Center for Drug Evaluation, National Medical Products Administration

　　May 2022

I. Introduction

　　In recent years, the rapid development of biotechnology has promoted the research and development of immune cell therapy products ，and provided a new therapeutic means for some serious and refractory diseases. In 2017, the former China Food and Drug Administration issued the Technical Guidelines for the Research and Evaluation of Cell Therapy Products (Trial), which comprehensively described the pharmaceutical technical requirements for cell therapy products under study and application in accordance with relevant regulations for drug management. Due to the great differences in cell source, type and in vitro operation among different immune cell therapy products, the quality study and quality control are more complex than traditional drugs. This guideline is developed to standardize and guide the R & D and evaluation of immune cell therapy products in accordance with Good Manufacturing Practice.

　　Based on the current scientific knowledge, the Guidelines provide general technical principles and recommendations for pharmaceutical study of immune cell therapy products, without mandatory content. Applicants/holders may also use other effective methods to carry out the study based on the specific circumstances of the product and explain the rationality. With the development of technology, the deepening of cognition and the accumulation of experience, the technical requirements of related products will be gradually revised and improved.

II. Scope

　　In the Guidelines, immune cell therapy products refer to the immune cell therapy products that are derived from human (autologous/allogeneic) cells or human-derived cell lines and undergo in vitro operation, including but not limited to separation, purification, culture, expansion, induced differentiation, activation, genetic modification, establishment of cell bank (line), cryopreservation and resuscitation, and then infused or implanted into the patient's body to treat diseases by inducing, enhancing or inhibiting the body's immune function, such as chimeric antigen receptor T cells (CAR-T), dendritic cells (DC), etc.

　　For the somatic cells such as islet cells and chondrocytes, as well as the cellular parts of combination products of cells and non-cellular components, may also refer to the Guidelines. For cell-derived products, such as exosomes, cell lysates, inactivated cells and other products, pharmaceutical research on the cell part may also be applicable. For gene-modified immune cell therapy products (such as CAR-T, etc.), the cell part may refer to the Guidelines, and gene-modified part may refer to other relevant technical guidelines. The Guidelines are unapplicable to stem cell , the hematopoietic stem cells for blood transfusion or transplantation , germ cells, as well as the tissue-like or organoid products composed of cells.

　　The Guidelines are applicable to the immune cell therapy products under R & D and registration application in accordance with relevant regulations for drug management, and mainly applicable to pharmaceutical study at the stage of marketing application.

III. General Principles

　　The immune cell therapy products developed and applied for registration as drugs shall meet the requirements in Drug Administration Law of the People's Republic of China, Provisions for Drug Registration, Pharmacopoeia of the People's Republic of China (hereinafter referred to as Chinese Pharmacopoeia) and other relevant laws and regulations. For immune cell therapy products, the manufacturing process should comply with the basic principles and relevant requirements of Good Manufacturing Practices (GMP). Biosafety shall comply with the requirements of relevant national laws and regulations. The source and treatment of human tissues, cells and genes shall meet the requirements of relevant laws and regulations of China for the administration of human genetic resources.

(I) Research and Development Rules

　　The CMC research of immune cell therapy products follows the general rules of drug research and development and runs through the whole life cycle of the product. Such products are highly personalized, complex and diverse processes, sensitive to the environment, and have a short shelf life under non-frozen state, while the cells themselves have the ability to survive in vivo, proliferate and/or differentiate autonomously, and interact between cells, etc. The CMC study shall fully consider the above basic characteristics and particularities of the product. While meeting the technical requirements at different stages, the CMC study shall be continuously optimized and improved to enhance the quality of the product.

　　1. Clinical Trial Stage under Application

　　The CMC technical requirements in the clinical trial stage under application should be evaluated and judged as a whole in combination with the characteristics of the product and the specific situation of the manufacturing process. To ensure the safety of subjects, the application for clinical trials usually focuses on the safety-related aspects, such as the quality control of raw materials for production, the measures to reduce the risk of confusion/contamination/cross-contamination, process stability, safety-related critical quality attributes, and the quality comparability between the samples of non-clinical study/non-registered clinical study (if applicable) and clinical trial samples. In addition, the production conditions of clinical trial samples should meet the basic principles of GMP.

　　In general, the following studies should be completed when applying for clinical trials: carry out adequate safety analysis on the raw materials and excipients used in the manufacturing process, especially human-derived/animal-derived materials, to evaluate the necessity and rationality of use. The manufacturing process shall be evaluated through transformation study from laboratory process to clinical trial process, to determine the steps and parameters of cell manufacturing process that are compatible with the clinical trial stage, as well as the control measures of manufacturing process, so as to support the rationality and stability of process, meet the capacity demand of samples for clinical trial and ensure the safety and quality controllability of the product. Complete the safety-related quality study, such as exogenous factors and impurities, and complete the relevant methodological validation. For quality control, set the quality standards adapted to clinical trial stage. Safety related quality control may be combined with quality study and may refer to existing safety standards or consensus standards for similar products. In addition, it is necessary to compare and analyze the similarities and differences of manufacturing process (broadly including raw materials, site, manufacturing process, scale, etc.) and sample quality between non-clinical study, non-registered clinical trial (if applicable) and clinical trial, and perform risk assessment and in-depth study when necessary. In the clinical trial stage under application, the stability study conditions for storage, transportation and use shall be representative, and the stability study data shall be able to support the actual storage / transportation of clinical samples. The materials in direct contact with samples shall be subject to safety and applicability evaluation.

　　2. Marketing Stage under Application

　　Based on adequate process development and research, a mature and stable commercial manufacturing process shall be established, which can consistently and steadily produce safe, effective and quality-controlled products. The commercial manufacturing process should be subject to comprehensive process validation, the quality of the materials for production should be strictly controlled, and the key manufacturing steps, range of key process parameters, key process control items and acceptance criteria should be specified. Establish reasonable quality standards through adequate quality study, methodological validation and stability study. According to the standard stability study and compatibility study of packaging materials, the shelf life of the product is established, the conditions and duration during the transportation and use are specified, and the appropriate packaging containers/materials are determined.

　　3. Process Change

　　The applicant/holder is encouraged to continuously improve and optimize the manufacturing process and continuously enhance the product quality. In case of process change, risk evaluation / corresponding comparability study shall be carried out according to the change, and the comparability of product quality before and after the change shall be analyzed to prove that the change has no adverse effect on the safety, efficacy and quality controllability of the product.

(II) Technical Considerations Related to Product Characteristics

　　1. Raw Materials for Production

　　The raw materials used for the production of immune cell therapy products have various sources and have different degrees of risks. The risk assessment and quality control should be conducted according to the relevant requirements of Chinese Pharmacopoeia, and a good and standardized quality management system for the raw materials used for production should be established. The raw materials with high specification level or low risk level shall be preferentially selected, to perform the biosafety assessment and control for the raw materials of human/animal origin, so as to reduce the risk of introduction or transmission of exogenous factors.

　　The cells for the production of immune cell therapy products may be from autologous source, allogeneic source, human cell line source, etc. For the collection of cells/tissues involved, it is necessary to establish reasonable and clear quality evaluation content, review and screening principles and criteria of medical institutions. It is recommended that applicants/holders should evaluate, review and screen the quality of cooperative medical institutions and ensure the standardization of the collection process by establishing and using the collection operation documents. In addition, in combination with the quality study, it is necessary to establish donor screening criteria and quality requirements for the collected cells/tissues, and establish clear operating specifications for the preservation, transportation and incoming inspection of the collected cells/tissues.

　　2. Manufacturing Process

　　The manufacturing process of immune cell therapy products is complex, without virus removal and terminal sterilization steps. The manufacturing should follow the principles and requirements of GMP. The manufacturing process should be validated and clear process control should be established. Special attention should be paid to personnel, premise and equipment, raw material control, environment and facilities in this process. The overall partition layout of the manufacturing plant should be reasonable. Each partition should be reasonably designed, laid out and operated according to the process steps and corresponding cleanliness level, which can meet the quality management requirements for the manufacturing of immune cell therapy products. It is recommended to use automatic, continuous, closed or semi-closed manufacturing equipment as far as possible and use special, product-specific devices that can meet the requirement to minimize the risk of contamination of microorganisms and various particles. Establish the site-opening and site-clearing system, and establish the whole-process control system to avoid the exogenous contamination or cross contamination that may be introduced in the raw materials for production and the manufacturing operation process. In the manufacturing process, attention should be paid to the operation time and spatial isolation of cells from different donor batches to avoid the confusion and cross-contamination of samples from different batches. Establish a product traceability management system to ensure the traceability of products from suppliers to recipients.

　　3. Quality control

　　The quality control strategy of immune cell therapy products includes material quality control, manufacturing process control, quality test of intermediate samples, release test of final products and sample retention test. In principle, each batch of products shall pass quality control and be released after passing inspection. However, considering the particularity of immune cell therapy products, on the premise of controlling risks to the greatest extent, reasonable and flexible quality control strategies may be developed in combination with the urgency of clinical use and the ways/times of product storage and transportation.

　　4. Storage and Transportation

　　The storage and transportation process of immune cell therapy products may involve freezing or refrigeration. The storage and transportation conditions, time and corresponding packaging shall be validated. For frozen products, attention should be paid to the effect of freezing and thawing on product quality, and the product quality after recovery should meet the requirements for clinical use. For fresh products requiring no freezing, the storage and transportation conditions and time should not only ensure product quality, but also meet the aging needs for clinical use.

IV. Risk Assessment and Control

　　Immune cell products are characterized by diversity, heterogeneity, complexity. Different types of such products may have varied degrees of risk. Therefore, it is necessary to carry out comprehensive risk assessment based on the characteristics of products, from raw materials, manufacturing process, product quality control, stability, clinical application process and other factors. Refer to the risk management concept of ICH Q9, scientifically use the risk assessment tools to identify, analyze and assess various risk factors of specific varieties, and formulate corresponding risk control measures according to the risk assessment results. Risk assessment and control runs through the whole product life cycle. With the deepening of research and the accumulation of product cognition, it is necessary to continuously track, analyze and update the risk factors, collect data to further determine their risk characteristics and formulate corresponding control strategies.

　　According to the current product research situation, the pharmaceutical risk factors of immune cell therapy products may be derived from the following categories:

　　(1) Source (such as autologous/allogeneic, human cell lines, etc.), acquisition mode, type and biological characteristics of cells (such as proliferation, differentiation and migration ability, cell function, secretion of active substances, ability to initiate/enhance/suppress immune responses, etc.).

　　(2) Safety risks of materials, such as the use of raw materials of human/animal origin.

　　(3) Manufacturing process of cells, such as equipment openness/airtightness, possible confusion in manufacturing process, endogenous and exogenous contamination/cross contamination; degree of operation on cells, such as in vitro culture/ expansion/ activation/ induction/ gene modification/ frozen storage/ recovery/ transportation; degree of effect of operation on cell characteristics, such as the effect of gene modification on cell function.

　　(4) Quality study and quality control, whether existing study and inspection means or methods can fully characterize the characteristics of the product and control its quality, such as biological activity and purity study (such as non-target cell population, impurity residue and non-cellular components). Whether the test method and test indicators are applicable, and whether the validation of the new test method is adequate, for example, whether the new test method is equivalent to the pharmacopoeia method, and the risk of false negative or false positive results with the new rapid test method.

　　(5) Storage, transportation conditions and time of cells for production and final cell products, airtightness and compatibility of storage containers.

　　(6) Formation of combination products with non-cellular materials (bioactive molecules or structural materials).

　　Other types of risk factors may include: administration method (such as systemic infusion, local application or surgical application); requirements for the quality, manufacturing cycle, storage method or transportation time of the product under different conditions of the recipient (such as whether pretreatment is required for the recipient, type, stage, severity or progression rate of the disease, etc.); and the reference of previous experience of similar products or relevant experience.

V. Materials for Production

　　Materials for production refer to all the raw materials, excipients and consumables used in the manufacturing process of immune cell therapy products. The source should be clear, the quality should be guaranteed, and special attention should be paid to prevent the introduction or transmission of exogenous factors. The material suppliers and contract manufacturers shall be subject to evaluation and audit. When necessary, quality risks shall be controlled by signing quality agreements.

(I) Raw Materials

　　Raw materials are directly related to the quality of the product. The risk assessment and quality control shall be conducted according to the requirements of "Quality Control of Raw Materials and Excipients for the Production of Biological Products" in Chinese Pharmacopoeia, and a good and standardized quality management system for raw materials shall be established. Based on the characteristics of immune cell therapy products and their manufacturing process, it is recommended to use the raw materials meeting pharmacopoeia standards or approved for human use as far as possible, otherwise the raw materials with high quality standard and low risk level shall be used as far as possible to ensure their safety and applicability. Raw materials include starting materials (such as cells for production, auxiliary cells for production, in vitro gene modification system) and other raw materials (such as culture medium, adding factors, other biochemical reagents, etc.).

　　1. Starting Raw Material

　　1.1 Cell for Production

　　According to the current development of biotechnology, the sources of cells for production include human donor sources (autologous cells, allogeneic cells) and human cell line sources. The source of donor cells shall meet the requirements of relevant national laws, regulations and ethics, and establish the management system of "informed consent and confidentiality". Cell lines shall have clear source, clear passage history and controllable safety risks.

　　1.1.1 Cells for Production from Donors

　　Selection of Donors:

　　In order to ensure the product quality as well as the biosafety of manufacturing environment and manufacturing personnel, based on the study, product risks and donor cell use needs, it is necessary to establish reasonable donor screening procedures and criteria and try to collect the relevant characteristics of donors, including but not limited to age, gender, previously known medication and radiation exposure, residence in epidemic area, past medical history, family history, pathogenic microorganism screening information, HLA (human leukocyte antigen) typing information, blood type and blood routine test. The criteria for donor screening vary according to product characteristics, but need to be reasonably set and the corresponding risks can be controlled.

　　In terms of pathogenic microorganism screening, allogeneic donors shall at least meet relevant national regulations for blood donation, such as screening donors for the presence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), treponema pallidum and other infections. According to the actual situation of the product, the corresponding detection items can also be added. For some specific products, the detection is required for those with clear risks and clear virus detection requirements. For example, in addition to the above pathogenic microorganisms, the donors of T cell therapy products are also recommended to perform the detection of human cytomegalovirus (HCMV), human Epstein-Barr virus and human T-cell lymphotropic virus (HTLV). Autologous donors are also required to conduct appropriate pathogenic microorganism screening to ensure that the manufacturing process and product use will not cause contamination or add additional risks to the patients themselves. According to the health/disease history of donors or the specific situation of living in epidemic areas, corresponding screening items may also be added in time, and the acceptance criteria and procedures shall be established. In order to ensure the sensitivity of the detection method and the reliability of the detection results, it is recommended to use the kits approved by the regulatory authorities and preferentially use the blood screening kit to detect pathogenic microorganisms. Allogeneic donors also need to consider window period for pathogenic microorganism screening.

　　In addition to the detection of pathogenic microorganisms, the screening items for donors may be added according to the clinical use and manufacturing needs. For example, for the products of allogeneic origin, it is recommended to timely evaluate the typing including polymorphisms, such as blood type, matching of major histocompatibility antigens (Class I and/or Class II HLA) between donors and recipients. In some cases, it may be necessary to pay attention to minor histocompatibility antigens, clarify and establish typing procedures and criteria.

　　Acquisition, processing and testing of cells/tissues:

　　In order to ensure that the quality of donor cells meets the manufacturing requirements, it is necessary to evaluate and review the medical institutions responsible for cell/tissue collection, select the medical institutions with relevant qualifications as the institutions for donor cell/tissue acquisition, establish the list of cooperative medical institutions, develop the corresponding operating specifications for cell collection, encourage the signing of relevant quality agreements, and regularly review, analyze and evaluate the quality of the donor cells/tissues collected by the medical institutions and the final products of clinically applied cells.

　　The acquisition process of cells/tissues needs to be fully studied. Determine the cell or tissue source, collection method, and other relevant identifying information, based on the characteristics of the product and based on the study, including but not limited to collection site/environmental requirements, equipment and procedures used, reagent and consumables used, blood volume collected, etc. Acquisition of cells/tissues includes single blood collection, peripheral blood collection, separation of lymphoid tissues, umbilical cord blood collection, tumor tissue separation and other collection methods. It is recommended to comprehensively consider cell types, health status of donors and cell requirements, and give priority to the collection methods that are easy to be standardized, such as blood component apheresis technology. The methods to obtain cells/tissues shall be studied and demonstrated, including but not limited to the type of related enzyme, anticoagulant, blood separation instrument and procedure (circulating blood volume, flow rate, etc.), surgical methods, etc. Minimize the collection of related impurities, such as cell debris, non-target cell content, etc., and consider reducing the degree of damage to donor tissues and organs. Avoid unnecessary or improper processing and handling steps to avoid damaging the integrity and/or function of cells, thereby reducing the risk of adverse reactions or treatment failure. The medical staff collecting cells/tissues must receive strict training, and can only operate after obtaining the corresponding qualification and authorization. The training shall be recorded. Collection operations should be performed in an environment that ensures the microbiological safety of the cells/tissues collected. The risk of microbial contamination, sample cross-contamination or mix-up should be controlled during collection.

　　If the collected cells/tissues need further processing, such as mixing, batch, packaging, preservation and transportation, corresponding study and validation shall be conducted, and appropriate storage conditions, transportation method and time shall be determined according to the study situation, and corresponding operating specifications shall be developed.

　　When the collected cells/tissues arrive at the factory, it is necessary to make inspection on appearance, package integrity and other aspects as well as confirm the transportation temperature, time and donor information. Prior to manufacturing, detection on cell type, quantity, phenotype, survival rate and microorganism may be performed according to process requirements and product characteristics. For example, cell type may be identified and confirmed by relevant genotype and/or phenotype markers, and the proportion of cells with positive markers may be used as the basis for the evaluation of expected cell population indicators. It is encouraged to study cell/tissue quality indicators related to the quality of the finished product and include them in the quality release criteria for cells/tissues collected.

　　1.1.2 Cell Line Derived Production Cells

　　For immune cell therapy products derived from human cell lines, the cell lines used should meet the requirements for clear sources, clear passage history, comprehensive and qualified test results. In principle, cell lines should be banked and managed hierarchically for production. The hierarchy of cell bank may be comprehensively considered according to the characteristics, manufacturing and clinical application of the cells; and the test criteria for cell bank may be established according to the relevant requirements of Chinese Pharmacopoeia, ICH Q5A and ICH Q5D, and the test results shall meet the requirements. Cell lines may be genetically modified for production. If possible, it is recommended to build and test cell lines after genetic modification.

　　1.1.3 Storage of Cells for Production

　　It is recommended to establish or adopt a stable and controllable cell storage system or platform, study and determine the appropriate storage conditions and packaging materials for donor cells or cell lines, properly preserve the cells without changing the cell characteristics, ensure that the risk of microbial contamination is not increased during the storage process, and that the cell survival rate, density, purity and biological function can meet the manufacturing requirements.

　　1.2 Manufacturing of Auxiliary Cells

　　Depending on the use or function, the manufacturing of auxiliary cells may be viral packaging cells, trophoblasts (Feeder cells), etc. The manufacturing of auxiliary cells shall meet the basic principles of clear source and culture passage history, controllable safety risks, grading management of cell bank (if applicable) and qualified test results. If expansion culture of manufacturing of auxiliary cells is required, it is recommended to complete expansion culture of final manufacturing of auxiliary cells at one time as far as possible, or ensure the consistency of each expansion culture process and quality, and assess whether new risks are introduced in the expansion culture process. If applicable, it is recommended to establish testing procedures at different manufacturing steps/stages, such as testing time, testing items, testing methods and acceptance criteria. Attention needs to be paid to their species-specific virus detection and possible safety risks introduced. The process involving inactivation treatment of trophoblasts, such as irradiation or addition of drugs, should be studied and validated.

　　1.3 Gene Modification System

　　If gene modification is involved, please refer to relevant technical guide for gene modification system, which will not be repeated in this paper.

　　2. Other Raw Materials

　　A variety of materials are also required in the collection, sorting, culture of cells and gene modification of cells, such as culture media, enzymes, antibodies, cytokines, serum, antibiotics, magnetic beads, other chemicals or solid supports (such as gel matrices), and the use of these materials may affect the quality of immune cell therapy products. The contents of risk assessment include the source, composition, function, use stage and quality control of raw materials. The relevant documents required include proof of source, COA, package insert and TSE/BSE-free statement, so as to prove that it meets the requirements for use and is suitable for its intended use. Based on the risk situation, the manufacturing process may refer to the relevant principles or requirements of GMP.

　　If antigens are required in the manufacturing process, the requirements for clear sources, risks and controllable quality shall be met. For recombinant expressed or synthesized antigens, the selection basis, antigen sequence, risk factors related to manufacturing process and quality control shall be clearly defined, and the impurity control of antigens (including exogenous viral agents) shall be clearly defined. For tumor cell lysate antigen, attentions shall be paid to the stability and quality-related risks of manufacturing process, such as tumorigenicity or contamination by exogenous factors. It is necessary to fully evaluate the immune-related risks possibly caused by antigen residue in the immune cell therapy products.

　　Minimize the use of potentially allergenic materials such as beta-lactam antibiotics (e.g., penicillin) during manufacturing process. Try to avoid using raw materials of animal origin, such as animal serum and protein of animal origin, and try to use non-animal derived materials with clear components instead. If raw materials of animal origin must be used, corresponding studies shall be carried out to prove the necessity and rationality of their use. A complete quality control system shall be established according to the species source, manufacturing region, manufacturing process and other characteristics of raw materials to assess the TSE/BSE safety risk, and detect the residue of raw materials of animal origin and carry out the safety risk assessment. It is strictly forbidden to use animal serum/plasma derived from epidemic areas, and serum/plasma without safety verification. If autologous serum or autologous plasma is used during the manufacturing process, it is needed to carry out the study on manufacturing process, quality, stability, packaging and storage, etc. During the manufacturing process, the use of non-pharmaceutical allogeneic human blood-derived materials shall be avoided as far as possible. If it is really necessary to use them, it is required to carry out the study on the contamination with exogenous factors, effectiveness and batch-to-batch consistency by referring to the relevant requirements for blood products and develop a reasonable internal control standard in combination with the manufacturer's release test standard.

(II) Excipients

　　For excipients, please refer to "Formulation and Process of Drug Product" in "Manufacturing Process".

(III) Consumables

　　The culture bottles, tubing, filters and other disposable consumables, culture and packaging containers used in the manufacturing process as well as the manufacturing equipment and materials in contact with the intermediate samples should be strictly screened, the applicability and biosafety should be evaluated, and the corresponding compatibility study should be carried out according to the evaluation results.

　　Immune cell therapy products may form combination products with other medical devices, matrices, microcapsules and other materials. For the cell part, refer to this guideline. The interaction and risk of cells with devices and other materials should be investigated and evaluated for the overall combination products.

VI. Manufacturing Process

　　The manufacturing process of immune cell therapy products in the manufacturing plant usually includes the whole process from the receipt of donor cells/tissues or initial culture of cell lines to the final cell harvest, preparation, storage and delivery. The overall manufacturing process shall be subject to adequate study and validation to determine a stable and feasible commercial manufacturing process, including but not limited to cell receipt, freezing of collected cells (if applicable), in vitro operation, preparation and product freezing, etc. The determined manufacturing process includes reasonable process operation steps and parameters, manufacturing process control and acceptance criteria, etc. The whole manufacturing process should be monitored, including the monitoring of process parameters and process control indicators. The study and validation of the manufacturing process of combination product shall also include all the process steps of the combination of individual components to form the final combination product, so as to ensure the feasibility and stability of manufacturing process.

(I) Process Study

　　With the deepening of research, the manufacturing process needs to be continuously optimized. In the process study, it is recommended to use the cells/tissues consistent with the actual source and quality of manufacturing as far as possible to carry out the study. If the amount of cells is limited (e.g., autologous cell products), studies with cells of similar characteristics, representative, and sufficient number may be considered.

　　1. Manufacturing Capacity and Lot Definition

　　The manufacturing capacity of immune cell therapy products directly affects the number of patients receiving treatment, the number of treatments and the quality of products, which is determined by various factors such as cell characteristics, manufacturing process, factory buildings, personnel, facilities and equipment, and clinical use, etc. The manufacturing capacity shall be validated through study. In the stage of transformation from laboratory preparation to industrial manufacturing, it is necessary to pay attention to the way of manufacturing capacity expansion and carry out research to ensure product quality. In the study on expansion of manufacturing capacity, if the manufacturing process and manufacturing volume of each batch of products remain unchanged at all times, but the manufacturing capacity is expanded by increasing the manufacturing batches, it is necessary to focus on the verification of raw materials and excipients, personnel, public facilities, equipment, manufacturing environment and quality monitoring and inspection, so as to ensure that the expansion of manufacturing capacity will not affect the product quality. If a new manufacturing process is introduced in the manufacturing capacity expansion study, such as the use of equipment such as multi-layer cell factory or cell reactor, it is necessary to focus on the possible impact of the changed process on the quality and carry out corresponding comparability study or evaluation.

　　The purpose of defining batches is to ensure the quality uniformity and traceability of immune cell therapy products. The products of the same batch shall have consistent sources and uniform quality, and shall be able to make quality evaluation on the whole batch of products after sampling inspection as required. Due to the diverse and complex processes of immune cell therapy products, appropriate batch definitions can be developed in combination with product process characteristics. According to the existing product process, the batch of immune cell therapy products may be considered to be defined as: a certain number of products with uniform quality produced under the same manufacturing conditions using the same manufacturing process in the same manufacturing cycle are considered as a batch. The total amount of all cells produced in this single batch is the batch size of this production.

　　2. Manufacturing Process Development

　　The process development of immune cell therapy products should be based on the target product quality profile, combined with physical and chemical characteristics and biological characteristics, rationally design of trials, continuously optimize process, and gradually establish stable manufacturing processes and key process parameters. For the research on the quality profile of target products, the characteristics of the products, such as surface markers, cell viability, purity, biological activity, target gene transduction efficiency, shall be analyzed from several aspects, and the key quality attributes that may affect the safety and effectiveness of the products shall be preliminarily obtained. Critical process parameters are determined based on the impact of process parameters on critical quality attributes, and matched parameter ranges are established and continuously optimized with process research and experience. Possible critical process parameters include but are not limited to: starting cell count, medium composition, and process parameters related to cell expansion, induction, and gene modification operations.

　　In the development of in vitro cell culture process, the condition of in vitro cell growth and the effect of any operation on the cells shall be considered to maintain the integrity and functional characteristics of the cells. The operation steps (liquid replacement, passage, activation, gene modification, induction, etc.), added components (culture medium, recombinant protein and related growth factors, serum substitutes, magnetic beads, viral vectors, nucleic acid substances, transduction/transfection reagents, etc.), culture vessels, culture conditions (such as temperature, dissolved oxygen, pH, etc.), impurity removal, culture time or the maximum passage number, culture scale and parameter settings shall be appropriately studied and validated. In order to monitor the quality of cells, it is recommended to establish test methods and criteria to continuously monitor the characteristics of cells during the manufacturing process, such as genotypic and/or phenotypic changes and functional changes of cells after cell culture expansion to determine or optimize the manufacturing process.

　　If the culture medium for cells is not liquid culture medium, but culture medium such as non-liquid matrix/device/stent (top), it is necessary to consider its effects on cell growth, function and integrity, such as the changes in cell environment (such as the changes in pH value, ion concentration and air-liquid interface) that may be caused by degradable biomaterials. In addition, it is necessary to consider the possible effects of cells on culture medium (such as degradation rate, medium morphology and medium composition).

　　If there is an in vitro cell induction operation, the induction method and conditions should be studied, and the study and validation should be performed in combination with the changes in cell growth characteristics, changes in cell phenotype and/or genotype, changes in cell function, residues of induced substances, and changes in the proportion of target cell population and non-target cell population, with continuous optimization.

　　If there is an in vitro cell gene modification operation, the operation method (such as electroporation, viral vector transduction, etc.) and conditions should be studied, such as the selection of transduction/transfection reagents, transduction equipment (such as electroporator), multiplicity of virus infection (MOI) and other conditions. The process can be studied, optimized and validated in combination with the transduction/transfection efficiency of target gene, integration of target gene in the chromosome, expression stability of target gene, changes in cell genotype and/or phenotype, function, residue and removal of risk genes for carcinogenicity, reverse mutation in virus replication capacity, insertion mutation or insertion site.

　　3. Formulation and Process of Drug Products

　　The overall goal of drug product studies is to ensure the reasonable dosage form and formulation, stable process, effective control of manufacturing process, and suitability for industrial manufacturing. In the study, the dosage form, formulation and prescription process of the product are determined according to its own characteristics and clinical application. Drug product studies generally include:

　　(1) Selection of Dosage Forms

　　Immune cell therapy products are usually injections. If other dosage forms are used, they should be selected rationally in combination with clinical use.

　　(2) Formulation Study

　　The formulation should be finally determined through formulation screening and optimization by reasonably design trials according to the characteristics of immune cell therapy products, stability research results, in combination with the characteristics of dosage form, usage and route of administration. Formulation studies focus on strengths, excipients composition and dosage, usage, as well as the stability manifestation of products in the process of storage, transportation and use. The formulation of the drug products should be adapted to the storage conditions, and refrigeration and/or freezing are often involved for immune cell therapy products. In the study, the effects of cryopreservation and/or freezing conditions and time on cell characteristics and viability should be validated so as to determine the formulation of drug products. The storage time of non-frozen immune cell therapy products is usually short, and its formulation of drug products should meet the requirements for stable product quality during storage, transportation and use.

　　The use, dosage and quality of excipients should be studied and validated to prove the necessity, safety and rationality of use. Excipients for pharmaceutical use or approved human use should be preferred, otherwise comprehensive studies and assessments are required. For novel excipients, in addition to the above studies, it is recommended to carry out appropriate non-clinical safety studies, specifically by referring to the relevant technical guidelines that have been issued. Cryoprotectants are commonly used for frozen immune cell therapy products, which are mainly divided into penetrating cryoprotectants (such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, etc.) and non-penetrating cryoprotectants (such as polyvinylpyrrolidone, albumin, sucrose, trehalose, etc.). In the selection of cell cryoprotectants, the following factors may be considered: toxicity and immunogenicity of cell cryoprotectants (such as DMSO, albumin, etc.), effects on cell characteristics, function and stability, acceptable criteria for removal methods or residual amount, cryoprotectant equipment and procedure, quality, source, composition, dosage and usage of cell cryoprotectants. The components, dosage and rationality of cell cryoprotectants (such as DMSO, etc.) should be verified in the study. In combination with the selected cryoprotectant, if the product needs to undergo physical state change, filtration, cleaning, container conversion, dose adjustment, combination with other materials and other operations before being used to the recipients, adequate study and verification should be performed.

　　(3) Process Study of Drug Products

　　Process study and validation should be conducted according to the characteristics of cell products, stability research results, in combination with manufacturing conditions and equipment, to determine the manufacturing process of drug products and establish appropriate process control standards. The process study of drug products can be carried out alone or in combination with formulation study at the same time. The risk prevention and control of confusion and contamination, the adsorption or effect of contact materials (containers) on cells during the drug product process, and the effect of shear force generated by filling on cells should be considered for the drug product process study, while ensuring that the cell number and density meet the requirements.

(II) Process Control

　　Good process control is the key to ensure product quality. Appropriately establish the sampling time points, test items and criteria for manufacturing process control or the output criteria of relevant process parameters, so as to ensure the stability of product manufacturing process and the consistency of product quality among different batches. For the closed cell culture system, the sampling operation of process control can be arranged as appropriate according to the characteristics of closed system structure and sampling process, so as to prevent contamination.

　　Establishing a reasonable process control strategy based on the characteristics of product and manufacturing process, it is recommended to pay attention to the following aspects: (1) monitor sample mix-up and cross-contamination, including donor materials, intermediate samples and products in the manufacturing process, pay special attention to the effective isolation of time/space for cell operations from different donors or cell lines; perform site-clearing after the end of each manufacturing, and perform cleaning and disinfection treatment using validated standard procedures. Prior to each manufacturing operation, the site-clearing shall be confirmed. (2) Monitor the contamination of microorganisms and their metabolites/derivatives (such as endotoxin). If applicable, it is recommended to carry out sterility, mycoplasma and other safety-related tests on appropriate intermediate samples at key time points or take relevant measures to control them. (3) Monitor the key process parameters or key quality attributes in the manufacturing process, such as cell survival rate, proliferation ability, cell phenotype, impurity content, biological activity, etc. In-process quality monitoring and release testing can be combined and supplemented. (4) Ensure the traceability of samples and manufacturing materials throughout the manufacturing process (including the whole process from cell/tissue collection, manufacturing, transportation to clinical application).

(III) Process Validation

　　Process validation of immune cell therapy products may follow the general principles for process validation of biological products and carry out process validation for each operating unit of the determined manufacturing process, storage conditions and time of intermediate samples, preparation and storage conditions of culture medium/buffer solution and transportation process. Process validation should demonstrate that the manufacturing process consistently produces a product meeting its intended use and regulatory requirements according to defined process parameters.

　　Under the condition of ethical and informed consent, it is recommended to use the cells similar to the clinical application scenario (such as patient-derived cells) to carry out the corresponding study in the process validation; under the condition of adequate process study, the autologous cell therapy products or other products with limited cell sources may also be considered to use the healthy donor cells that are considered to be representative after study and evaluation for relevant process validation after marketing.

　　In the validation work, it is necessary to pay attention to the study and validation of the maximum manufacturing capacity at the same stage at the same time. The maximum manufacturing capacity actually produced shall not exceed the validated maximum manufacturing capacity. The increase of manufacturing capacity shall be subject to appropriate validation. In the study, the support capacity for the maximum manufacturing capacity in terms of raw materials and excipients, personnel, facilities and equipment, environment, quality detection capacity and overall operation capacity should be considered, and the validation for the worst conditions should be considered. After completing the validation of commercial manufacturing process, continuous process study and validation should also be conducted to ensure that the process is in a controlled state.

VII. Quality Study and Quality Control (I) Quality Study

　　Quality study is the basis for process optimization and improvement, formulating overall control strategy and ensuring product quality, which runs through the whole life cycle of products. Comprehensive quality study is conducive to the determination of critical quality attributes and needs to be continuously supplemented and improved with the deepening of product understanding and the development of technology. For quality studies, representative samples from the appropriate study stage (e.g., nonclinical study batches, clinical trial batches, commercial manufacturing batches) or appropriate step (e.g., donor cells or cell line cells, in-process intermediate samples or finished products) should be used.

　　The quality study of immune cell therapy products generally includes safety study, purity and impurity study, functional study and study of other items. Other relevant studies may also be added according to the characteristics of the product.

　　1. Safety Study

　　Mainly include microbiological safety study and product related safety study. The former refers to the study on microbial contamination and microbial metabolite/derivative contamination, such as fungi, bacteria, mycoplasma, viruses, endotoxin, etc.; the latter refers to the study on the product itself which may lead to safety problems in addition to microbiological safety, such as malignant transformation of cells, non-target cell residues, etc. Carry out the safety study in the above two aspects according to the cell type, characteristics and sources, manufacturing process and relevant material characteristics. It is recommended to include at least the following aspects (if applicable):

　　Adventitious Agents: Cells used in manufacturing, manufacturing auxiliary cells, other human-derived/animal-derived raw materials, and adventitious agents may be introduced during manufacturing. Based on routine adventitious agents testing, specific adventitious agents can be tested in combination with in vivo and in vitro methods depending on the adventitious agents that may be introduced. For example, if bovine serum is used in the manufacturing, the detection of bovine specific virus is required; if porcine trypsin is used, the detection of porcine specific virus is required; if trophoblasts are used, the detection of cell species-specific virus is required. For allogeneic therapy products, when carrying out human-derived virus detection, attention should be paid to the possibility that the donor is in the window period of infection, and secondary sampling and detection should be timely carried out.

　　Replication competent virus (RCV): RCV is generated by reverse mutation of viral vector and is an important test item related to product safety. It needs to be studied by applicable detection methods.

　　Malignant transformation of cells: In some cases, the cells in the product have the possibility of malignant transformation (including but not limited to tumorigenicity, pro-tumorigenicity, etc.). In this case, the possibility of malignant transformation of cells can be studied and evaluated in combination with in vivo and in vitro experiments according to the source of immune cells, the characteristics of target cells in the product or residual impurities and other factors.

　　Gene insertion site and copy number: As it is related to the safety and efficacy of the product, it is necessary to use an applicable detection method for study and explore its correlation with the safety and efficacy.

　　Abnormal immune response: It is recommended to select appropriate method for immunological response detection for the cell products of allogeneic origin.

　　Non-target cell and impurity studies: See below for details.

　　2. Purity and Impurity Studies

　　The quality and biological activity of immune cell therapy products are often related to the purity of the target cells in the product. The actual situation may be complicated: on the one hand, different types of products have different requirements for the purity of target cells; on the other hand, different cell populations in the same type of product or have different effects on the biological activity of the product, and it is necessary to study the proportion of cells with different cell populations or. Cell purity studies may include, but are not limited to:

　　Proportion of viable cells: The proportion of viable cells needs to be investigated using an appropriate method. When an immune cell therapy product is a single cell species and homogeneous, the purity of the product can be studied by directly detecting the proportion of viable cells in the product.

　　Cell population or subsets proportion: When an immune cell therapy product is a mixture of cells of different types or different genotypes/phenotypes, it is recommended to study the composition and proportion of cells in the sample, such as the composition and proportion of cell population or subsets. For example, depending on the stage of maturation (naive, senescent, exhausted, etc.), immune cell populations or subsets are studied.

　　Proportion of target cells: The purity of the product can be studied by detecting the proportion of target cells. For example, in CAR-T products, after CAR transfer operation, the target cell population for purity analysis should select the target cells that can correctly express both CAR and T cell surface markers, excluding T cells that do not express CAR and cells that express CAR but have incorrect T cell surface markers.

　　Proportion of non-target cells: Non-target cells may adversely affect product quality. Therefore, cell purity studies include qualitative and/or quantitative studies of non-target cells. For example, the residues of non-target cells such as tumor cells and iPS cells have a high safety risk, so it is necessary to study their proportion and carry out strict control. After study, when non-target cells have no effect on product safety and efficacy, their composition and proportion should be studied, and batch-to-batch consistency should be controlled when necessary.

　　Impurities:

　　Process-related impurities: refer to the impurities introduced in the process, such as residual proteases, induction reagents, transduction/transfection reagents, serum, viral vectors, and residual magnetic beads, fibers and plastic microbodies, trophoblasts, etc., which need to be studied by appropriate methods.

　　Product-related impurities: such as non-target cells, unexpected cell expression products, residual dead cells, cell debris and other possible degradation products, the applicable methods should be used for the study.

　　For the possible high-risk impurity components in the product, the removal method and the quantitative detection method for residues should be established and specified. If the impurity components cannot be effectively removed, the safety and toxicity assessment should be performed in the animal model or other systems, and the residue limit should be set according to the results of the maximum human exposure dose or in vivo safety study.

　　3. Functional Study

　　Functional study is a study to evaluate whether immune cell therapy products have the expected biological function through in vivo/in vitro functional analysis experiments. According to the properties, characteristics and intended use (indications) of cell products, especially the specific mechanism and indicators to achieve clinical therapeutic effect, establish and validate appropriate in vivo/in vitro functional analysis methods and carry out functional studies. Their functional studies may include, but are not limited to the following:

　　Differentiation/developmental potential: It can cover the possible differentiation/developmental direction of cells in the product. Among them, differentiation/developmental function related to the safety and effectiveness of clinical application is recommended to be included in quality control as a representative evaluation content.

　　Qualitative and quantitative study on expression products: When the functions of immune cell therapy products involve the expression of endogenous or exogenous gene products, the study on expression products shall be carried out, such as the type, characteristics, expression level, modification degree (such as glycosylation, phosphorylation, etc.) and polymerization (such as homopolymer or heteropolymer, etc.) of expression products.

　　Response to exogenous stimulation: cytological response can be studied after related factors act on cells, such as changes in cell morphology, cell proliferation, secretion of cytokines, phenotype, signaling pathways, and metabolism, etc.

　　Biological activity: According to the product characteristics and mechanism of action, carry out the study on the biological activity corresponding to the intended function of the product in vivo, such as the effect of target cells (such as lysis reaction, induction of apoptosis or proliferation), secretion of specific factors. If functional cells are obtained after stimulation with added components (such as induction, antigen load, etc.) or gene modification (such as gene editing, exogenous gene expression, etc.) during the manufacturing process, it is necessary to carry out applicable biological activity study on the cells before and after stimulation or operation and compare and analyze the function of cells before and after stimulation or operation. When direct research assays are limited by the number of cells required or other conditions, a reasonable alternative assay may be developed and implemented.

　　4. Studies in Other Items

　　In terms of physical and chemical properties, the appearance, pH, osmotic pressure and obvious visible particles can be studied according to the product characteristics and dosage form.

　　In terms of cell survival rate and proliferation ability, applicable detection methods can be used, such as viable cell count, cell doubling time analysis, cell cycle analysis and colony formation rate analysis.

　　The manufacturing and/or use of certain immune cell therapy products may require blending of many different cell types, allowing quality studies of the blending characteristics of the product and identifying critical quality attributes of the blended product. Prior to blending, relevant quality studies should be conducted separately for each individual immune cell therapy product to determine their respective critical quality attributes.

(II) Quality Control

　　1. Specification

　　The specification shall be established based on comprehensive risk analysis, accumulated manufacturing and clinical trial experience as well as statistical analysis (if applicable), reliable scientific knowledge and in combination with the results of quality study and stability study. The specification includes test items, test methods and standard limits. The stage of sample testing may be reasonably set according to the testing requirements, such as intermediate sample, release test and reserved sample, so as to effectively control the product quality. Inspection items generally include identification, biological activity, purity, impurities, transgene copy number (if applicable), cell number (viable cell number, functional cell number/proportion, etc.) and general detection (such as pH, osmotic pressure, sterility, mycoplasma, bacterial endotoxin, appearance, obvious visible foreign matter, etc.).

　　(1) Test Item and Test Method

　　Cell identification: It is recommended to use applicable and specific detection methods and multiple methods for identification when necessary. The methods used may be cell morphology, HLA analysis, genetic polymorphism analysis, karyotype analysis, STR analysis, metabolic enzyme subtype profiling, cell surface marker and specific gene expression product analysis.

　　Purity: In combination with quality study, select the indicators for purity test according to product characteristics, such as cell surface marker and specific biological activity.

　　Sterility and mycoplasma: carry out the test for bacteria, fungi and mycoplasma according to the sterility test method and mycoplasma test method specified in Chinese Pharmacopoeia. Under special circumstances, such as limited sample size or the need for rapid release, if pharmacopoeia methods cannot be met, the development of new sterility and mycoplasma test methods for release testing may be considered, but new test methods should be fully validated. At the stage of accumulating data, new and fully validated methods and compendial methods can be employed in parallel.

　　Bacterial endotoxin: The bacterial endotoxin shall be tested according to the bacterial endotoxin test method in Chinese Pharmacopoeia, or other applicable validated methods shall be adopted.

　　Cellular endogenous and exogenous viral agents: The endogenous and exogenous viral agents that may be introduced in the manufacturing process of immune cell therapy products and their products are determined according to the results of quality study. On this basis, the appropriate methods such as cell culture, nucleic acid or protein detection, fluorescent antibody detection are selected for the detection of endogenous and exogenous viral agents.

　　Replication competent virus (RCV): For the immune cell products genetically modified with viral vectors, RCV is an important safety risk concern. In addition to the detection and control at the viral vector stage, it is also necessary to establish perfect detection and risk control strategies for the final cell products. When rapid RCV test methods are used for cell release testing, sample retention is recommended. In the clinical trial stage or early marketing stage, indicator cell culture method is used to retain samples for concurrent detection and analysis and accumulate data; in the post-marketing maturation stage, retained samples can be used as necessary study and analysis.

　　Biological activity: It is recommended to select the method that can characterize the biological activity of the product and is suitable for release testing. In some cases, if the product has multiple mechanisms of action, or a single test method cannot fully reflect its mechanism of action, more than one method may be considered for biological activity testing.

　　Impurity control: According to the results of manufacturing process and quality study, define the residual process-related impurities and product-related impurities in the product manufacturing process that may affect the product quality, and select the applicable methods for detection and control. If it has been adequately validated that general process-related impurities can be effectively and consistently removed by process, they can be controlled in combination with the process.

　　Tumorigenicity/Oncogenicity: If applicable, inclusion in the tumorigenicity/oncogenicity control program may be considered based on the results of quality studies.

　　In addition to the test items and methods listed above, reasonable adjustment may be made in combination with the actual study situation.

　　(2) Standard Limit

　　The specification limit may be established based on the product R & D related data, non-clinical study batch test data, clinical trial batch test data, process validation data and stability study data, while taking into account the product characteristics and current scientific understanding and consensus. It is recommended to establish the standard limit based on the test data of clinical trial batches.

　　2. Validation of Test Method

　　Test methods should be studied and validated, especially for self-built product-specific methods. Suitability should be confirmed for methods included in pharmacopoeias. When methods of pharmacopoeias are revised or substituted, their rationality should be validated.

　　Methodological validation study should pay attention to the rationality of positive control, negative control, inhibition control (if applicable), sampling representativeness and sampling size of test article, detection indicators and judgment criteria.

　　For products with short shelf life or small sample size, new rapid and trace detection method can be used. New test methods should be adequately validated, compared and assessed with compendial test methods, as appropriate.

　　In case of any change in the detection method during the R & D process, the changed method shall be evaluated and studied to prove that the proposed change method is superior to or equivalent to the pre-change method.

　　3. Standards/Controls

　　Where possible, standards/controls can be established as needed to meet the needs of the test and help determine that the equipment and reagents work within the specified range, improving the reliability and accuracy of the test results.

　　Established standards/controls should have a clear intended use, be tested and calibrated using validated detection methods, and the standards/controls used at each stage of development should be traceable and corresponding stability studies should be conducted.

　　4. Other Conditions

　　Product release testing is an important guarantee to ensure that product quality meets clinical application, but some immune cell therapy products may not complete all release testing before clinical use due to their short aging. In such cases, release for use may be considered before the results of full release testing are available if the risk is assessed by adequate studies and demonstrated to be manageable (release for use); release for use is not recommended when the risk is not assessed by adequate studies or assessed to have potentially serious and irreparable consequences.

　　In order to strengthen quality control and reduce risks, it is recommended to consider the following measures:

　　(1) When the time for release testing is limited, it may be considered to strengthen the quality control and process control of raw materials and combine them with release testing to control the risks.

　　(2) In terms of detection methods, rapid alternative detection methods can be used for detection to minimize risks. Before the validation of alternative test methods is fully completed, parallel testing of pharmacopoeias methods and alternative test methods is required to accumulate data and continuously optimize them in the study.

　　(3) Full release testing should continue to be completed while release for use. It is necessary to take full consideration of relevant risks, formulate measures in advance and initiate emergency plans applicable to relevant risks in case of abnormal or nonconforming release test results.

　　5. Quality Approval before Use

　　The product shall be subject to quality approval before administration, especially when there are operations such as cell recovery and dilution before use. The approved contents may include but not limited to: label verification; review of storage and transportation conditions; review of operating procedures; observation of appearance and obvious visible particles; observation of cell morphology (if applicable); determination of viable cell count and proportion (if applicable); rapid sterility test (if applicable).

VIII. Stability Study

　　The stability study of immune cell therapy products is to obtain the law of the change of its quality attributes over time under the influence of various environmental factors (such as temperature, freezing and thawing, etc.) through the design of trials, which is an important basis for the development of product specifications and product shelf life (or temporary storage period of intermediate samples). Meanwhile, it can also be used to judge whether the process parameters, formulation of drug products and packaging materials are reasonable.

(I) Basic Principles and Storage Stability Study

　　Immune cell therapy products may refer to general requirements for the stability study of biological products, and a reasonable study protocol should be designed based on the product characteristics, needs of clinical medication, packaging, storage and transportation.

　　Samples for study: According to the needs of specific manufacturing process and the availability of corresponding cells, representative samples will be selected to carry out the study, including collected starting cells, in-process intermediate samples, finished cell products, samples in clinical use, etc. The manufacturing, use and quality (such as total cell density and volume range, etc.) of samples for study should be representative of the actual situation. For the packaging container and closure system of sample, it should select small-size packaging container and closure system which are the same or of the same material as those for actual storage. For autologous immune cell therapy products, if the use of patient cells is limited, studies can be performed using healthy donor cells that are assessed to be representative.

　　Investigation condition: Select reasonable and comprehensive stability investigation conditions according to the actual situation and possible exposure conditions of the product during storage, transportation and use. For example, for the finished cell products, starting cells or intermediate samples requiring frozen storage, the changes in cell quality (such as cell number, survival rate, appearance integrity, function, etc.) under freezing and recovery conditions should be studied; when needed, the effect of multiple freezing and thawing can also be studied. In addition, according to the strenuous conditions that the product may be exposed to during storage, transportation and use, the study on such influencing factors as high temperature, irradiation and oscillation may be conducted.

　　Test indicators and test methods: Generally, reasonable and comprehensive test indicators are set in combination with product characteristics, including but not limited to physical and chemical properties, cell survival rate, proportion of target cells, biological activity, microbial safety indicators. The study should reasonably set the frequency of investigation of various indicators, such as sterility test or alternative test (such as container/closure integrity test) at least at the early and late stage of the proposed shelf life. If the stability data suggest that the excipients may have oxidation and degradation during the shelf life, which may adversely affect the product quality, it is necessary to monitor the content of excipients or related activities in the stability test. The detection method used should be subject to validation study, which can sensitively reflect the change trend of product stability.

(II) Transportation Stability Study

　　Cold chain transportation is generally required for immune cell therapy products, and corresponding stability simulation validation study should be conducted for the transportation process of products. For the stability study, it is necessary to take full consideration of various factors such as transportation route, transportation vehicle, distance, season, time and condition (such as temperature, radiation and vibration conditions), product packaging (such as secondary package and primary package), product placement and monitor condition (such as the quantity and position of temperature monitor). It is suggested to simulate the worst conditions for actual transportation to carry out the study. For the finished cell products, starting cells or intermediate samples stored in suspension in liquid, it is necessary to pay attention to the placement direction of product (such as upright, inverted or horizontal placement) and the effect of oscillation on the cells in the study. Transportation stability studies confirm that the stability of the product is maintained under the proposed storage conditions during transportation. It is also recommended to assess the impact of product quality during short periods of time out of the intended storage conditions (e.g., temperature, number of times, total time product is out of the cold chain).

(III) In-use Stability Study

　　The in-use stability study should be designed to consider the impact of clinical practice scenarios on product quality, such as the type of syringe and needle, aspiration and injection and drip rate, the type of infusion tube for intravenous drip, infusion pressure, and administration environment conditions (such as temperature, light, etc.) and time. For samples requiring recovery, dilution, mixing and/or temporary storage during use, studies are required to support the stability of the product during the in-use storage period. In the in-use stability study, it is also necessary to pay attention to the risk of microbial contamination during operation. The hold conditions and time after product thawing or clinical compatibility are justified based on the use of stability study data.

(IV) Identification of Storage Conditions

　　According to the results of stability study, storage conditions and shelf life of the product should be specified in the package insert and/or label. Products that cannot be frozen should be indicated separately. If the product requires radiation protection or freeze-thaw prevention, it is recommended to specify in the label of various container packages and package insert.

IX. Packaging and Sealing Container Systems

　　Safety assessment, compatibility study and functional suitability study are required to avoid unintended effect of materials, storage containers and packaging materials contacting samples during manufacturing on the quality of immune cell therapy products.

　　In the aspect of safety assessment, it is necessary to make adequate safety assessment on the material composition as well as its source, the risks possibly introduced in the manufacturing process (if applicable) and quality control. The results of basic performance test and biosafety assessment made by the supplier on the packaging materials can be used as reference basis.

　　For the compatibility study, the basic principles may refer to the requirements for the compatibility study on the packaging materials of general biological products. Based on a risk assessment, extractables/leachable studies may be conducted on materials or containers that come into direct contact and a safety assessment performed. The interaction between components with higher compatibility risk (such as excipient DMSO) and materials or containers shall be fully assessed in the study.

　　In terms of functional applicability, studies on container tightness/airtightness and freezing applicability can be generally considered.

　　In addition, the validation study shall also be carried out for the secondary packaging container (not in direct contact with cells) or material for transportation, including but not limited to heat preservation, airtightness, mechanical pressure resistance and light shading (if necessary).

X. References

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