Expert Consensus on Quality Control and Preclinical Evaluation of Antibody-Drug Conjugates

Transcription

1 Expert Consensus on Quality Control and Preclinical Evaluation of Antibody-Drug Conjugates National Institutes for Food and Drug Control July 20 th, 2018

2 Contents 1. Overview Manufacturing Basic Requirements Process Validation Reference Material Control of Engineering Cells Control of Raw Materials for Production MAbs Linkers Small Molecule Drugs Control of Production Process Production of MAbs Conjugation Process for ADCs Extraction and Purification of ADCs ADC Drug Substance Final ADC Product Quality Control Quality Control of MAbs Quality Control of ADC Characterization Product Testing Stability Evaluation, Storage, and Shelf-life Preclinical Pharmacodynamics (PD), Pharmacokinetics (PK) and Safety Evaluation PD Evaluation of ADCs Investigations for Targeting Effects of ADCs Investigations for Endocytosis of ADCs by Tumor Cells Investigations for Antitumor Effect of ADCs Investigations for Antibody-mediated Effector Functions of ADCs / 39

3 5.1.5 Evaluation for Anti-tumor ADCs not Targeting Directly Tumor Cells Pharmacokinetic (PK) Evaluation of ADCs Preclinical Safety Evaluation of ADCs Formulation Analysis for ADC Test Substance Selection of Animal Species for ADC Safety Study General Toxicity Study for ADCs Tissue Cross Reaction of ADCs Toxicokenetic (TK) Studies of ADCs Immunogenicity Study of ADCs Genotoxicity Studies of ADCs Safety Pharmacology Study of ADCs Other Safety Studies of ADCs Closing Reference Drafters Advisers / 39

4 1. Overview Antibody-Drug Conjugate (ADC) is comprised of a monoclonal antibody (MAb) conjugated to a biologically active small molecule drug via a linker. Currently, most of ADCs are generated by the conjugation between an antibody targeting tumor antigen and a highly cytotoxic small molecule drug through a linker, which takes advantage of the specific binding of the antibody to the target antigen to deliver the small molecule drug selectively to tumor cells for its tumor killing effect. Compared with therapeutic antibodies, ADCs generally kill target cells with increased efficacy. However, it is undoubtedly more complicated to design ADCs than antibodies. It should consider each component of the ADCs: the antibody, the linker, the small molecule drug, and their rational combinations. Selection of an antibody is the start of an ADC design and also determines the potential indications of an ADC. The target antigen should generally have the characteristics of high-level expression related to tumors or diseases; the linker should be sufficiently stable in systematic circulation of the ADC and also capable of releasing the small molecule drugs effectively in the highly active form after the ADC reaches the target cell surface or enters target cells. The small molecule drug should be highly effective in killing tumor cells. Due to the complexity and unique properties of ADCs, drafting this Expert Consensus on Quality Control and Preclinical Evaluation of ADCs is of great significance to standardize and improve production and quality control of ADCs and thus, to ensure the safety, efficacy and quality of ADCs in China. This Expert Consensus provides guidance for the production, quality control, and preclinical evaluation of ADCs for human use. It is intended to provide technical reference for developers of ADCs. As innovative therapeutic antibodies, ADCs should be investigated in a phase appropriate manner in accordance with relevant technical requirements for novel drug development and regulatory application in China and other countries provided that the basic clinical safety is ensured. Companies should 3 / 39

5 adopt appropriate development strategies with science-based and risk-based approach during the life cycle of ADCs from development stages to market approval. In addition, the production and quality control involving MAb should also comply with the General Monograph of Recombinant DNA Technology Products, the General Monograph for Monoclonal Antibodies for Human Use and relevant requirements in the current edition of Chinese Pharmacopoeia. This Expert Consensus is applicable to ADC products of tumor indications due to the fact that current ADCs are mainly developed as anti-tumor products. For other types of ADCs, whether the Expert Consensus is applicable depends on the product features and assessment of relevance. It should be noted that the Expert Consensus will be revised accordingly with the continuous development and advancement of ADC technologies and products in order to gradually refine it as a technical consensus on quality control and preclinical evaluation for ADC products. 2. Manufacturing 2.1 Basic Requirements Manufacturing of ADCs mainly includes preparation of MAbs, linkers, and small molecule drugs, conjugation and purification of ADC drug substances, and production of final drug products. In some cases, the linker and drug may be produced together as a single linker-drug entity. In such a case, the requirements for the linker and small molecule drug should be applied to the combined moiety. The principles and concepts of Quality by Design and Risk Assessment should be adopted with the full understanding of complex quality attributes and clinical applications of ADCs. Appropriate control strategies of processes and quality should be determined with full process development and optimization (e.g., use of Design of Experiment, i.e. DOE method) conducted for each process step. Critical process steps should be identified and ranges for process parameters should be determined with the understanding of 4 / 39

6 impact on key quality attributes. Moreover, an effective quality management system should be established to ensure the safety and efficacy of products Process Validation Process validation for ADC manufacturing should consist of the validations for the processes of MAbs, linkers, and small molecule drugs, as well as ADC drug substances and final drug products. In the IND stage, the process of ADCs should be adequately understood and preliminary process validation for ADCs should be conducted. Prior to application for marketing, process validation for ADCs should be completed in a systematic and integrated manner to confirm critical process steps and critical process parameters with appropriate control of critical quality attributes, therefore to ensure reproducibility of the process, and controllability and batch-to-batch consistency of the product quality. The process validation for MAbs should refer to General Monograph for Monoclonal Antibodies for Human Use, including consistency of production processes, inactivation or removal of infectious agents, removal of product-related and process-related impurities, acceptable limits for reusability of materials for purification (such as column packing materials), batch-to-batch consistency of product quality, and monitoring of disposable materials required for production. The production process of linkers and small molecule drugs can be validated by referring to relevant requirements specified in ICH Q7, including the consistency of production processes, impurity limits, acceptable limits of organic reagent residues, and cleaning validation of reaction vessels. The process validation for ADC drug substances should be conducted by considering cross conjugation of the MAb and small molecule drug from different batches. It should also take into consideration of the consistency of processes, residuals of product-related and process-related impurities, acceptable limits of critical process parameters (e.g., input amount, reaction time, reaction temperature, and stirring speed, 5 / 39

7 etc.), batch-to-batch consistency of product quality, purification and ultrafiltration processes, and cleaning validation. It is particularly important to focus on the removal effectiveness and residual limits of free small molecule drugs, and establish in-process control testing (such as endotoxin and bioburden, etc.) for critical process steps. For the validation of the manufacturing process for ADC final products, critical process steps and their operation ranges should be confirmed for the filling and lyophilization (if applicable) to ensure controllability and batch to batch consistency of the final product quality Reference Material One (or more) batch (es) that has been shown to be sufficiently stable and suitable for clinical studies, or a representative batch of clinical study materials, can be selected as the reference material of the ADC. The reference material is used for analyses involving identity, physicochemical properties, biological activity and others. Comprehensive analytical characterization should be conducted according to the requirements for ADC characterization as described in Control of Engineering Cells The engineering cell management for ADCs should follow the relevant guidance documents such as General Monograph for Monoclonal Antibodies for Human Use. The source, management, and verification of cell strains should comply with Regulations for Bacteria and Virus Strains Used for Production and Quality Control of Biologics, Requirements for Preparation and Control of Animal Cell Matrix Used for Production and Quality Control of Biologics and current Good Manufacturing Practice. 6 / 39

8 2.3 Control of Raw Materials for Production MAbs The MAbs for ADCs should be produced and controlled in accordance with General Monograph for Monoclonal Antibodies for Human Use and Technical Guidelines for Quality Control of Monoclonal Antibodies for Human Use. Current state-of-the-art analytical methodology should be utilized for comprehensive analysis of the product from physicochemical, immunological, and biological perspectives. Detailed product information should be provided to adequately reflect the quality attributes of the target product. Characterization of the antibody should at least include physicochemical heterogeneity, structural integrity, amino acid sequence, higher-order structure, glycosylation, disulfide bonds, biological activity and immunological properties Linkers Preparation of Linkers It is applicable to the development of total chemically synthesized or semi-synthesized linkers. The development process should be controllable, stable, and scalable for industrial production while ensuring stability and batch-to-batch consistency of the product quality. The quality of the linker, such as correct structure, purity and types of impurities may impact its conjugation with the monoclonal antibody or small molecule drug. Comprehensive studies should be conducted for the key process parameters with reference to Technical Guidelines for Drug Substance Preparation and Structural Elucidation Study for Chemical Drugs and the relevant contents in ICH Q11. Such studies should at least include: selection of process, control of process parameters, requirements of starting materials and reagents, accumulation of process data, optimization and scale-up of processes, and removal and control of impurities. 7 / 39

9 Quality requirements of the starting materials should be developed based on their impact on the quality of the linker or linker-small molecule drug from the study results of the designed processes. For the impurities and isomers introduced from the starting materials, relevant studies should be performed and associated quality control methods should be provided when necessary. For chiral starting materials, enantiomer or diastereoisomer impurities should be monitored, and the appropriate control points and acceptable limits should be determined with full understanding of the process Quality Control of Linkers Quality control testing of the linkers should be developed with specifications reasonably set based on the characteristics of the linker process and quality control of the final product. In general, quality control testing should include appearance/description, structure elucidation, physicochemical properties (such as melting point, boiling point, specific rotation, solubility, etc.), purity (such as related substances, isomers), assay, bacterial endotoxin, and residual organic solvents. For the linker containing chiral centers, chirality studies should be conducted according to the relevant requirements. Limits of unknown impurities should be controlled. Additionally, stability study of the linker is also important for establishing appropriate storage condition and expiry date for the linker Small Molecule Drugs Preparation of Small Molecule Drugs It is applicable to the development of small molecule drugs that are total chemically synthesized or semi-synthesized or extracted from animals, plants, and microorganisms. The development process and requirements are generally consistent with those for preparation of linkers as described in Additionally, appropriate protective equipment, protection and emergency actions, and waste disposal plans should be developed considering the toxicity of small molecule drugs and associated reagents. 8 / 39

10 Quality control of Small Molecule drugs Quality control strategies should be formulated according to the structural characteristics, and physicochemical properties of small molecule drugs as well as the quality control need for the final products. It should take into consideration of the impacts on the quality of the final product imposed by the starting materials and reagents, process intermediates, by-products, organic solvents and other elements. The small molecule drug and the linker-small molecule drug should be confirmed for chemical structures or components, on which respective quality studies should be conducted. Structural elucidation studies may include FTIR, UV, NMR (13C spectrum, 1H spectrum, and 2D correlation spectrum if necessary) and MS methods. Assessment of the quality attributes should include physical state, identity, purity, assay and others. Special attention should be paid to conjugatable impurities, chiral isomers and setting limits of unknown impurities. For small molecule drugs with chiral centers, it is critical to control the starting materials and process intermediates appropriately, and analyze chiral isomers in the final products as necessary. Additionally, stability studies of the small molecule drug are also important for establishing appropriate storage condition and expiry date Physical attributes Descriptions of the appearance, including physical state (e.g., solid or liquid) and color. If any of the properties changes during storage, investigations should be conducted and appropriate measures taken. Optical rotation or specific optical rotation measurement should be performed for optically active small molecule drugs as appropriate Identity Methods used should be highly specific, sensitive and repeatable and easy to operate. Common methods include chemical reaction, chromatography and spectroscopy Inspection 9 / 39

11 Generally, it should take into account the effects of the small molecule drug on the safety, efficacy and stability of ADCs. The impurity testing may consider general impurities (such as chlorides, sulfates, heavy metals, arsenic salts, and residues on ignition, etc.), related substances, solvent residues, crystal forms, loss on drying or water content, and isomers. The impurities that may be generated in the process of production as per established processes and during normal storage, such as process impurities, degradation products, isomers and solvent residues, need to be well characterized and controlled Content assay Methods of content assay should be highly specific, precise and accurate, and can reflect the stability of the product during appropriate stages of development. 2.4 Control of Production Process The production process of ADCs is complicated. In order to ensure the ADC products with controllable quality and high batch-to-batch consistency, the quality standard and expiration should be established for the key materials, including MAbs, linkers and small molecule drugs. If intermediates are to be stored, corresponding stability study should be conducted on the storage conditions for the intermediates Production of MAbs The guidance document General Monograph for Monoclonal Antibodies for Human Use can be referred to for the requirements of cell culture and harvesting, purification and production of drug substance of the MAb Conjugation Process for ADCs Antibody Structure Modification The structure modification of an antibody refers to the formation of a specific 10 / 39

12 activating group on the antibody by biological or chemical reaction to facilitate the subsequent conjugation reaction. There are typically two types of conjugation between antibody and small molecule drug: non-specific conjugation and site-specific conjugation. The former is generally the conjugation with the small molecule drug via the amino group of lysine or the sulfhydryl group of cysteine on the antibody molecule. The latter is achieved by modification of antibody molecules to facilitate site-specific linking of small molecule drugs, and such modifications include, for example, introducing cysteine or non-natural amino acids of special structures at a specific site or forming a specific conjugating site through deglycosylation with biological enzymes. Depending on the types of modification reactions and the impacts of various factors on the final product, process parameters such as reaction concentration, amount of input materials, temperature, time, buffer system, ph, type or amount of organic co-solvent should be carefully studied. With accumulation of process and product knowledge, appropriate in-process control testing (e.g., bacterial endotoxin, etc.) and operation ranges should be established to finalize the process conditions. Compatibility of the reaction system to the selected containers should also be studied in the appropriate stage of process development Conjugation Reaction The conjugation reaction refers to the process of forming an intact ADC molecule by linking the small molecule drug to the reactive group on the antibody through biological or chemical reactions. As described in the process of antibody structure modification in , reaction concentration, reactant ratios, temperature, time, buffer system, ph, type and amount of organic co-solvent in the reaction system should be investigated. Appropriate in-process control testing (e.g., DAR, SEC, bacterial endotoxin etc.) and operation ranges should be established in the finalized process conditions through continuous accumulation of process and product knowledge. Compatibility of the reaction system to the selected containers should also 11 / 39

13 be studied in the appropriate stage of process development Extraction and Purification of ADCs The crude conjugation product should be extracted and purified with the processes proven to effectively remove process-related and product-related impurities from the conjugation reaction. Process-related impurities include residual reducing agents, oxidants, catalysts, reactive enzymes and organic solvents, etc. Product-related impurities include free linkers, small molecule drugs, linker-small molecule drug intermediates, unconjugated antibodies, and ADC aggregates, etc. Attention should also be paid to the control of microbial contamination in the process (such as bacterial endotoxins, etc.). Compatibility assessment should be conducted between purification buffers and contact materials in appropriate stage of the process development ADC Drug Substance The ADC drug substance refers to the purified ADC stored in an intermediate storage container after formulation and sterile filtration. For the drug substance storage, studies to assess the compatibility of the drug substance with its container, and stability of the drug substance should be performed to determine the appropriate storage conditions and expiry date Final ADC Product The drug substance or the semi-finished product will constitute the final product once it is filled and stored in sterile containers after sterilization, filtration and packaging. The container closure system should be assessed for sterility to prevent contamination. If freeze drying is required, it should be lyophilized first and followed by sterile closure. 12 / 39

14 3. Quality Control Compared with MAbs, ADCs have more complicated structures and more special quality attributes. The quality control strategy should be formulated comprehensively based on the quality evaluation of key raw materials (MAbs, linkers and small molecule drugs, etc.), the understanding of key quality attributes of final product, and the continuous accumulation of process knowledge, in combination with risk assessment methods. 3.1 Quality Control of MAbs Relevant technical requirements should refer to the Technical Guideline for Quality Control of Monoclonal Antibodies for Human Use and the General Monograph for Monoclonal Antibodies for Human Use. In general, the antibody must be well-characterized. Appropriate quality control strategies should be developed based on critical quality attributes, accumulated MAb quality and process knowledge, and risk assessments. In addition, quality attributes that may impact the conjugation process need to be well-studied and appropriately controlled. 3.2 Quality Control of ADC Characterization Comprehensive characterization of an ADC should include the determination of physicochemical properties, immunological properties, biological activity and impurities, etc., by appropriate and advanced analytical techniques in a comprehensively detailed manner. Detailed information should be provided as much as possible based on the full understanding of relevant characteristic changes before and after conjugation by characterization of naked antibodies to reflect the quality attributes of the final product and to provide the reference for establishing specifications for the product. The characterization, at a minimum, should include the 13 / 39

15 following categories: Physicochemical Properties It is known that MAbs commonly display complicated heterogeneity (e.g., glycosylation and other post-translational modifications). ADCs have a greatly increased level of complexity that arises from the heterogeneity of the conjugation, layered on top of the variability associated with MAbs. The increased heterogeneity associated with ADCs, even site-specifically conjugated ones, requires comprehensive characterization using robust methods with sufficient resolution to characterize and measure the diversity of drug product-related species. Selection of the most appropriate characterization methods will depend on the chemical properties of the small molecule drug and linker, the mode of conjugation (amino conjugation, sulfhydryl conjugation, and site-specific conjugation, etc.) and the resulting complexity of the product. The physicochemical characterization for an ADC generally includes assessing the impact of the conjugation process on antibody primary structure, a determination of primary sites for drug conjugation, drug-to-antibody ratio (DAR) and drug load distribution, size and charge variants, and higher order structure analysis Primary Structure and Drug Conjugation Site Impact of the conjugation process on the MAb primary structure (e.g., integrity of the amino acid sequence, disulfide bridges, glycosylation and post-translational modifications) are assessed by suitable methods such as peptide mapping and mass spectrometry analysis. If the conjugation process is demonstrated not to affect glycosylation, characterization associated with glycosylation may not be necessary for conjugated ADCs. In addition, mass spectrometry may be used to identify and analyze the primary conjugation sites of small molecule drug to MAb in the ADC DAR DAR is one of the important quality attributes of an ADC and directly affects both 14 / 39

16 safety and efficacy of the ADC. It represents the average count of small molecule drugs conjugated to each antibody molecule. Depending on the chemical properties of the linker and the small molecule drug and the mode of conjugation, DAR may be analyzed by common methods, including ultraviolet and visible spectrophotometry (UV), hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC), reversed phase-high performance liquid chromatography (RP-HPLC), and mass spectrometry (MS), etc Drug Load Distribution ADCs, especially non-site specific ADCs, are typically mixtures containing ADC molecules linking to different numbers of small molecule drugs. Drug load distribution refers to the proportion of ADC molecules with different numbers of small molecule drugs to the total ADC molecules, respectively. The distribution of components with different numbers of the drugs (e.g., fraction of antibodies containing 0, 1, 2 n drugs) should be characterized by an appropriate method, such as HIC-HPLC, RP-HPLC, capillary electrophoresis (CE), or mass spectrometry (MS), etc Molecular Size Variants As with Mab for human use, the molecular size variants (i.e., aggregates and fragments) of ADCs should be appropriately characterized using a combination of methods such as size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), non-reduced and reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) and analytical ultracentrifugation (AUC), etc. Particular attention needs to be paid to aggregates because many of small molecule drugs that are conjugated to antibodies are highly hydrophobic and can increase the likelihood of aggregate formation during production and storage Charge Variants 15 / 39

17 For MAbs, charge variants are commonly determined by a suitable method, such as capillary zone electrophoresis (CZE), ion exchange-high performance liquid chromatography (IEX-HPLC), capillary isoelectric focusing (CIEF), or imaging capillary isoelectric focusing (icief), etc. The applicability of these methods to ADC analysis will rely on the characteristics of the linker-drug (especially the charge), along with the choice of conjugation site (such as lysines, inter-chain sulfhydryls, carbohydrates, etc). Although IEX-HPLC is a common method to characterize charge variants of antibodies, it may not be applicable to the analysis of charge variants of ADCs with non-specific conjugation due to the potential of multi-site conjugation of small molecule drugs and the possible non-specific interactions with the column stationary phase. In some cases, charge-based analysis may not be possible or meaningful after conjugation to lysine residues. For example, each uncharged linker-drug molecule conjugated through lysine residues decreases the net positive charge by one for each ADC molecule. In this case, charge-based separation results in profiles that characterize the drug load of the lysine conjugated ADC, and thus cannot reflect the charge heterogeneity of the antibody itself. Characterization methods such as peptide mapping may be needed to assess the impact of conjugation on the charge heterogeneity of the antibody Higher-order Structure The higher-order structures of proteins are dictated by both the amino acid sequence and post-translational modifications. Therefore, the confirmation of primary structure of a protein is fundamental to the characterization of its structural properties. Physicochemical methods that provide a direct assessment of the covalent structure of an ADC molecule are described in sections to Classical biophysical tools, e.g., circular dichroism (CD), differential scanning calorimetry (DSC), dynamic light scattering (DLS), and Fourier transform infrared spectroscopy (FTIR), may be used to characterize the biophysical properties of an 16 / 39

18 ADC. However, antibodies or ADCs are biomacromolecules which often limits the ability of these tools (e.g., CD and FTIR) to detect the structural heterogeneity with desired sensitivity. The analysis results may be complicated by the presence of each component when studying the ADC products. In addition, higher-order structure of the ADC may be confirmed by biological function (section ). Biological activity is the validation for higher-order structure, and additional in vitro or in vivo assays that illustrate the functional activity of the product may serve as a supplemental confirmation of the higher-order structure Immunological Properties and Biological Activity For the ADC product, impact of conjugation on the immunological properties (e.g., antigen binding) of the antibody are assessed by appropriate methods, e.g., enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR), etc. The ADC should maintain the specificity of its binding to the target antigen and the consistency of its binding activity from batch to batch. The primary mechanism of action of ADCs is to bind to an extracellular target antigen, be internalized, and then kill the cell with the small molecule drug. Target-dependent cytotoxicity (the biological effect) should be demonstrated by a cell-based assay to confirm this mechanism of action. For antibodies, Fc-mediated effector function may play a role in the mechanism of action and have an impact on the product safety and efficacy. For ADCs, Fc-mediated effector functions may not contribute significantly to the anti-tumor activity because of the competition with internalization. However, if the Fc-mediated effector functions are shown to be possibly relevant for clinical activity of ADCs, a cell-based bioassay or another assay that reflects effector functions should be performed Process-related Impurities and Contaminants Process-related Impurities 17 / 39

19 Potential process-related impurities (e.g., free small molecule drug and its related substances, residual solvents, and heavy metals) should be identified, and evaluated qualitatively and/or quantitatively, as appropriate. One approach to residual free drug analysis for ADC product is to precipitate the proteins (including the small molecule drug conjugated on ADC molecule) and then analyze the residual free small molecule drug in the resulting supernatant using a method that is capable of detecting the small molecule drug. Other approaches to prepare samples may be used as well. Rational limit standards should be set for the amount of residual free small molecule drug based on related small molecule drug s pharmacological and toxicological properties and the maximum usage dose of the drug Contaminants Contaminants, which include all adventitiously introduced materials and not intended to be part of the manufacturing process (e.g., microbial species, bacterial endotoxins) should be strictly avoided and/or suitably controlled Content It is recommended that the content is determined using appropriate physical, chemical or immunological assays. For example, after determination of extinction coefficient of the ADC, spectrophotometry can be used to determine the protein concentration at 280 nm. For ADCs, in addition to the polypeptide backbone, potential contribution of the small molecule drug or linker-drug to the measured absorbance at 280 nm should be considered. If significant interference is detected, appropriate correction factor should be incorporated into the calculation of the test substance concentration Product Testing Similar to other recombinant DNA proteins for human use, quality control testing of ADC drug substances and products needs to account for its identity, purity, content, and potency. However, some specific features need special consideration because of the structural complexity of ADCs and the presence of small molecule drugs. Some 18 / 39

20 quality attributes derived from the antibody (e.g., glycosylation and other post-translational modifications) are most appropriately controlled at the point of manufacturing and control of the antibody. Routine quality control release testing and acceptable limits for ADCs should be determined comprehensively by combining the data from multiple representative batches, batch-to-batch consistency data, and stability study data. The quality control testing of ADC products should include at least the following items Identity The identity test(s) is highly specific and based on unique aspects of the product s molecular structure and/or other specific properties. Methods used to determine identity must be adequately specific for an ADC to confirm that the product contains both essential components (the antibody and the small molecule drug). Depending on the product characteristics, one or more than one physicochemical, biological and/or immunochemical test method may be required for identification DAR and Drug-load Distribution The DAR should be determined and acceptance criteria are established accordingly. The testing results should be within the specified range. Qualitative assessment of the drug-load distribution profile should be included Purity and Product-related Impurities As noted in the characterization section, ADCs may display a complex purity/impurity profile that is assessed by a combination of orthogonal methods. Individual and/or collective acceptance criteria should be established for product-related variants. Molecular size variants are determined by accurate and robust methods to measure the extent of aggregation and fragmentation in ADCs. If possible, charge variants should also be determined by a suitable method Process-related Impurities 19 / 39

21 The control methods of process-related impurities should be included in the control strategy. In some situations, and where appropriately demonstrated, the control of key impurities from the conjugation process may be achieved at an appropriate step. Control of free small molecule drug and residual conjugation solvent is typically part of the product specification. Testing results should be within the specified limit Potency Potency is the quantitative measure of biological activity based on an attribute of the product which is linked to the relevant biological properties. A potency assay should be part of the specifications for drug substance and drug product and reflect the clinical relevant biological activity as much as possible. Target-dependent cytotoxicity should be demonstrated by a cell killing-based potency assay for ADCs based on the mechanism of action of ADCs. A target binding assay (e.g., ELISA, SPR, etc.) may be required as a part of routine quality control release testing after validation if it provides more information about product quality. The potency of each batch of drug substance and drug product should be determined using an appropriate national standard, international standard, or reference. In the absence of national or international standard, approved and well-characterized in-house reference standard may be used. The establishment and preparation of standard and reference should comply with the Regulations for Preparation and Calibration of National Standard Substances for Biological Products Content The content of the drug substance and final drug product should be determined using an appropriate assay Safety Testing Testing for sterility, bacterial endotoxins and bioburden should comply with the requirements (see General Monograph for Monoclonal Antibodies for Human Use ). 20 / 39

22 The toxicological data and clinical dose of ADCs should be considered for the abnormal toxicity testing to assess the applicability, rationality and operability of the testing Other Testing Appearance (such as physical state, color, clarity), visible particles, ph, osmolality, content uniformity, and insoluble particles should be conducted as appropriate. In the case of lyophilized products, reconstitution time, and water content, etc, should also be considered. 4. Stability Evaluation, Storage, and Shelf-life The stability evaluation for ADCs should be conducted based on Technical Guidelines for Stability Study with Biologics. It should also comply with the Regulations for Storage and Transportation of Biologics. The types of stability evaluation include long-term stability, accelerated stability, and stress testing. Since the study conditions for long-term stability are the actual storage conditions for the ADC product and the degradation pathways and degradation products (including MAb portion and small molecule drug portion) that occur in the process are also actually present in the product, the long-term stability testing is considered the most basic stability study. The storage conditions and the shelf life should be specified according to the results of the long-term stability study. Products should be stored and transported under the specified environmental conditions. It should be subject to the approved shelf life from the date of manufacture. 5. Preclinical Pharmacodynamics (PD), Pharmacokinetics (PK) and Safety Evaluation The structure of the ADC is complicated and ADC design varies significantly for 21 / 39

23 different types. Even if the ADC acts on the same target, its mechanism of action, plasma stability, in vivo metabolic process, and toxic side reactions may vary due to differences in its recognized epitopes, conjugation sites, linkers, and the conjugated small molecule drugs. Therefore, targeted preclinical studies and evaluations should be conducted according to the structural characteristics of ADCs and expected biological process in a case-by-case manner. This section is based on domestic and international guidelines for biologics and anti-tumor drugs and published literature, as well as expert consensus, to provide recommendations for the specific conduct of preclinical studies with anti-tumor ADCs. Primary considerations in preclinical studies of ADCs include: 1) antibody characteristics in the ADC, e.g., clear target, definite pathophysiological function of the target with specific distribution on the disease sites, target or non-target tissue-specific binding, and stability of linkages; 2) characteristics of linkers, such as the type of linkers, the stability in systematic circulation, and the ability of the effective release of the small molecule drug in the highly active form when entering cells, as well as the mode of cytotoxicity of the delivered small molecule drug; 3) characteristics of small molecule drugs, for example, whether it is a new compound, whether the toxicity characteristics are known or reported in the literature, and whether the mechanism of action is clear. 5.1 PD Evaluation of ADCs The mechanism of action of ADCs relies on the specific recognition of target tumor cell surface antigens through the targeting of the MAb and the endocytosis mediated by the target antigen to allow ADCs to enter tumor cells for hydrolyzing or enzymatic hydrolyzing and then releasing small molecule drugs so as to kill tumor cells. PD studies and evaluations should be conducted according to the structural characteristics of drug and the technical characteristics of product Investigations for Targeting Effects of ADCs ADCs act by MAb providing targeting effects for small molecule drugs to enter tumor 22 / 39

24 cells. The affinity of MAb to the target may be affected after conjugated to the linker and small molecule drug. As a result, the overall targeting effect of ADCs is critical in the development of ADCs. Full evaluations are recommended to be conducted with in vitro and in vivo methods, and other methods, as well as design protocol. The binding activity (affinity) of ADCs to target antigens can be tested in vitro with methods such as Biacore, competitive ELISA, flow cytometry, etc. The enrichment of ADCs in tumor tissues can be investigated in vivo by tissue-distribution test in tumor-bearing mice with tumor cell highly expressing target antigens (evaluations may be conducted by using radio-immunoassay or determining the small molecule drug if the routine ELISA cannot be used due to interference of the antigen). In vivo and in vitro targeting trials may use cells or tumor-bearing mice not expressing the target antigens or ADCs without specific targets as controls, and the targeting characteristics of ADCs are deployed in a better manner compared with the control, while considering the addition of a small molecule drug group, to further demonstrate the targeting of the ADC. In addition, after the MAb and the small molecule drugs form ADCs, the potential changes of spatial structure may affect the binding specificity to the target antigen. Therefore, it is necessary to investigate the specific binding of the ADC to the target antigen, and it may be accessed by some non-marker techniques such as ELISA or SPR, etc Investigations for Endocytosis of ADCs by Tumor Cells The ADC-induced endocytosis is also the key to the efficacy of certain ADCs and constitutes important part for investigation in the development stage of ADCs. It is affected by a variety of factors, such as the selectivity of epitopes, the affinity of the antibody binding to the antigen, and the mode of transport of the ADC drug into tumor cells, etc. Therefore, it is necessary to screen and select the ADC with the most appropriate endocytosis in the early stage of development. For IND application, further investigation such as endocytic dynamics is recommended. The in vitro techniques such as flow cytometry or immunofluorescence confocal microscopy can 23 / 39

25 be used to investigate the endocytosis of ADCs. Cellular pharmacokinetics is also a good method to determine the efficiency of endocytosis through comparison of the amount of small molecule drug in negative and positive cells. Certainly, this investigation can be ignored for ADCs exerting efficacy without endocytosis Investigations for Antitumor Effect of ADCs The specific in vitro and in vivo evaluation methods and models are similar with those used for other anti-tumor drugs. Various conventional or validated methods and tumor models can be used to detect ADC antitumor activity in vivo and in vitro. Grouping design is particularly critical in a specific trial. Several critical control groups are recommended other than common active control groups: 1) MAb controls: for MAb with either antitumor effects or only targeting effects, the set of MAb controls allows the investigation on whether the ADC has a stronger antitumor effect to show its superiority; 2) irrelevant MAb ADC controls: the ADC molecule is a macromolecule drug, and if in a solid tumor-bearing model, develops the enhanced permeability and retention effect (EPR) of the solid tumor in blood circulation, and the irrelevant ADC shows some inhibiting effects on tumor growth. The set of irrelevant ADC controls allows investigations on the specificity of the target ADC; and 3) small molecule drug controls with molar content equivalent to the target ADC: which allow an investigation on the high efficacy and low toxicity of the target ADC. Studies on the antitumor effects of ADCs require multiple pre-trials and the design of control groups, which may be considered overall based on pilot mechanism studies, in vitro tests, or early exploratory efficacy studies in vivo Investigations for Antibody-mediated Effector Functions of ADCs The effector functions, namely ADCC and CDC effector functions differ for different subtypes of IgG antibodies. IgG1 subtype has stronger ADCC and CDC effector functions, while IgG2 and IgG4 subtypes have weaker ADCC and CDC effector 24 / 39

26 functions. The antibody s effector function is required for some ADCs to exert greater anti-tumor effects; while for some other ADCs, potential non-targeted toxicity caused by the effector function should be avoided. In addition, the effector function of antibody may change after conjugation with small molecule drugs. Therefore, it is necessary to test the effector function of ADCs in which the naked antibody has the Fc effector function to determine the presence of the expected effector function. However, the effect of antibody-mediated ADCC and CDC in ADCs is no longer a major contributor, and consideration can be given to whether or not to reduce the investigation on antibody-mediated effector functions Evaluation for Anti-tumor ADCs not Targeting Directly Tumor Cells Some tumor-specific antigens are expressed on new vasculature of tumor tissues or stromal cells in tumors instead of on tumor cells, and special considerations should be given to the PD evaluations for anti-tumor ADCs designed for these antigens. Common tumor cell proliferation inhibition in vitro assay does not apply to evaluations for such ADCs, for which PD evaluations may be conducted with other appropriate assays designed based on the mechanism of action. HUVEC proliferation inhibition assay and chorioallantoic membrane vascular assay may be considered if the ADC targets endothelial cells to inhibit angiogenesis provided with related models of pharmacological effect. In addition, in vivo PD evaluation with surrogate molecule or PD with target antigen humanized animal models are recommended, if the antibody in an ADC binds specifically to human target antigens only rather than to murine target antigens. 5.2 Pharmacokinetic (PK) Evaluation of ADCs The complexity and diversity of the structure, the low level of small molecule drug delivered in biological samples and other specificities of ADCs pose great challenges to the PK studies, mainly on the complexity and diversity of PK profiles, PK-PD correlation, target analytes, methods of biological analysis and data interpretation. 25 / 39

27 Relevant animal species are used for PK studies of single and multiple doses according to the specificity of ADCs. PK studies may be conducted only in non-rodents if there is no relevant species for the test substance ADC, or only non-rodents are the relevant species. The PK differences between different species of animals result in significant differences in predicting the dose-effect relationship in toxicity tests. A specific test should be conducted following the case-by-case principle based on the drug characteristics. The formulation, concentration, and mode of administration of test ADC for PK studies should be consistent with those used for safety evaluation tests or clinical trials as much as possible, with the concentration to be confirmed for the formulations to be administered in studies. The analytes that are commonly used to characterize the PK of ADCs include: 1) complete ADCs and/or total antibodies (antibodies that are conjugated and unconjugated are total antibodies and antibodies that are conjugated with at least one drug), and if antibodies compete with ADCs for target binding, the detection of antibodies is also of some significance; 2) free small molecule drugs and/or naked antibodies. Primary tests in PK studies of ADCs include: ADCs stability, the area under the concentration-time curve, absorption, distribution, metabolism, and excretion (ADME) process. If the small molecule drug is a new compound, it is recommended that in vitro and in vivo methods, qualitative and/or quantitative methods be used to conduct detailed studies on the systemic exposure, plasma protein binding and excretion profile, uptake/distribution characteristics of tumors and normal tissues of small molecule drugs. Systemic exposures, metabolite profiles, distributions, shedding patterns, and breakpoints may be systematically studied for small molecule drug s metabolites, if necessary. In vitro study of plasma stability and metabolic stability in animals may be conducted for ADCs by the measurement of small molecule drug s shed amount. In addition, it is recommended to consider the impact of the stability of the linker on the unexpected early release of the small molecule drug and on PK and 26 / 39

28 PD, and toxicity. PK analytical methods of ADCs include ligand binding assays for the determination of antibody and LC-MS/MS for the determination of small molecule drug and metabolites. In some cases, traditional methods of detection are difficult to meet the need for accurate biological analysis of antibody molecules in ADCs and lower limit of detection for in vivo assay for concentration of small molecule drugs, and it requires effective biological analysis methods with high sensitivity and wide linear detection range to evaluate the stability and metabolic characteristics of ADCs in vivo. The effect of DAR on the results of detection should be investigated for the establishment and validation of bioanalytical methods with the monitoring of changes of DAR in vivo over time when necessary. The detection of ADA is recommended to be conducted during the PK study of ADC to help interpret data from PK study. 5.3 Preclinical Safety Evaluation of ADCs In general, the preclinical safety study on ADCs should be conducted under GLP conditions in accordance with Good Laboratory Practice. For some exploratory studies in early development or tests that are difficult to conduct under GLP, it also may be conducted under non-glp condition, impact on the quality of study to be clarified, if necessary Formulation Analysis for ADC Test Substance The test substances for nonclinical studies should be samples with adequate representation of the quality attributes of the proposed samples for clinical trials. The test report of physical and chemical properties of test substance should cover the information such as source, batch number, purity, concentration, and composition, etc. If the test substance is to be administrated after dissolution (mixture/suspend/ dissolution), it is also necessary to test and verify the stability and homogeneity of the solution of the test substance (the range of concentrations should cover those in all of 27 / 39