufacturing process of naked antibodies is similar to conventional antibody drugs, but some genetically engineered manufacturing process for targeted conjugation may differ due to the characteristics of their modified groups. Therefore, in addition to focusing on the risks of conventional antibody production, for naked antibodies, it needs to focus on the impact on the ADC DS manufacturing process and the quality of the final ADC product from the entire process of ADCs designing and producing. For example, the control of the types and levels of aggregates needs to take into account changes in aggregates during the production of ADC DS; for ADCs made by lysine-mediated conjugation technology, it is necessary to consider the level of N-terminal lysine residues of naked antibodies and amino acid components in naked antibody solutions; for ADCs made by cysteine-mediated conjugation technology, attention should be paid to the levels of cysteine-related variants (such as disulfide bond, trisulfide bonds, free radicals, etc.) of naked antibodies, etc. In the process development and control of naked antibodies (especially bispecific antibodies or multispecific antibodies), it is also necessary to pay attention to the impact of product-related impurities (such as disulfide bond mismatched impurities) on the safety and efficacy of the final ADC product. For the development of naked antibody solution components, in addition to ensuring the stability of the naked antibody itself, it is also necessary to consider the compatibility with the subsequent conjugation process to avoid affecting subsequent ADC manufacturing (including conjugation and formulation, etc.); if it will have an impact on subsequent ADC manufacturing, buffer replacement and other steps need to be performed before conjugation to reduce the impact on subsequent processes.

　　3. ADC DS

　　The manufacturing process development of ADC DS can refer to the ICH Q8 concept of Quality by Design for process design and development.

　　The manufacturing process for ADC DS usually includes steps such as antibody modification (if applicable), conjugation reaction, and ADC purification.

　　The antibody modification part may vary according to the conjugation technology used. The main purpose of antibody modification is to introduce reactive chemical groups into the antibody, such as opening the antibody disulfide to produce an active free thiol group through a reducing agent, or the sugar chain can also be structurally modified through biological enzymes to introduce active reactive groups into the sugar chain. Antibody modification is a key step in determining ADC drug load and drug load distribution, so it is necessary to develop an appropriate manufacturing process, select and set a reasonable in-process control strategy according to conjugation technology and modification reaction characteristics. If the naked antibodies after modification requires purification, a reasonable purification process should also be introduced; for example, if the reagent in the modification process is at risk of introducing adventitious viral agents, the purification process should take into account the removal of adventitious viral agents and carry out virus clearance studies with reference to ICH Q5A; if the modified naked antibodies are managed according to intermediates, reasonable intermediate specifications should also be formulated and corresponding stability studies should be carried out to set a shelf life, etc.

　　Compared with the antibody modification step, there are multiple similar technology platforms for conjugation reactions. Usually, a rapid chemical reaction or enzymatic reaction is used to conjugate the small molecules to the active site of the modified naked antibody, or the specificity of the chemical reaction and enzymatic reaction is used to directly conjugate the small molecules to a specific site of naked antibody. During this process, the different physical and chemical properties of the naked antibody, linker, and payload must be taken into account. For example, antibodies are more stable in buffered aqueous solutions, while most small molecules are hydrophobic, so organic solvents (such as dimethyl sulfoxide, dimethylacetamide, acetonitrile, etc.) are usually used to assist in improving the solubility of small molecules in aqueous solutions to improve the efficiency of conjugation reaction, but the use of organic solvents may lead to instability of the naked antibodies. In conjugation reaction, special attention should be paid to parameters such as the selection of organic solvents, small molecules feeding ratio, pH, temperature and concentration of naked antibody etc., to control the generation of aggregates and nonspecific conjugation. Considering the high heterogeneity of ADCs, it is recommended to focus on the reducing sites and reduction proportion of antibody disulfide bonds in the conjugation process (if applicable), the control of conjugation sites and number of binding, distribution of payloads, impurity conjugation profiles, etc., so as to determine reasonable process parameters. For site-specific conjugation, attention should be paid to the probability of occurrence of possible non-target-site conjugation and other side reactions based on the principles of site-specific conjugation. If enzyme-catalyzed reactions are involved, attention should also be paid to the safety risks of introduction of catalyst enzyme and their residues in the ADC DS. If overage occurs during the conjugation reaction, corresponding process research needs to be carried out, and risk assessment must be conducted in conjunction with downstream processes. If a multi-step conjugation reaction is involved, attention should be paid to the parameters that affect the conjugation efficiency of each step (such as feed ratio, etc.) to avoid high proportions of unloaded linker- conjugated antibodies and low Drug-to-Antibody Ratio (DAR) and high free payload residue due to low conjugation efficiency or insufficient feed. For dual-payload ADC products, the conjugation process is more complicated. During the conjugation process, attention need to be paid to whether the DAR values of the two payloads reach the target value. At the same time, attention should also be paid to the conjugation of the target site and non-target site; if overage involved, attention should also be paid to the impact on the drug loading distribution and the control of the DAR values of the two payloads.

　　The main purpose of purification is to remove process-related impurities introduced from the manufacturing process and generated product-related impurities. Process-related impurities mainly include residual modification reagents (for example, reducing agent, etc.), catalysts, reaction enzymes and organic agents; product-related impurities mainly include aggregates, fragments, free small molecules and their derivatives and side reaction products generated during the manufacturing process, etc. In some manufacturing process, DAR and drug load distribution of ADCs may be adjusted by purification process. Therefore, purification methods and technologies should be selected according to the purpose of purification, and a comprehensive evaluation of the impurities that may be introduced or generated during the manufacturing process should be performed to determine the purification effect.

　　4. ADC DP

　　The selection of dosage forms and formulation for ADCs requires the development of dosage forms and formulation according to the complexity and specificity of ADCs. Since ADC is prone to degradation and aggregation, etc., under solution storage conditions, sufficient research should be conducted to select the appropriate dosage form. At present, freeze-dried preparation is generally chosen. However, in order to improve the convenience and safety of clinical medication, the development of ADC liquid dosage forms is also the research direction of ADC preparations based on continuous research and optimization of the stability of naked antibodies and small molecules.

　　When developing ADC prescriptions, a scientific formulation development strategy should be established based on the in-depth understanding of the physical and chemical properties of the naked antibody, payload, linker and ADC using appropriate methods (such as design space, etc.) and taking into account the stability of naked antibody and small molecule. In this way, appropriate DP formulation can be determined to maintain the stability of the ADC DS and DP. For example, the conjugation of hydrophobic small-molecule drugs with hydrophilic antibodies may cause ADC aggregation or other physico-chemical instability. Therefore, ADC products usually need to select the appropriate type and concentration of surfactants to reduce risks such as aggregation and degradation caused by heterogeneity and hydrophobicity..

　　The type, usage and specification of excipients are determined based on the formulation development of the drug products. Excipients shall meet the relevant requirements of “Quality Control of Raw materials and Excipients Used in the Production of Biological Products” in the general principles of the “Chinese Pharmacopoeia”. If novel excipients are used, comprehensive study data should be submitted. In the absence of human safety data, studies should be carried out in accordance with the “Guidelines for Non-clinical Safety Evaluation of Novel Pharmaceutical Excipients”.

　　The manufacturing process of ADC DP generally includes steps such as DS thawing, aspetic filtration, aseptic filling, and freeze-drying (if applicable). The manufacturing scale and batch size are determined according to the study characteristics. The definition of batches should be clarified. Attention should be paid to the matching of upstream and downstream scales, etc. Due to the high heterogeneity and complex processes of ADC products, mixed batch operations should be avoided as much as possible during the production process. If the formulation process involves a freeze-drying process, the parameters of freeze-drying process should be studied.

　　5. Process optimization

　　During process development, manufacturing process changes are carried out with continuous optimization of process procedures and parameters, scale amplification, improvement of production quality or stability, etc., and implementation of the changes shall be based on adequate comparability studies. Comparability studies can refer to the requirements of relevant guidelines, such as ICH Q5E, etc., to establish a comparability study protocol and bridging program based on the risk assessment results of changes. During the process of scale-up and transfer of manufacturing process, it is necessary to fully evaluate the impact of process scale-up and transfer on the critical quality attributes of the product, and carry out risk-based comparability studies based on the stage of drug development and with reference to relevant guidelines. For the amplification and transfer process of ADC DS manufacturing process, special attention needs to be paid to critical quality attributes before and after amplification and transfer, such as drug loading distribution, comparability of DAR value and conjugation site, impurity removal ability, biological activity, etc. In the early development stage, since the manufacturing process is still being developed and optimized, and the knowledge, experience, and number of batches are limited, comparability studies can be carried out based on the study data of limited batches, but attention should be paid to quality attributes related to safety and efficacy (such as impurity profiles, DAR values, biological activity, etc.). In the later stage of development, with the accumulation of production experience and in-depth understanding of product technology and quality, comprehensive and sufficient comparability studies should be carried out to set acceptable criteria supported by sufficient production experience and clinical data.

　　The complexity of ADCs manufacturing process and supply chains poses an new challenges to evaluating the potential impact of changes. The comparability assessment of naked antibody and small molecules also requires consideration of whether to conduct comparative studies or production-scale process qualification of ADC DS or DP. Attention should also be paid to whether differences in quality attributes resulting from changes in naked antibody and small molecule will adversely affect the subsequent conjugation process or critical quality attributes of the ADC DS.

　　(II) Qualification and validation of manufacturing process

　　During the process development, qualification and validation studies of manufacturing process should be conducted according to the different purposes of different development stage. The purpose of process validation is to prove whether the determined manufacturing process can continuously and stably produce products that meet the expected specifications according to the proposed process conditions. The influence of each process parameter on product quality or process performance should be identified through process characterization, and the process control strategies should be developed to ensure the robustness of the process. After the commercial manufacturing process is determined, at least 3 batches of representative products manufactured by commercial manufacturing processes should be used for standardized process validation before marketing. Process validation of ADC products should include process validation of naked antibodies, small molecules, and ADC DS and DP. After marketing, continuous process qualification should be carried out in the commercial manufacturing process.

　　The process validation of naked antibodies and small molecules can be carried out in accordance with the requirements of the “Chinese Pharmacopoeia” and relevant domestic and international guidelines, focusing on the robustness of the process and the inter-batch consistency of product quality attributes.

　　For Process validation of ADC DS and DP, the critical process parameters and control strategies of the manufacturing process should be determined on the basis of a full understanding of the relationship of manufacturing process and critical quality attributes, with the representative commercialized manufacturing process to carry out standardized process validation. The validation content should be determined according to the manufacturing process and risk assessment of the product, generally including the consistency of the process, the stability of the process parameters, the removal of product-related impurities and process-related impurities and inter-batch consistency of product quality attributes. In addition, validation studies may also include (but are not limited to) sterile process validation, sterilizing filtration validation, lifespan studies of fillers and membranes, transportation validation, cleaning validation, facility and equipment validation, etc. If there are intermediate products in the manufacturing process, the temporary storage conditions and time limit of intermediates should also be studied. Due to the structural characteristics of ADC molecules, it is necessary to simultaneously investigate the strategy for conjugation of different batches of naked antibody and small molecule during qualification and validation. In addition, according to product characteristics, it is also necessary to focus on the removal effect and residue limit of small molecules-related impurities.

　　VII. Quality Study and Specification

　　(I) Quality study

　　In quality studies, representative batches (such as non-clinical study batches, clinical study batches and/or (or commercialized batches, etc.) and/or samples at appropriate production stages should be selected as study subjects, and the studies should be conducted with advanced methods. Study items should be comprehensive and thorough, covering all items that may be related to product safety and efficacy to the extent possible. The analytical method should also pay attention to the processing and analysis process of the samples, so as to avoid the impact of the analysis process such as sample pretreatment on the testing results, resulting in the analysis result not being able to represent the actual quality of the sample.

　　The ADC product quality study includes the small molecule, the naked antibody, and the ADC DS/DP. If there is a difference in quality characteristics between the DS and the DP, they should be sampled separately for the study.

　　1. Small molecule

　　Quality studies of small molecule can refer to chemical drug-related guidelines, such as “Technical Guideline for Manufacture and Structure Elucidation of Chemical Drug APIs”, “Technical Guideline for Impurity Study of Chemical Drugs”, “Technical Guideline for Residual Solvent Study of Chemical Drugs”, “Technical Guideline for Standardized Process of Establishment of Chemical Drug Specifications”, “Technical Guideline for Quality Control of Chiral Drugs”, ICH and other related guidelines.

　　In addition to conventional impurity analysis, the impact on subsequent processes and final product quality should also be studied and analyzed in conjunction with the structure of impurities. Based on the type, structure and other characteristics of impurities combined with comprehensively risk assessment such as fate (whether the impurity participates in the conjugation reaction) studies and purge (whether the impurity is removed through subsequent processes) studies, a reasonable impurity control strategy can be formulated. Since the conjugatable impurities are extremely difficult to quantify and difficult to remove after the conjugation reaction is completed, it is necessary to focus on the risks of conjugation impurities. Strict control criteria should be formulated and reasonable basis should be provided. For non- conjugatable impurities, a reasonable control strategy can be established based on subsequent process removal capabilities, etc., and sufficient research data should be provided.

　　2. Naked antibody

　　Quality study of naked antibodies can refer to the “Chinese Pharmacopoeia” and related guidelines, such as the “Guidelines for Quality Control of Human Monoclonal Antibodies” and ICH. In principle, the quality study requirements for naked antibody are basically the same as those for antibody drugs. In addition, quality attributes that may affect conjugation process, such as the oxidation level of the antibody, need to be fully studied and appropriately controlled. For antibody modifications that introduce cysteine or non-natural amino acids containing active reactive groups at specific sites of the antibody through genetic engineering technology, the modification site needs to be confirmed. Although Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement Dependent Cytotoxicity (CDC) or Antibody Dependent Cellular Phagocytosis (ADCP) are generally not the main mechanisms of action of ADC products , but it may also have certain effects. Fc segment function studies should be carried out based on antibody type, structure modification, etc., and if it affects the safety or efficacy of the final product, appropriate control should be carried out.

　　3. ADC DS/DP

　　ADC products have the characteristics of the targeting of antibody drugs and the strong potency advantages (such as cytotoxic effects) of small-molecule drugs, but the conjugation of the two also changes each other's physico-chemical properties, which may cause changes in drug structure, charge, etc. Therefore, for ADCs, in addition to the need to pay attention to the quality attributes of macromolecular proteins and the quality attributes related to small molecules, it is also necessary to add critical quality attributes caused by conjugation, such as DAR, drug load distribution, conjugation sites (including non-target conjugation sites), unconjugated naked antibody, free small molecule and their derivatives (such as degradation products and/or reaction products with quenchers), and heterogeneity. Appropriate and advanced analytical techniques should be used to perform comprehensive characterization from the perspectives of structural confirmation, physico-chemical properties, biological activity and impurity studies, etc., and to fully understand the relevant characteristic changes before and after conjugation (such as high-order structure, post-translational modifications, molecular size variants, charge variants, antigen binding activity, Fc functional activity, etc.), and provide as detailed information as possible to reflect the quality attributes of the final product. Quality study should at least cover the following areas:

　　3.1 Structural confirmation and physicochemical properties

　　Structural confirmation studies should combine the structural characteristics of ADCs and use appropriate analytical methods to characterize primary structure, secondary structure, high-order structure, conjugation sites and conjugation proportions at each sites, etc. Furthermore, during structural elucidation of ADC, it is necessary to use appropriate analytical methods (such as peptide mapping and mass spectrometry, etc,) and taking into consideration the mechanism of the conjugation reaction to evaluate the effects of the conjugation process on the structure of naked antibodies (such as completeness of amino acid sequence coverage, disulfides and thermal stability, etc.), glycosylation modifications, and other post-translational modifications, etc. Particular attention should be paid to key points affecting product functionality, especially complementarity-determining regions.

　　Given the complexity of the ADC molecular structure, enzymatic hydrolysis, reduction and other methods combined with liquid chromatography-mass spectrometry or tandem mass spectrometry (LC-MS/MS) technology can be used to reduce the complexity of ADC molecular structure step by step and to improve resolution of analytical methods and reliability of results. For products where modifications occur at cysteine residues or where reducing agents are used in the process, disulfide bonds, trisulfide bonds, free sulfhydryl groups, and sulfhydryl oxidation should also be considered.

　　Antibodies usually have complex heterogeneity (such as charge variants, molecular size variants, glycosylation, and other post-translational modifications, etc.), and the heterogeneity of naked antibodies themselves combined with heterogeneity caused by conjugation greatly increases the complexity of ADC. ADCs with high heterogeneity (including products using site-specific conjugation technology) require comprehensive characterization using reliable analytical methods with sufficient resolution to clarify the diversity of product-related substances. During the study, it is necessary to select the appropriate characteristic analysis method according to the chemical properties of the payload and the linker, the conjugation method, as well as the heterogeneity of the product.

　　3.1.1 Primary structure and drug conjugation sites

　　With appropriate analytical methods, such as peptide mapping and mass spectrometry, evaluate the effects of the conjugation process on primary structure, glycosylation modifications, and other post-translational modifications. Conjugation sites may affect PK/PD and molecular stability, and the conjugation sites can be identified and analyzed by enzymolysis with LC-MS method.

　　3.1.2 Higher Order Structure

　　Higher order structure is the basis for the stability and effectiveness of antibody drugs. The characterization methods of higher order structure generally include circular dichroism (CD), fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy, differential scanning calorimetry (DSC), analytical ultracentrifugation (AUC), etc. Compared with conventional antibodies, the higher order structural analysis of ADCs may become more complicated due to conjugation with chemical drugs. If the conjugated payload may produce an infrared, ultraviolet, or fluorescent response, it may affect the analysis of the results. If there are differences in the higher-order structures before and after conjugation, while comparing the structural differences using biophysical methods, it is necessary to focus on the impact of the differences on biological activity.

　　3.1.3 DAR value

　　DAR indicates the average number of payloads conjugated to each antibody molecule, directly related to the efficacy and safety of the product, which is the critical quality attribute of ADCs. Depending on the chemical properties of the linker and payload, the conjugation method, and the degree of DAR heterogeneity, commonly used methods include ultraviolet-visible spectrophotometry (UV), hydrophobic interaction high performance liquid chromatography (HIC-HPLC), reverse phase high performance liquid chromatography (RP- HPLC), MS, etc. Among them, the hydrophobicity and absorbance contribution of small molecule may interfere with the determination of DAR values and should be considered when analyzing the results. For dual payload ADC, in addition to the total DAR value, the DAR values for each payload also should be characterized.

　　3.1.4 Drug load distribution

　　ADCs , especially those with the non-site-specific conjugation method, are usually ADC molecular mixtures containing different point conjugation and different amounts of payload. The drug load distribution indicates the proportion of ADC molecules conjugated with different amounts of payloads to the total number of drug molecules. Appropriate methods, such as HIC-HPLC, RP-HPLC, capillary electrophoresis (CE), or MS, should be used to determine the different drug load distributions.

　　3.1.5 Molecular size variants

　　Molecule size variants directly affect efficacy and safety of products. Compared with antibody drugs, ADCs may have a stronger aggregation tendency due to possible increase of hydrophobicity, changes in surface charge distribution, and reduced thermal stability due to conjugation with small molecules. Appropriate methods should be used, such as size exclusion chromatography (SEC-HPLC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion multi-angle light scattering (SEC-MALS), AUC, dynamic light scattering (DLS), particle measurements, and LC-MS to study the molecular size variants of ADCs. Currently, SEC-HPLC and CE-SDS are commonly used test methods for releasing. SEC has a good resolution for aggregation, while the CE-SDS method has a good resolution for fragments, thus the two methods have a certain degree of complementarity. Molecular size variants can be fully characterized using a variety of analytical methods based on product structural characteristics.

　　3.1.6 Charge variants

　　Conventional antibody drugs usually use methods such as capillary zone electrophoresis (CZE), ion exchange chromatography (IEX-HPLC), capillary isoelectrofocused electrophoresis (CIEF), or whole-column imaging capillary isoelectrophoresis (iCEF) to study charge variants. For ADCs, the development of charge variant analytical methods requires considering product characteristics, such as small molecule characteristics (especially charge) and conjugation site selection (such as lysine, interchain thiol, cysteine, etc.). It should be considering that small molecules may have non-specific interactions with separation media. One or more appropriate methods can be selected for analysis. Furthermore, the conjugation process may consume the charge on the naked antibody (such as lysine conjugation) or introduce charged groups from the payload-linker. Some small molecules have special structural characteristics, such as strong hydrophobicity, dynamic transformation between different structures, etc., so a single analytical method may not fully reflect charge heterogeneity. The analytical results should be interpreted scientifically on the basis of a full understanding of the deep principles of the analytical method. If necessary, other analytical methods can be used for supplementary analysis. For example, the peptide mapping method is used to analyze different ion exchange collection components of samples, and the impact of conjugation on the charge heterogeneity of the antibody itself is indirectly evaluated. For dual-load ADC products, the complexity and detection difficulty of charge heterogeneity may be higher. iCIEF or other analysis methods can be used to monitor the batch-to-batch consistency of charge heterogeneity.

　　3.2 Biological activity

　　Biological activity is an important indicator of product quality and clinical efficacy. Biological activity analytical methods that reflect the mechanism of action in vivo can be established based on product characteristics, mechanism of action, etc., to study the functional activity of products. The main mechanism of action of ADCs is that naked antibodies bind to target antigens, release payloads inside or outside target cells, and exert their biological activity. Appropriate analytical methods (such as ELISA, surface plasmon resonance or biolayer interferometry, etc.) should be used to evaluate the binding activity with the target antigen. Select appropriate cell lines expressing target antigens to develop cell-based biological activity methods. Biological activity should be able to reflect the specific killing activity of ADC. If the Fc-mediated effector unction (ADCC, CDC, ADCP, etc.) may affect the efficacy of the drug or show nonspecific toxicity associated with the effector binding function, a corresponding biological activity assessment should be carried out.

　　3.3 Impurities analysis

　　3.3.1 Product related impurities

　　A product-related impurity is an unintended product that arises during manufacturing or storage. Potential product-related impurities in ADCs generally include aggregates, fragments, disulfide mismatched variants (if applicable), unconjugated naked antibodies, impurity conjugated ADCs (if applicable), free small molecules and their derivatives (the source may be additives or degradation products in the process), side reaction products, etc. In order to control product quality, it is recommended to use appropriate methods to separate and/or identify impurities related to various products, evaluate their safety risks by referring to the ICH Q6B concept, and comprehensively consider the impurity control strategy based on the evaluation results.

　　Unconjugated naked antibodies compete with ADC final products to bind to target cells and ultimately reduce the amount of drugs delivered to the target cells, which may directly affect the efficacy of the product, and changes in unconjugated naked antibody content may cause changes in efficacy. In release testing and stability studies, appropriate analytical methods should be selected to monitor the percentage of unconjugated naked antibody based on the properties of the conjugated payload (such as charge, hydrophobicity).

　　3.3.2 Process related impurities

　　Process-related impurities refer to impurities introduced during the manufacturing process. Manufacturing raw materials, production processes, etc. should be considered when identifying potential process-related impurities, and qualitative and/or quantitative studies should be carried out according to the situation to assess the safety risk of residual impurities. If necessary, impurity with potential safety risks should be included in the specification for control. Organic solvents, enzymes (if applicable), elemental impurities, conjugation reagents, nitrosamines (if applicable), etc. may be included.

　　3.3.3 Contaminants

　　Contaminants refer to particulates, microorganisms or related components introduced during manufacture, such as bacterial endotoxins, etc. During the manufacturing process, measures should be taken to avoid the introduction of contaminant and corresponding controls should be carried out. At the same time, necessary indicators should be monitored accordingly during release testing and stability studies of DS and DP.

　　3.4 Assay

　　Use appropriate physical, chemical, or immunological methods to determine the assay. For example, when use spectrophotometry to determine the protein concentration at 280 nm, the potential contribution of small molecules, buffer components, etc. to absorbance measured at 280 nm should also be studied. If significant interference occurs, appropriate correction factors or other quantitative methods should be introduced in the concentration calculation of test solution.

　　3.5 Other characteristics

　　Other characteristics analysis needs to be controlled in combination with product type and dosage form, which may include appearance, foreign particulate matter, abnormal toxicity (if applicable), subvisible particles, pH value, osmolarity, filling volume, reconstitution time (if applicable), moisture (if applicable) ), content of excipients, etc.

　　(II) Specification

　　As an important part of product quality control, specifications are generally determined based on product characteristics and quality study. Given the complex structure and special quality attributes of ADCs, quality control strategies should be comprehensively formulated through risk assessment methods based on development experience of naked antibodies and small molecule, quality studies, understanding of the critical quality attributes of the final products, and continuous knowledge accumulation of process, combined with non-clinical and clinical data.

　　The control strategys for naked antibodies and small molecule is basically the same as the routine control strategy for antibody drugs or chemical DS alone, so it will not be indicated in details.

　　1. Test items

　　Test items in the specification are usually determined on the basis of sufficient quality study, and are quality attributes that have an important impact on ensuring product safety and efficacy. Specifications for ADC DS and DP generally include appearance (such as appearance, color), identification, DAR and drug load distribution, purity and impurities, biological activity, assay, general tests, etc. For special dosage forms, additional test items are required according to the characteristics of the dosage form. Specific test items should also be determined based on product type, manufacturing process, stability and risk assessment, etc.

　　2. Limits

　　Limits of the specifications are usually based on the consideration of safety and efficacy, and should be formulated with a reasonable basis. Generally, product characteristics, non-clinical exposure and clinical trial exposure, data of batches used for each stage of the study, inter-batch consistency data, and stability should be considered. Additionally, the sensitivity and variability of the analytical methods used should also be considered.

　　3. Analytical methods

　　The development and validation of analytical methods should follow the general methodological rules of drug development, and should be gradually optimized, improved and validated with the accumulation of experience and continuous deepening of studies to meet the quality control requirements in all stages. New methods based on specific products should be fully validated; applicability of pharmacopoeia methods should be verified; If a modified pharmacopoeial method or an alternative pharmacopoeial method is used, comparative studies should be carried out to confirm the rationality. Based on the needs of product quality control and comparative studies at different stages of development, qualification studies of analytical procedures of safety-related test items and biological activity should be completed before clinical trials are initiated. Full methodological validation should be completed before applying for marketing. It is recommended that the development and validation of methodology be completed before the conduct of confirmatory clinical trials as much as possible to ensure that the quality control of samples used in confirmatory clinical trials is consistent with the quality control of commercial products. If methodological changes or transfers occur during research and development, corresponding methodological bridging studies and methodological qualification or validation should be carried out.

　　4. Standards/Reference

　　For the establishment and preparation of standards/reference, please refer to the relevant requirements of the “Preparation and Calibration of National Standard Substances for Biological Products” in the “Chinese Pharmacopoeia”. It is recommended to use representative batches to establish standard/reference, carry out the calibration of activity and assay according to the application, and carry out corresponding stability studies.

　　VIII. Stability study

　　Stability studies are important in overall drug development, marketing, and post-marketing quality studies, which is the basis for setting product shelf life and re-test date, providing a basis for manufacturing process, formulation, packaging materials, etc., and is also the basis for setting product specifications.

　　Stability studies can be carried out with reference to the relevant requirements of the “Technical Guideline for Stability Study of Biological Products (Trial)” and ICH Q5C, ICH Q1A and other guidelines, and should comply with the relevant provisions of “Storage and Transportation Regulations for Biological Products” in the “Chinese Pharmacopoeia”. The stability study subjects of ADCs generally include naked antibodies, small molecules, ADC DS, and ADC DP. The study protocol should be designed based on the product’s own characteristics, manufacturing process, clinical medication scheme, etc., and generally include long-term stability, accelerated stability, influencing factor stability, transportation stability, and in-use stability studies. The study conditions should fully consider the situations that may be encountered in manufacturing, storage, transportation and use. The test items should be comprehensive and reasonable. According to the characteristics of the product dosage form, test items that have important indicative significance for the safety and efficacy of the product should be selected. The test method should have the ability to indicate stability and be able to fully detect the change in product quality.

　　Payloads and linkers usually have highly active groups. In addition to long-term and accelerated stability studies, stability studies under high temperature, high humidity, light, oxidation and other conditions should also be carried out based on product characteristics, paying attention to the stability of small molecules during manufacturing, transportation and storage.

　　Due to the special and complex molecular structure of ADC DS and DP, there is a high risk of protein instability, so during stability studies, close attention should be paid to the aggregation and particle formation of proteins in the drug product, payload dropping and the adsorption of drugs in containers (such as infusion bags).

　　IX. Packaging and container closure systems

　　Packaging and container closure systems generally include packaging containers used for storing naked antibody, small molecule, ADC DS and DP. The hydrophobicity of ADC products may cause non-specific adhesion to containers. Therefore, appropriate packaging containers should be selected based on product characteristics. In order to avoid unintended effects of storage containers or closure systems on product quality, compatibility studies on container closure systems and sealing studies on container closure can be carried out with reference to relevant domestic and international guidelines. For packaging materials with special functions (such as light-shielding materials), their functions should be studied and validated.

　　X. Terminology

　　Naked antibodies: Antibodies or antibody fragments that target specific antigens, such as monoclonal antibodies (including monoclonal antibodies, bispecific antibodies or multispecific antibodies, nanobodies, etc.), antibody fusion proteins, Fab/ScFv fragments, etc.

　　Payload: a small molecule drug that works after an ADC drug reaches a tumor cell, tumor microenvironment, or other target cell. Currently, the most common payloads are small-molecule cytotoxic drugs.

　　Small molecules: In ADC products, the linker, payload or payload-linker linked to the naked antibodies. If the naked antibodies first reacts with the linker and then further reacts with the payload to form the ADC DS, then both the linker and payload are small molecules. If the payload-linker complex is first synthesized and then further reacted with naked antibodies to obtain the ADC DS, the payload-linker is a small molecule.

　　XI. Reference

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　　[10] ICH Q5E. Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process es. 2004.

　　[11] ICH Q5C. Stability Testing of Biotechnological and Biological Products. 1996.

　　[12] ICH Q6B. Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. 1999.

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　　[16] ICH Q11. Development and Manufacture of Drug Substances (Chemical Entities and Biotechnological/Biological Entities). 2011.